Engineered RNA ligases

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Engineered RNA ligases with tailored amino acid sequences improve the efficiency and versatility of oligonucleotide synthesis, overcoming the limitations of chemical methods by efficiently ligating modified polynucleotides, thereby facilitating large-scale production with reduced environmental impact.

AU2025205747A1Pending Publication Date: 2026-07-16CODEXIS INC

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Patent Information

Authority / Receiving Office: AU · AU
Patent Type: Applications
Current Assignee / Owner: CODEXIS INC
Filing Date: 2025-01-03
Publication Date: 2026-07-16

Application Information

Patent Timeline

03 Jan 2025

Application

16 Jul 2026

Publication

AU2025205747A1

IPC: C12N9/00; C12N15/52

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Technology Topics

RNA Ligase (ATP)Polynucleotide

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AI Technical Summary

Technical Problem

Existing chemical synthesis methods for oligonucleotide therapeutics face challenges such as low efficiency, difficulty in scale-up, and environmental impact, particularly when producing modified oligonucleotides with non-standard internucleoside linkages and nucleotide analogs.

Method used

Development of engineered RNA ligases with specific amino acid sequences and substitutions to enhance the efficiency and promiscuity in ligating polynucleotides, including those with modified sugar residues and nucleobases, offering improved nucleic acid synthesis capabilities.

Benefits of technology

The engineered RNA ligases provide enhanced efficiency and versatility in synthesizing modified oligonucleotides, addressing the limitations of chemical synthesis and facilitating their large-scale production with reduced environmental impact.

✦ Generated by Eureka AI based on patent content.

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Abstract

The present disclosure relates to engineered RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA ligase polypeptides. The present disclosure also provides methods of using the engineered RNA ligase polypeptides or compositions thereof for molecular biological, diagnostic, and other purposes.

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Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63 / 618,203, filed January 5, 2024, U.S. Provisional Application 63 / 554,938, filed February 16, 2024, U.S. Provisional Application 63 / 646,753, filed May 13, 2024, and U.S. Provisional Application 63 / 718,750, filed November 11, 2024, all of which are incorporated by reference herein. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX10-262WO5_ST26.xml, created on January 3, 2025, with a file size of 6,025,556 bytes, is part of the specification and is incorporated by reference herein. TECHNICAL FIELD

[0003] The present disclosure provides engineered RNA ligase polypeptides and compositions thereof, and polynucleotides encoding the engineered RNA ligase polypeptides. The disclosure further provides methods of using the recombinant RNA ligase or compositions thereof for the synthesis of polynucleotides and as molecular biological tools. BACKGROUND

[0004] Oligonucleotide therapeutics, either as synthetic single stranded or double stranded polynucleotides, represent a class of therapeutics that exert their biological effects by modulating gene expression. The oligonucleotides are designed primarily to target pre-mRNA, mRNA, or non-coding RNA (e.g., miRNA) to promote RNA degradation, modulate splicing, interrupt translation, or in some instances activate expression. Small interfering RNAs (siRNA) are a class of oligonucleotide therapeutics that are typically double stranded oligonucleotides that act via RISC (RNA-induced silencing complex) pathway, where the strand complementary to an RNA target, also referred to as the guide strand, targets the RNA for degradation or translation inhibition. To improve in vivo stability, the oligonucleotides have modifications at the 2’-position of the sugar residue, such as 2’-O-methyl, 2’-O-ethyl, 2’-O-methoxyethyl. or 2’-fluoro, and contain at the 5’-and 3’-ends non-natural intemucleoside linkages, such as a phosphorothioate linkages. Conjugating targeting moieties to the oligonucleotide, such as GalNac and lipid groups, can enhance delivery to cells and tissues.

[0005] Modified oligonucleotides are generally synthesized chemically, such as by solid-phase synthesis using phosphoramidite chemistry. However, chemical synthesis has several disadvantages, including low efficiency in synthesis of long oligonucleotides, difficulty in scale-up of manufacturing, and toxic chemical waste and solvent consumption, e.g., toluene and acetonitrile. To circumvent some of the limitations in chemical synthesis, RNA ligase 2 can be used to ligate shorter oligonucleotides to generate the oligonucleotide therapeutic. Double stranded RNA ligases, also referred to as RNA ligase 2 or RNA ligase II, preferentially join a nick in an RNA duplex. In view of the potential for use of RNA ligase 2 for the synthesis of modified oligonucleotides, desirable are RNA ligases that exhibit increased efficiency and promiscuity in ligating polynucleotides that contain nucleotide analogs, such as modified sugar residues, modified nucleobases, and non-standard internucleoside linkages. SUMMARY

[0006] The present disclosure provides engineered RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA ligase polypeptides. The present disclosure also provides methods of using the engineered RNA ligase polypeptides and compositions thereof for nucleic acid synthesis, diagnostic assays, and other purposes.

[0007] In one aspect, the present disclosure provides an engineered RNA ligase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1470 and 1478-3058. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862. 956, 1088. 1416, 1612, 1840, 1866, 1954. 2062. 2674, 2754, or 2786, or relative to a reference sequence corresponding to SEQ ID NO: 2, 54. 244, 648, 862, 956. 1088, 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754. or 2786.

[0008] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612. 1840, 1866, 1954, 2062, 2674. 2754. or 2786. or relative to the reference sequence corresponding to SEQ ID NO: 2, 54. 244, 648, 862, 956. 1088. 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754. or 2786.

[0009] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0010] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, or 956, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0011] Tn some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0012] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 15, 17, 18, 21, 23, 25, 26, 28, 29, 30, 32, 33, 37, 38, 39, 40, 41, 42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 81, 83, 84, 86, 87, 89, 90, 91, 93, 95, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 116, 117, 119, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 144, 148, 150, 154, 156, 157, 158, 159, 160, 161, 162, 165, 169, 170, 171, 173, 175, 177, 178, 179, 181, 183, 184, 185, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 212, 213, 214, 215, 218, 223, 224, 226, 228, 229, 230, 232, 236, 238, 241, 243, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 264, 265, 266, 267, 268, 270, 271, 272, 274, 276, 277, 280, 282, 283, 286, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301, 302. 305, 306, 307, 310, 311, 315, 316, 317. 318, 323, 324. 325, 326, 327, 330. 331, 332, 334, 335. 336, 337, 338, 341. 342, 344, 345, or 346, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 17, 21, 23, 25, 26, 28, 29, 30, 32, 37, 38, 40, 41, 42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 83, 84, 87, 90, 91, 93, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 148, 150, 157, 158, 160, 161, 162, 165, 169, 170, 173, 178, 179, 184, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 213, 215, 218, 223, 228, 229, 232, 236, 238, 241, 243, 248, 249, 250, 251, 252, 253, 254, 255, 258, 261, 264, 265, 267, 268, 270, 271, 272, 274, 276, 277, 282, 286, 289, 290, 292, 294, 295, 297, 298, 299, 300, 301, 302, 305, 311,318, 323, 324, 325, 327, 332, 336, 338, 341, 344, or 346, or a substitution 18A / H, 33A / I / T / V, 39P, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 95A / C / G / 1.116L. 117A / Q. 119L. 144L. 154S / V. 156G / H / S, 171S, 175A, 177K, 181E / N / P / R / S, 183L, 224A / Q / R, 212E / G / K / L. 226R, 230T / V. 247I / L / R / S, 256E / G / M / S / W. 257D / E / G. 260G, 280L / W, 283C7V. 288L / R / W. 291S, 296E / G / L / Q / W, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 315E, 3161 / Y, 317E / E / G / Q / V / W, 326C / L, 330A / D / E / G / K, 331D / G / S, 334E, 335Q, 337A / V, 338G / L / M, 342R / S / T, 345Q / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 86, 89, 93, 101, 122. 143, 157, 183, 192, 194, 198, 212, 214, 230, 241, 248, 249, 250, 251, 254, 266, 268, 280, 291, 295, 298, 307, 316, 318, 326, 330, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11,12, 23, 25, 29, 37, 38, 75, 93, 101, 122, 143, 157, 192, 194, 198, 241, 248, 249, 250, 251, 254, 268, 295, 298, or 318; or substitution 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y. 183L. 212E / G / K / L, 214A / C / H / K / L / Q / Y. 226R, 230T / V. 266C / I, 280L / W, 291H / L / Q / R / S / T / W, 307K / R, 326C / L, 330A / D / E / G / K, or 331D / G / S: or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered RNA ligase further comprises at least a substitution at amino acid position 2, 14, 15, 63, 69, 81, 85, 95, 138, 142, 149, 159, 171, 186, 195, 202, 266, 275, 283, 285, 303, 314, 333, or 339; a substitution 181, 33L, 39Q / S, 86G, 89G / H / I / M / W, 116N, 117D, 119T, 144W, 154C, 156A / L / N / Q / T, 175K, 177P, 181D, 183V, 185G / K, 2121, 2141, 224Y, 2261, 230D, 247K, 256A, 257L, 260S, 280G / N, 288E, 291P, 296K, 306L, 307E / Q, 310K, 315S / T, 316L, 317A, 326R, 330M / R, 331L / R / W, 334R, 335D / H. 337L. 3421, or 345V; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0017] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 256, 258, 254, 251, 248, 37, 54, 232, 15, 71, 26, 116, 38, 25, 106 / 251, 52, 33, 247, 37 / 249 / 266 / 316 / 330 / 334 / 335, 21, 11 / 37 / 249 / 266 / 316, 261, 11 / 266 / 316, 11 / 248 / 249 / 266, 331, 11 / 248 / 249, 255, 37 / 249 / 266 / 316 / 334, 266, 11 / 37 / 156 / 249 / 335, 37 / 156 / 248 / 249 / 266 / 334,334, 156, 11 / 266 / 316 / 334, 11 / 39 / 156 / 248 / 316, 11 / 249, 37 / 156, 107 / 251, 156 / 249 / 316, 37 / 156 / 316, 11 / 37 / 156 / 249, 11 / 37 / 334, 194, 37 / 316, 175, 11 / 156 / 249 / 316, 11 / 37, 252, 12, 37 / 266, 11 / 37 / 156 / 266, 11 / 37 / 156 / 248 / 266, 37 / 236 / 248 / 249, 37 / 156 / 248 / 249, 11 / 266 / 330 / 335, 249, 11 / 156 / 248 / 249 / 316, 37 / 249 / 316, 11, 11 / 156, 11 / 37 / 249, 330,316, 156 / 249 / 266 / 335,249 / 266 / 316 / 330 / 335, 11 / 156 / 249,266 / 316 / 330 / 334 / 335, 156 / 248 / 249 / 316 / 334, 37 / 156 / 248 / 249 / 316, 156 / 266, 248 / 249, 156 / 249 / 316 / 330 / 335, 218, 37 / 156 / 248 / 249 / 266, 11 / 37 / 156 / 248 / 249 / 316, 37 / 156 / 316 / 330, 11 / 37 / 248 / 249 / 316 / 330, 11 / 37 / 248 / 249, 249 / 330 / 335, 11 / 37 / 156 / 330 / 334.40, 11 / 37 / 148 / 249 / 316, 83, 323, 193, 335, or 257, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least one substitution provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1,10.1,11.1, 12.1,13.1, 14.1, 15.1, 16.1,17.1, 18.1, 19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0019] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set of an RNA ligase variant provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1, 10.1, 11.1, 12.1,13.1,14.1,15.1, 16.1,17.1,18.1,19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0020] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising a substitution or substitution set provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1, 10.1,11.1,12.1,13.1, 14.1,15.1, 16.1,17.1,18.1,19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0021] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0022] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058.

[0023] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0024] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0025] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12. 13, 15, 17, 18, 21, 23, 25, 26, 28, 29, 30, 32, 33. 37, 38, 39, 40, 41, 42, 45, 52, 54, 56, 57. 58, 60, 61. 62, 64, 65, 67, 71, 73, 74. 75, 77, 80. 81, 83, 84. 86, 87, 89, 90, 91, 93, 95,97,99, 100, 101, 102. 103, 104, 105, 106. 107, 109, 113, 114. 116, 117, 119, 121. 122, 123, 125, 126. 127, 128, 129, 131, 135, 137, 140, 141, 143, 144, 148, 150, 154, 156, 157, 158, 159, 160, 161, 162. 165, 169, 170, 171, 173, 175, 177, 178, 179, 181, 183, 184, 185, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 212, 213, 214, 215, 218, 223, 224, 226, 228, 229, 230, 232, 236, 238, 241, 243, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 264, 265, 266, 267, 268, 270, 271, 272, 274, 276, 277, 280, 282, 283, 286, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301, 302, 305, 306, 307, 310, 311, 315, 316, 317, 318, 323, 324, 325, 326, 327, 330, 331, 332, 334, 335, 336, 337, 338, 341, 342, 344, 345, or 346, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0026] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12. 13, 17, 21, 23, 25, 26, 28, 29, 30, 32. 37, 38, 40. 41,42, 45, 52, 54, 56, 57, 58, 60, 61, 62. 64, 65, 67. 71, 73, 74, 75, 77, 80, 83, 84, 87, 90. 91, 93, 97. 99, 100. 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 148, 150, 157, 158, 160, 161, 162, 165, 169, 170, 173, 178, 179, 184, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 213, 215, 218, 223, 228, 229, 232, 236, 238, 241, 243, 248, 249, 250, 251, 252, 253, 254, 255, 258, 261, 264, 265, 267, 268, 270, 271, 272, 274, 276, 277, 282, 286, 289, 290, 292, 294, 295, 297, 298, 299, 300, 301, 302, 305, 311, 318, 323, 324, 325, 327, 332, 336, 338, 341, 344, or 346; a substitution or amino acid residue 18A / H, 33A / UT / V, 39P, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 95A / C / G / I, 116L, 117A / Q, 119L, 144L, 154S / V, 156G / H / S, 17IS, 175A, 177K, 181E / N / P / R / S, 183L, 212E / G / K / L, 224A / Q / R, 226R, 230T / V, 247VL / R / S, 256E / G / M / S / W, 257D / E / G, 260G, 280L / W, 283C / V, 288L / R / W, 291S, 296E / G / L / Q / W, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 315E, 316I / Y, 317E / F / G / Q / V / W. 326C / L, 330A / D / E / G / K, 331D / G / S, 334E, 335Q, 337A / V, 338G / L / M, 342R / S / T, or 345Q / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674. 2754. or 2786.

[0027] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 86, 89, 93, 101, 122. 143, 157, 183, 192, 194, 198, 212, 214, 230, 241, 248, 249, 250, 251, 254, 266, 268, 280, 291, 295, 298, 307, 316, 318, 326, 330, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0028] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23. 25, 29, 37, 38, 75, 93, 101, 122, 143. 157, 192, 194, 198. 241, 248, 249, 250, 251, 254, 268, 295. 298, or 318; a substitution or amino acid residue 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 183L, 212E / G / K / L, 214A / C / H / K / L / Q / Y. 226R. 230T / V, 266C / I, 280L / W, 291H / L / Q / R / S / T / W, 307K / R, 326C / L, 33OA / D / E / G / K, or 331D / G / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956. 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0029] In some embodiments, the amino acid sequence of the engineered RNA ligase further comprises at least a substitution at amino acid position 2, 14, 15, 63, 69, 81, 85, 95, 138, 142, 149, 159, 171, 186, 195, 202, 266, 275. 283. 285, 303, 314, 333, or 339; a substitution or amino acid residue 181, 33L, 39Q / S, 86G, 89G / H / I / M / W, 116N, 117D, 119T, 144W, 154C, 156A / L / N / Q / T. 175K. 177P. 181D. 183V. 185G / K, 2121. 2141, 224Y. 2261, 230D, 247K, 256A, 257L, 260S, 280G / N, 288E, 291P, 296K, 306L, 307E / Q, 310K, 315S / T. 316L. 317A. 326R. 330M / R, 331L / R / W, 334R, 335D / H. 337L. 3421, or 345V; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862. 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062. 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416, 1612, 1840, 1866, 1954, 2062, 2674. 2754, or 2786.

[0030] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or to the reference sequence corresponding to SEQ ID NO: 54, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or relative to the reference sequence corresponding to SEQ ID NO: 54.

[0031] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 220-630, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 220-630, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or relative to the reference sequence corresponding to SEQ ID NO: 54.

[0032] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 156 / 232 / 334 / 335, 12 / 254 / 256, 12 / 230, 12 / 230 / 251, 12 / 230 / 251 / 254 / 256, 12 / 38 / 230, 230 / 251, 12 / 38 / 254, 230 / 256, 254, 230, 12 / 230 / 254, 12 / 38 / 230 / 251 / 256, 12 / 251, 12 / 251 / 254 / 256, 38, 12 / 230 / 256, 12, 12 / 25 / 38 / 230 / 251, 12 / 38 / 102 / 230 / 251 / 256, 12 / 256, 38 / 230 / 256, 256, 12 / 230 / 251 / 256, 230 / 251 / 254, 230 / 254, 251 / 254, 12 / 25 / 230. 38 / 230, 38 / 251, 12 / 38 / 230 / 256, 102, 251, 12 / 25 / 251 / 256, 12 / 102 / 256, 12 / 38 / 230 / 251 / 254 / 256, 12 / 251 / 256, 102 / 230 / 251 / 254 / 256, 12 / 38 / 251, 12 / 230 / 251 / 254, 156 / 248 / 334 / 335, 12 / 25 / 38, 12 / 102 / 230 / 251 / 254, 12 / 38 / 251 / 254, 12 / 251 / 254, 12 / 102, 38 / 230 / 251 / 254 / 256, 12 / 25 / 230 / 251 / 256, 12 / 230 / 311, 12 / 254, 38 / 230 / 251 / 254, 12 / 38 / 251 / 256, 156 / 334 / 335, 257 / 258 / 334, 12 / 25 / 38 / 251 / 256,334, 12 / 25 / 230 / 254 / 256,38 / 251 / 256, 12 / 38 / 230 / 251 / 254, 12 / 38, 257 / 258 / 334 / 335, 12 / 38 / 254 / 256, 232 / 248 / 257 / 258 / 334 / 335, 156 / 258 / 334 / 335, 156 / 249 / 334 / 335, 12 / 102 / 230 / 254, 25, 156 / 257 / 258 / 334 / 335, 12 / 102 / 230, 156 / 248 / 257 / 258 / 334, 248 / 249 / 334 / 335, 232 / 257 / 258 / 334 / 335, 334 / 335, 156 / 334, 12 / 38 / 102 / 256, 156 / 257 / 334 / 335, 12 / 140 / 251 / 254. 251 / 254 / 256, 248 / 334 / 335, 156 / 258 / 334, 156 / 248 / 330 / 335, 12 / 38 / 256, 232 / 334 / 335, 156 / 249 / 334, 330 / 334 / 335, 156 / 248 / 334, 156 / 335, 257 / 334, 248 / 334, 232 / 248 / 334 / 335, 12 / 25 / 38 / 230 / 251 / 256, 156 / 248 / 249 / 334 / 335, 330 / 335, 248 / 257 / 334 / 335, 232 / 248 / 334, 156 / 232 / 248 / 334 / 335, 232 / 257 / 330 / 334, 156 / 248 / 291 / 334 / 335, 156 / 232 / 248 / 257 / 258 / 334, 156 / 232 / 249 / 334 / 335, 12 / 25, 156 / 248 / 330 / 334 / 335, 156 / 248 / 249 / 334, 232 / 248 / 330 / 335, 156 / 232 / 330 / 334 / 335, 197, 338, 327, 192, 331, 326, 345, 101, 100, 141, 198, 64. 306, 317, 265, 296, 194, 62, 90, 307, 103, 210, 105, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or relative to the reference sequence corresponding to SEQ ID NO: 54.

[0033] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or to the reference sequence corresponding to SEQ ID NO: 244, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or relative to the reference sequence corresponding to SEQ ID NO: 244.

[0034] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 632-820. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 632-820, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or relative to the reference sequence corresponding to SEQ ID NO: 244.

[0035] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 141 / 197 / 326 / 327, 100 / 101 / 338, 141 / 198 / 326, 64 / 100 / 101, 100 / 338, 101 / 141 / 198 / 326, 101 / 198 / 326 / 327, 198 / 326,64 / 100 / 101 / 338 / 345, 197 / 326, 198 / 327, 141 / 198 / 327, 198 / 326 / 327, 197 / 326 / 327, 64 / 100 / 338, 64 / 100 / 101 / 156 / 197 / 338, 64 / 100 / 101 / 338, 100 / 197 / 338, 197 / 338, 101 / 197 / 326, 64 / 100 / 197 / 338, 101 / 198, 141 / 197 / 327, 64 / 100 / 101 / 197 / 338, 101 / 326, 101 / 197 / 326 / 327, 197 / 327, 197, 141 / 197, 101, 101 / 198 / 326,64 / 197 / 198 / 338, 141, 101 / 141, 100 / 101 / 197 / 338, 141 / 326, 338, 326, 197 / 198 / 338, 64 / 101 / 338, 64 / 197 / 198, 101 / 197, 100 / 101, 100 / 109 / 197 / 338, 101 / 197 / 338, 197 / 198, 141 / 326 / 327, 100 / 197, 64 / 100, 64 / 197 / 338, 100 / 197 / 198, 101 / 338, 141 / 327, 101 / 306, 64 / 100 / 101 / 197, 101 / 326 / 327, 100, 327, 326 / 345, 64 / 100 / 101 / 197 / 198, 326 / 327, 64 / 101, 100 / 101 / 197, 101 / 327, 64 / 100 / 197 / 198 / 338, 306, 64 / 338, 101 / 141 / 326 / 327, 64 / 101 / 197 / 338, or 101 / 198 / 306 / 327, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or relative to the reference sequence corresponding to SEQ ID NO: 244.

[0036] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or to the reference sequence corresponding to SEQ ID NO: 648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or relative to the reference sequence corresponding to SEQ ID NO: 648.

[0037] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 822-954. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 822-954, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or relative to the reference sequence corresponding to SEQ ID NO: 648.

[0038] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 194 / 330 / 331, 298, 331, 192 / 193 / 194 / 298, 330, 192 / 330 / 331, 192 / 193 / 194 / 331,298 / 330 / 331, 194 / 298 / 330 / 331, 192 / 193 / 330 / 331, 192 / 298 / 331, 192 / 193, 194 / 330, 60 / 100 / 156, 193 / 296 / 331, 298 / 331, 193 / 194 / 330 / 331, 194, 194 / 298 / 331, 193 / 298, 193 / 330, 192, 192 / 193 / 194 / 298 / 331, 192 / 193 / 194 / 298 / 330, 192 / 193 / 298 / 331, 100 / 156, 62 / 100,298 / 330, 60 / 100, 192 / 193 / 298 / 330 / 331, 100, 193 / 194 / 298, 338, 62, 247, 18, 37, 254, 33, 261, 251,260, 12, 117, 256, 255, 38, 29, 25, or 252, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or relative to the reference sequence corresponding to SEQ ID NO: 648.

[0039] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or to the reference sequence corresponding to SEQ ID NO: 862, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or relative to the reference sequence corresponding to SEQ ID NO: 862.

[0040] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 956-1078, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 956-1078, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or relative to the reference sequence corresponding to SEQ ID NO: 862.

[0041] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 254, 18 / 254, 247 / 317, 37 / 317 / 342, 33. 18 / 156 / 254, 18, 156, 254 / 338, 254 / 307, 33 / 37 / 317, 37, 37 / 156 / 317, 156 / 265 / 317, 265 / 317, 156 / 254. 33 / 156 / 317, 317, 156 / 317, 37 / 317, 18 / 338, 338. 156 / 247 / 317, 33 / 317. 37 / 247 / 265 / 317, 33 / 37 / 156 / 317, 156 / 247 / 296 / 317, 18 / 307, 156 / 296 / 317, 156 / 247, 37 / 247 / 317. 18 / 254 / 307, 307. 265, 33 / 37, 18 / 156, 33 / 156 / 296 / 317, 18 / 156 / 254 / 338, 37 / 265 / 317, 33 / 37 / 156 / 247 / 317, 296, 33 / 296 / 317, 156 / 296, 247 / 296 / 317, 37 / 296 / 317, 247 / 296, 33 / 37 / 156, 296 / 317, 156 / 254 / 307. 18 / 156 / 307, or 33 / 37 / 265, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or relative to the reference sequence corresponding to SEQ ID NO: 862.

[0042] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or to the reference sequence corresponding to SEQ ID NO: 956, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

[0043] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1080-1374, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1080-1374, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

[0044] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 12 / 25 / 29 / 156. 12 / 255 / 256 / 257, 12 / 156 / 255, 247 / 252, 12 / 25, 12, 12 / 25 / 29 / 156 / 255,256, 12 / 156, 12 / 25 / 156 / 255 / 256 / 257 / 261, 18 / 247 / 251,25 / 255 / 256 / 257, 12 / 25 / 257, 255 / 257, 12 / 25 / 29 / 255 / 257,256 / 257, 12 / 255 / 257, 12 / 156 / 255 / 256 / 257,25, 18 / 38 / 251 / 252, 12 / 256 / 257 / 261, 12 / 29, 12 / 25 / 255 / 256 / 257 / 261, 38, 12 / 25 / 29 / 256 / 257, 29 / 257, 12 / 261, 251, 249 / 251, 12 / 255, 12 / 29 / 156 / 256 / 257, 252, 255 / 257 / 261, 18, 29 / 255 / 257, 255 / 256 / 257, 12 / 25 / 29 / 156 / 256 / 257, 25 / 255 / 256 / 257 / 261, 29 / 255 / 256 / 257 / 261, 25 / 256, 18 / 38 / 247 / 252 / 327, 18 / 247, 25 / 29, 12 / 25 / 156 / 255 / 257, 18 / 156 / 251 / 252, 12 / 255 / 261,25 / 255, 12 / 255 / 256 / 261, 156 / 255 / 257, 156 / 257, 12 / 156 / 256 / 257, 25 / 156 / 256 / 257, 12 / 25 / 29 / 156 / 257 / 261,25 / 261, 12 / 156 / 255 / 256 / 261, 12 / 25 / 29 / 255 / 256 / 257, 38 / 252, 255, 249 / 252, 12 / 156 / 255 / 256, 12 / 25 / 29 / 261,257, 12 / 255 / 256, 12 / 29 / 255, 12 / 255 / 257 / 261, 12 / 156 / 257, 192 / 249 / 251 / 252, 12 / 25 / 29 / 255 / 261, 12 / 156 / 255 / 257, 12 / 29 / 255 / 257, 12 / 29 / 256 / 257, 18 / 252, 249, 156 / 250, 38 / 251 / 252, 156 / 255 / 256 / 257, 25 / 257, 12 / 255 / 256 / 257 / 261, 25 / 156 / 261, 156 / 252, 12 / 25 / 255 / 256 / 257, 12 / 25 / 29 / 156 / 255 / 257, 29 / 156, 251 / 252, 256 / 257 / 261, 25 / 156 / 255 / 256 / 257, 156 / 249 / 251 / 252, 25 / 29 / 255, 12 / 29 / 156, 255 / 256, 38 / 250 / 252, 247 / 249 / 252, 12 / 256 / 257, 60, 12 / 256, 12 / 156 / 256, 25 / 255 / 257 / 261, 12 / 25 / 29 / 255 / 256 / 261, 12 / 29 / 156 / 255 / 256, 12 / 25 / 156 / 255 / 256, 247 / 251 / 252, 256 / 261, 38 / 251, 12 / 25 / 29 / 156 / 261, 12 / 257, 156 / 255 / 256 / 261, 12 / 25 / 156 / 255, 12 / 25 / 29 / 156 / 255 / 257 / 261, 12 / 29 / 256 / 261, 29 / 156 / 261, 25 / 29 / 156 / 257 / 261, 12 / 25 / 255 / 256 / 261, or 12 / 156 / 255 / 261, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

[0045] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 157, 183, 95, 185, 181, 213, 89, 28, 190, 214, or 143, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

[0046] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or to the reference sequence corresponding to SEQ ID NO: 1088, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or relative to the reference sequence corresponding to SEQ ID NO: 1088.

[0047] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1376-1434, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1376-1434, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or relative to the reference sequence corresponding to SEQ ID NO: 1088.

[0048] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 95 / 157, 95 / 183 / 185 / 214, 89 / 183, 28 / 89 / 143 / 183, 28 / 143 / 157, 181 / 183, 143 / 181 / 183 / 214, 143 / 181 / 183, 89 / 143 / 183, 28 / 143 / 181 / 183, 143 / 157, 95 / 181 / 183, 157, 143 / 183, 89 / 143 / 157 / 183, 89 / 95 / 157, 89 / 157, 89 / 95 / 143 / 181, 95 / 143, 89 / 157 / 183 / 214, 143 / 214, 143 / 181 / 183 / 185, 28 / 95 / 143, 143 / 183 / 214, 28 / 95 / 183 / 214, 89 / 143 / 157, 28 / 95, 95 / 143 / 183 / 185, or 95 / 143 / 157 / 183, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or relative to the reference sequence corresponding to SEQ ID NO: 1088.

[0049] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416. or to the reference sequence corresponding to SEQ ID NO: 1416, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

[0050] Tn some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1436-1470. 1478-1766, and 1908-1946. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1436-1470, 1478-1766. and 1908-1946, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

[0051] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 83 / 137 / 150, 137 / 213, 137, 150 / 190, 143 / 150, 104 / 150, 60 / 143 / 150 / 190, 60 / 150 / 190 / 213, 104 / 137 / 154, 123 / 137, 137 / 150 / 154 / 190, 123 / 143 / 150, 60 / 104 / 143 / 150 / 213, 60 / 95 / 143 / 190, 123 / 137 / 190, 60 / 123, 104 / 123, or 60 / 123 / 143, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

[0052] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38, 249, 255, 23, 33. 25, 226, 29, 37, 10. 250, 75, 230, 77, 251. 228, 81, 224, 64, 254, 268, 65, 232, 332, 331, 330, 101, 307, 137, 223, 280, 289, 288, 342, 93, 296, 86, 241, 126, 277, 87, 247, 243, 84, 83, 192, 105, 25 / 29, 25 / 29 / 250, 25 / 29 / 75 / 77 / 250 / 255, 25 / 75 / 230, 25 / 77 / 226 / 255, 25 / 230, 250 / 255, 29 / 75 / 226 / 230 / 255, 29 / 75 / 77, 29 / 75 / 77 / 226, 29 / 75 / 77 / 230 / 255, 29 / 226, 29 / 226 / 230, 29 / 230 / 255, 33 / 228, 33 / 249, 33 / 37 / 228, 33 / 37 / 38 / 228, 33 / 37 / 38 / 228 / 249, 33 / 38, 75 / 250 / 255, 75 / 226, 75 / 226 / 250 / 255, 75 / 226 / 230 / 250 / 255, 75 / 77, 75 / 77 / 255, 75 / 77 / 250 / 255, 75 / 77 / 226 / 255, 75 / 77 / 226 / 250 / 255, 75 / 77 / 230, 75 / 77 / 230 / 250, 75 / 230, 75 / 230 / 255, 75 / 230 / 250, 226 / 255, 226 / 250, 226 / 230, 226 / 230 / 255, 37 / 228, 37 / 228 / 249, 37 / 38, 37 / 38 / 228, 37 / 38 / 228 / 249, 37 / 38 / 249, 38 / 249 / 291, 10 / 228, 10 / 228 / 249, 10 / 33 / 249, 10 / 33 / 37, 10 / 33 / 37 / 38 / 249, 10 / 33 / 38 / 249, 10 / 33 / 38, 10 / 33 / 249 / 251, 10 / 33 / 37 / 228, 10 / 33 / 37 / 249, 10 / 33 / 37 / 38 / 228 / 249, 10 / 33 / 37 / 38, 10 / 249, 10 / 37, 10 / 37 / 228, 10 / 37 / 228 / 249, 10 / 37 / 249, 10 / 37 / 38 / 228, 10 / 37 / 38 / 228 / 249, 10 / 37 / 38, 10 / 38 / 249, 10 / 38, 10 / 38 / 228, 77 / 255, 77 / 250, 77 / 250 / 255, 77 / 226, 77 / 226 / 255, 77 / 230, 77 / 230 / 255, 230 / 255, 230 / 250, 230 / 250 / 255, 23 / 25 / 226 / 255, 23 / 25 / 226 / 230, or 23 / 25 / 230, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416. or relative to the reference sequence corresponding to SEQ ID NO: 1416.

[0053] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 86, 255, 306, 250, 75, 318, 335, 241, 248, 344, 38, 272, 83, 257, 258, or 323, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

[0054] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or to the reference sequence corresponding to SEQ ID NO: 1612, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or relative to the reference sequence corresponding to SEQ ID NO: 1612.

[0055] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1768-1840. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 768-1840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or relative to the reference sequence corresponding to SEQ ID NO: 1612.

[0056] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 101, 291, 291 / 307, 331, 33 / 75, 33 / 75 / 249 / 291 / 331, 33 / 38, 33 / 93 / 249, 38 / 75 / 291 / 307, 38 / 75 / 249 / 250, 38 / 75 / 93 / 271, 38 / 93, 10 / 75, 10 / 38 / 75, 10 / 38 / 75 / 291, 10 / 38 / 75 / 249, 10 / 38 / 75 / 93, 64 / 224, 224, 23 / 101 / 307, 23 / 268, 23 / 37 / 64 / 224 / 254, 23 / 64 / 81, 23 / 224 / 268, 23 / 224 / 254, 23, 23 / 64 / 224, 64, 75 / 249 / 307, 93, 33 / 38 / 75 / 249, 75 / 249, 25 / 38 / 75 / 93 / 291, 33 / 38 / 75, 75, or 23 / 254. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or relative to the reference sequence corresponding to SEQ ID NO: 1612.

[0057] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or to the reference sequence corresponding to SEQ ID NO: 1840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or relative to the reference sequence corresponding to SEQ ID NO: 1840.

[0058] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1842-1906, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1842-1906, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or relative to the reference sequence corresponding to SEQ ID NO: 1840.

[0059] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 65 / 192, 38 / 65, 38 / 65 / 224, 38 / 64, 93 / 307, 38, 38 / 224, 38 / 192 / 224, 65, 192 / 224, 75 / 307, 75 / 250 / 268 / 307, 250 / 268 / 307 / 342, 224, 65 / 192, 268, 64 / 192, 250 / 268 / 291 / 342, 75 / 93 / 249 / 307 / 342, 250 / 291. 38 / 64 / 192. 38 / 64 / 224, 93 / 249 / 307. 38 / 64 / 86 / 192 / 241, 38 / 342, 248 / 250 / 307, 75 / 77 / 250 / 268 / 307 / 342, 93 / 268, 192, 38 / 192, or 64, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or relative to the reference sequence corresponding to SEQ ID NO: 1840.

[0060] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or to the reference sequence corresponding to SEQ ID NO: 1866, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

[0061] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1948-2038 and 2130-2620, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1948-2038 and 2130-2620, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

[0062] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 248 / 251, 38 / 248 / 250 / 251 / 277, 248 / 250 / 251, 38 / 93 / 192 / 248 / 251, 38 / 192 / 248 / 251, 250 / 251, 38 / 248 / 250 / 251, 248 / 251 / 344, 192 / 248 / 250 / 251, 38 / 248 / 250, 38 / 93 / 250 / 251 / 277, 38 / 255 / 280 / 318, 248, 38 / 86 / 241 / 255 / 280 / 318 / 335, 38 / 255, 38, 318, 38 / 101 / 255 / 280, 38 / 318 / 335, 38 / 255 / 318, 192 / 250 / 251 / 344, 38 / 335, 318 / 335, 38 / 277, 38 / 93 / 251, 192 / 251, 255, 38 / 64 / 318, 38 / 64 / 241 / 255 / 318, 251, 64 / 101 / 255 / 280 / 318, 38 / 101 / 255 / 280 / 318 / 335, 38 / 255 / 280, 38 / 251 / 277, 64 / 318, 38 / 64 / 255 / 318, 241 / 255 / 280 / 318, 38 / 86 / 101 / 241 / 318, 38 / 241, 38 / 255 / 318 / 335, 38 / 64 / 101 / 318, 38 / 64 / 255 / 280 / 318, 241 / 255 / 318, or 38 / 241 / 335, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

[0063] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 89, 122, 181, 28, 125, 157, 238, 330, 212, 143, 208, 290, 161, 214, 84, 270, 91, 295, 316. 286, 301, 170, 264. 289, 190, 183, 128, 60, 310. 131, 302, 267, 288. 185, 291, 129, 280, 298, 158 / 215, 28 / 122, 276, 274, 271, 215, 294, 106, 58, 135, 45, 299, 315, 204. 332, 169, 305, 154, 158, 297, 95, 337, 324, 150, 179, 283, 206, 236, 266, 213, 336, 178, 99, 184, 282, 57, 177, 109, 191, 107, 160, 144, 56, 159, 341, 325, or 91, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

[0064] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954. or to the reference sequence corresponding to SEQ ID NO: 1954. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0065] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2040-2128, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2040-2128, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954. or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0066] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 93 / 232 / 291 / 306, 93 / 232 / 280 / 306, 93 / 241 / 257 / 306 / 335, 232 / 280, 93 / 291 / 306, 93 / 257, 93 / 306, 93 / 280, 192 / 318, 93 / 280 / 291, 93 / 241 / 291 / 306, 192, 257 / 291, 241 / 257 / 306, 280 / 335, 93 / 291 / 306 / 335, 93 / 335, 93 / 232 / 335, 64 / 318, 93 / 241, 93 / 232 / 241 / 280, 64 / 192 / 318 / 323, 232 / 280 / 306, 93 / 232, 291 / 335, 257 / 258, 93 / 232 / 241 / 257 / 258 / 280 / 335, 318 / 323, 93 / 241 / 291 / 335, 83 / 86 / 323,257 / 258 / 291, 101 / 224 / 318, 83 / 192 / 255 / 296, 171 / 232 / 257 / 258 / 280, 83 / 224 / 255 / 318, 64 / 192 / 250 / 318, 101 / 296, 232 / 241 / 280, 64 / 255 / 318, 86 / 250 / 255, 83 / 86 / 101, 83 / 86, 93 / 241 / 258 / 335, 250 / 296 / 323, or 257 / 258 / 280, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0067] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062, or to the reference sequence corresponding to SEQ ID NO: 2062, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062, or relative to the reference sequence corresponding to SEQ ID NO: 2062.

[0068] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2622-2746. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2622-2746, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062, or relative to the reference sequence corresponding to SEQ ID NO: 2062.

[0069] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 83 / 84 / 330, 91 / 270 / 290, 83 / 181, 161 / 290 / 330, 181 / 330, 161 / 270 / 330, 270 / 290, 83 / 84 / 270 / 318 / 330, 64 / 83 / 84 / 161 / 192 / 290 / 318,91 / 161 / 290 / 318 / 330, 161 / 330, 161 / 214 / 270 / 290 / 330, 192 / 270 / 318,91 / 161 / 192 / 330, 161 / 270,181 / 270 / 330,91 / 181 / 192 / 290, 161 / 290, 83 / 161 / 270, 83 / 192 / 318, 214 / 330, 83 / 161 / 290 / 330, 64 / 83 / 161 / 290 / 330, 83 / 161 / 192 / 214 / 318, 122 / 143 / 212 / 295, 161 / 192 / 270 / 290, 181 / 290, 91 / 161 / 214 / 270 / 290 / 318, 91 / 192 / 214 / 270, 214 / 290, 64 / 83 / 161 / 270 / 318 / 330, 91 / 161 / 181 / 330, 161 / 318 / 330, 91 / 270 / 290 / 330, 64 / 181 / 290 / 318, 181 / 270 / 290 / 318, 64 / 83 / 161 / 192 / 270 / 290 / 318 / 330, 64 / 83 / 161 / 214 / 290 / 318, 122 / 192 / 212, 64 / 83 / 91 / 192 / 214 / 330, 83 / 91 / 161 / 181 / 290 / 318, 91 / 161 / 192 / 214 / 270 / 290, 91 / 161 / 181 / 290 / 330, 83 / 91 / 125 / 161 / 270, 28 / 212, 122 / 212, 28 / 64 / 83 / 122 / 143 / 192 / 212 / 295 / 318, 122 / 295, 91 / 161 / 192 / 318, 28 / 122 / 143 / 212, 64 / 161 / 290, 64 / 122 / 208 / 318, 64 / 83 / 192 / 290 / 318, 64 / 83 / 161 / 290, 181 / 192 / 318, 83 / 161 / 192 / 318, 214 / 270, 83 / 122, or 192 / 212, 28 / 122, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062, or relative to the reference sequence corresponding to SEQ ID NO: 2062.

[0070] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674. or to the reference sequence corresponding to SEQ ID NO: 2674, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

[0071] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2748-2782, 2822-2914, and 2916-3012, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2748-2782, 2822-2914, and 2916-3012, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

[0072] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 28 / 291 / 295 / 298 / 315, 28 / 95 / 291 / 295 / 298 / 315, 143 / 295 / 298 / 315, 291 / 295 / 298, 28 / 143 / 295 / 298, 28 / 291 / 295 / 298, 143 / 185 / 291 / 295 / 298, 28 / 143 / 185 / 291 / 298 / 336, 28 / 298, 280 / 295, 143 / 298 / 336, 143 / 291 / 295 / 298, 291 / 295 / 298 / 315 / 336, 143 / 295 / 298, 28 / 143 / 295 / 298 / 336, 95 / 143 / 295 / 298, 143 / 280 / 290, or 143 / 291 / 298, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

[0073] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 208, 25, 90, 141, 258, 248, 318, 117, 299, 33, 80, 253, 83, 254, 256, 30, 327, 73, 100, 97, 119, 265, 296, 32, 156, 40, 105, 107, 41 / 114, 41, or 64, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

[0074] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162, 208, 25, 103, 346, 42, 74, 113, 13, 228, 318, 121, 299, 33, 342, 253, 127, 254, 292, 30, 67, 86, 165, 173, 300, 317, 326, 11, 229, 97, 71, 17, 210, 241, 271, 40, 56, or 95, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

[0075] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or to the reference sequence corresponding to SEQ ID NO: 2754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or relative to the reference sequence corresponding to SEQ ID NO: 2754.

[0076] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2784-2820. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2784-2820, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or relative to the reference sequence corresponding to SEQ ID NO: 2754.

[0077] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 106 / 264 / 280, 280 / 330, 28 / 106 / 264 / 291, 28 / 264 / 280 / 330, 57 / 60 / 135 / 264 / 291, 28 / 135 / 264 / 280, 106 / 264 / 280 / 291,28 / 106 / 264 / 280, 280 / 299, 28 / 57 / 89 / 106 / 264 / 280 / 291, 264, 185 / 291, 28 / 106 / 299, 28 / 214 / 264 / 299, 264 / 330, 28 / 89 / 214 / 291, 28 / 185 / 271 / 310, 122 / 271, or 89 / 106 / 135 / 264 / 280 / 330, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or relative to the reference sequence corresponding to SEQ ID NO: 2754.

[0078] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or to the reference sequence corresponding to SEQ ID NO: 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2786.

[0079] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 3014-3058. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 3014-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2786.

[0080] Tn some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 25 / 228 / 248 / 264, 32 / 113 / 228 / 248, 25 / 32 / 113 / 241 / 248 / 264 / 299, 25 / 113 / 248 / 264 / 318, 25 / 127 / 228 / 264 / 299 / 318, 25 / 113 / 228 / 248 / 327, 228 / 241 / 264, 264 / 299 / 318 / 327, 25 / 113 / 241 / 248 / 264 / 318, 25 / 127 / 248 / 299 / 318, 113 / 127 / 228 / 264 / 299, 25 / 127, 25 / 228 / 248 / 264 / 327. 25 / 113 / 264 / 299, 32 / 113 / 264 / 318, 25 / 32 / 241 / 264 / 327, 113 / 264 / 327, 25 / 127 / 228 / 248 / 318, 327, 32 / 113 / 254 / 264, 25 / 228 / 241 / 264, 25 / 113 / 228 / 241 / 318, or 113 / 327, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2786.

[0081] Tn some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least one substitution provided in Tables 6.1, 6.2, 7.1, 8.1, 8.2, 9.1,10.1,11.1,12.1,13.1,14.1,15.1,16.1,17.1,18.1, 19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54. 244, 648, 862.956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244. 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0082] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set of an RNA ligase variant provided in Tables 6.1, 6.2, 7.1, 8.1, 8.2, 9.1, 10.1, 11.1,12.1,13.1,14.1,15.1,16.1, 17.1,18.1,19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0083] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or substitution set provided in Tables 6.1, 6.2, 7.1, 8.1, 8.2, 9.1, 10.1,11.1, 12.1.13.1, 14.1, 15.1,16.1,17.1,18.1,19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0084] In some embodiments, the engineered RNA ligase comprises an amino acid sequence comprising residues 12 to 346 of an even numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or an amino acid sequence comprising an even numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally has 1, 2, 3, 4. 5, 6, 7, 8, 9, or up to 10 substitutions.

[0085] In some embodiments, the engineered RNA ligase comprises an amino acid sequence comprising residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or an amino acid sequence comprising SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally has 1,2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0086] In some embodiments, the engineered RNA ligase has RNA ligase 2 activity. In some embodiments, the engineered RNA ligase has RNA ligase 2 activity and at least one improved property as compared to a reference RNA ligase.

[0087] In some embodiments, the improved property of the engineered RNA ligase is selected from i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased activity on polynucleotides ligase substrates with phosphorothioate internucleoside linkages, vi) increased activity on oligonucleotides with 2’-modifications, particularly at the ligation junction, vii) increased substrate tolerance, or any combination of i), ii), iii), iv), v), vi), and vii), compared to a reference RNA ligase. In some embodiments, the improved property of the engineered RNA ligase is in comparison to the reference RNA ligase having the sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or the sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0088] In some embodiments, the engineered RNA ligase is purified. In some further embodiments, the engineered RNA ligase is provided in solution, as a lyophilizate, or immobilized on a substrate, such as solid substrates, porous substrates, membranes or particles.

[0089] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered RNA ligases disclosed herein.

[0090] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 75%, 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 53, 243, 647, 861. 955, 1087. 1415, 1611, 1839, 1865, 1953, 2061, 2673, 2753, or 2785, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 53, 243, 647, 861,955,1087, 1415, 1611, 1839, 1865,1953, 2061,2673,2753, or 2785, wherein the recombinant polynucleotide encodes an RNA ligase.

[0091] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057, wherein the recombinant polynucleotide encodes an RNA ligase.

[0092] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide encoding an engineered RNA ligase is codon optimized for expression in an organism or cell type thereof, for example a bacterial cell, a fungal cell, insect cell, or mammalian cell.

[0093] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1038 of SEQ ID NO. 53, 243. 647, 861, 955, 1087, 1415, 1611. 1839. 1865. 1953, 2061, 2673, 2753, or 2785, or a polynucleotide sequence comprising SEQ ID NOs: 53, 243, 647, 861,955, 1087, 1415, 1611, 1839. 1865, 1953,2061,2673, 2753. or 2785.

[0094] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1038 of an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057, or a polynucleotide sequence comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057.

[0095] In a further aspect, the present disclosure provides expression vectors comprising at least one recombinant polynucleotide provided herein encoding an engineered RNA ligase. In some embodiments, the recombinant polynucleotide of the expression vector is operably linked to a control sequence. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter.

[0096] In another aspect, the present disclosure also provides a host cell comprising at least one expression vector provided herein. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell. In some preferred embodiments, the host cell is a bacterial cell, such as E. coli. or B. subtilis.

[0097] In a further aspect, the present disclosure provides a method of producing an engineered RNA ligase polypeptide, the method comprising culturing a host cell described herein under suitable culture conditions such that at least one engineered RNA ligase is produced. In some embodiments, the method further comprises recovering or isolating the engineered RNA ligase from the culture and / or host cells. In some embodiments, the method further comprises the step of purifying the engineered RNA ligase.

[0098] In another aspect, the present disclosure provides a composition comprising at least one engineered RNA ligase disclosed herein. In some embodiments, the composition comprises at least a buffer. In some embodiments, the composition further comprises a nucleotide substrate (e.g., ATP or dATP) and one or more of polynucleotide substrates, such as synthetic polynucleotides, particularly modified polynucleotides.

[0099] In a further aspect, the present disclosure provides a method of ligating at least a first polynucleotide strand and a second polynucleotide strand, comprising contacting the first polynucleotide strand and a second polynucleotide strand with an engineered RNA ligase described herein in presence of a nucleotide substrate under conditions suitable for ligation of the first polynucleotide strand to the second polynucleotide strand, wherein the first polynucleotide strand comprises a ligatable 5’-end and the second polynucleotide strand comprises a 3’-end ligatable to the 5-end of the first polynucleotide strand. In some embodiments, the first polynucleotide strand and / or second polynucleotide strand comprises RNA or a mixture of RNA and DNA.

[0100] In some embodiments, the method further comprises a third polynucleotide strand, wherein the first polynucleotide strand and second polynucleotide strand hybridize adjacent to one another on the third polynucleotide to position the 5’-end of the first polynucleotide strand adjacent to the 3’-end of the second polynucleotide strand to form a nick. In some embodiments, the third polynucleotide strand is continuous with the first polynucleotide strand or second polynucleotide strand. In some embodiments, the third polynucleotide strand is continuous with the first polynucleotide strand and second polynucleotide strand to form a single continuous polynucleotide substrate.

[0101] In some embodiments, 3’-end region of the second polynucleotide strand that hybridizes to the third polynucleotide strand is at least 4, 6, 8 or more base pairs in length. In some embodiments, the 5-end region of the first polynucleotide strand that hybridizes to the third polynucleotide strand is at least 4, 6, 8 or more base pairs in length.

[0102] In some embodiments of the method, the third polynucleotide strand comprises a splint or bridging polynucleotide, wherein the 5’-terminal sequence of the first polynucleotide strand and the 3’-terminal sequence of the second polynucleotide strand hybridize adjacent to one another on the splint or bridging polynucleotide to position the 5’-end of the first polynucleotide strand adjacent to the 3’-end of the second polynucleotide strand.

[0103] In some embodiments, the first and third polynucleotide strands hybridize to each other to form a first double stranded polynucleotide fragment, and the second polynucleotide strand hybridizes to another polynucleotide strand to form a second double stranded fragment, wherein the first and second double stranded fragments have complementary ends that can base pair to form substrates for the engineered RNA ligase.

[0104] In some embodiments, the engineered RNA ligase is used in a method to synthesize RNA or DNA / RNA polynucleotides by ligation of shorter RNA or DNA / RNA oligonucleotides. In some embodiments, the engineered RNA ligase is used to repair nicks in dsRNA or dsRNA / DNA. In some embodiments, the engineered RNA ligase is used to ligate the 3' OH of RNA to the 5' phosphate of DNA or RNA. In some embodiments, the engineered RNA ligase is used to ligate the 3' OH of RNA to the 5' phosphate of DNA in a double-stranded-format NGS RNA library construction. In some embodiments, the engineered RNA ligase is used in a method of preparing RNA rings.

[0105] In a further aspect, the present disclosure also provides a kit comprising at least one engineered RNA ligase disclosed herein. In some embodiments, the kit further comprises one or more of a buffer, nucleotide substrate (e.g., ATP or dATP), ligation enhancing agent, and / or one or more polynucleotide substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG.l shows the output of a Gaussian Process Regression (GPR) analysis for two recombinant single stranded RNA ligase for a RNA ligase substrate for ligase reaction variables of MgCh, ATP, and oligonucleotide substrate concentration. DETAILED DESCRIPTION

[0107] The present disclosure provides engineered RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA ligase polypeptides. The disclosure also provides methods of using of the engineered RNA ligase polypeptides and compositions thereof for molecular biological, diagnostic, and other purposes. In some embodiments, the engineered RNA ligase polypeptides display, among others, increased activity, increased stability, increased product yield, and / or increased activity on polynucleotides containing one or more modified nucleotides or nucleotide analogs. Abbreviations and Definitions

[0108] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0109] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide.

[0110] Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0111] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0112] “About” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

[0113] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0114] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0115] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0116] “Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0117] “Amino acids” and “amino acid” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (CH). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0118] “Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).

[0119] ‘‘RNA ligase” refers to enzymes that covalently joins the 5’-phosphoryl termini of RNA or DNA to the 3’-hydroxyl termini of RNA or DNA. Two families of RNA ligases are known to occur in nature. RNA ligase 1 catalyzes the covalent joining of single stranded 5’-phosphoryl termini of RNA or DNA to single stranded 3-hydroxyl termini of RNA or DNA. RNA ligase 2 also catalyzes the covalent joining of a 3’-hydroxyl terminus of RNA to a 5’-phosphorylated RNA or DNA but shows preference for double stranded substrates. In some embodiments, RNA ligases include those enzymes classified in EC 6.5.1.3.

[0120] Without being bound by any theory of operation, although some DNA ligases are capable of acting on either DNA or RNA as the 3’-hydroxyl strand substrate, an RNA ligase 2 acting at a duplex nick preferentially acts on 3’-hydroxyl strand of RNA but is agnostic to whether 5-phosphoryl strand is DNA or RNA. Moreover, in some embodiments, the strand with the 3’-hydroxyl may include deoxyribonucleotides if sufficient number of ribonucleotides are present at the 3’-hydroxyl terminus. For example, the RNA specificity of T4 RNA ligase 2 is affected by the 3’-hydroxyl strand, and specifically by the two terminal ribonucleotides of the 3-‘hydroxyl side of the nick (see, e.g., Nandakumar et al., Mol. Cell., 2004, 16:211221). It is to be understood that the ligation reaction is not limited to naturally occurring RNA and DNA substrates also includes polynucleotide substrates that contain modified nucleotides and / or nucleotide analogs.

[0121] “Polynucleotide,” “nucleic acid,” or “oligonucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T): and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2’-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’-deoxyribonucleosides on an individual basis or on an aggregate basis. When a polynucleotide, nucleic acid, or oligonucleotide sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. The polynucleotide or nucleic acid may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions.

[0122] In some embodiments, the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide or nucleic acid or oligonucleotide, which include, among others, nucleosides linked together via other than standard phosphodiester linkages, such as non-standard linkages of phosphoramidates, phosphorothioates, amide linkages, etc.; nucleosides with modified and / or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’-O-alkyl, 2’-halo. 2.3-dideoxy. 2’- halo-2’-deoxy. p-D-ribo LNA, a-L-ribo-LNA (e.g., locked nucleic acids), etc.: and / or 5’-phosphate analogs, including, among others, phosphorothioate. phosphoacetate, phosphoramidate, monomethylphosphate, methylphosphonate, or phosphonocarboxylate.

[0123] “Duplex” and “ds” refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule comprised of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs to T or U, C pairs to G), arranged in an antiparallel 5’ to 3’ orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).

[0124] “Complementary” is used herein to describe the structural relationship between nucleotide bases that are capable of forming base pairs with one another. For example, a purine nucleotide base present on a polynucleotide that is complementary to a pyrimidine nucleotide base on a polynucleotide may base pair by forming hydrogen bonds with one another. Complementary nucleotide bases can base pair via Watson / Crick base pairing or in any other manner than forms stable duplexes or other nucleic acid structures.

[0125] “Watson / Crick Base-Pairing” refers to a pattern of specific pairs of nucleobases and analogs that bind together through sequence-specific hydrogen-bonds, e.g. A pairs with T or U, and G pairs with C.

[0126] “Annealing” or “Hybridization” refers to the base-pairing interactions of one nucleobase polymer (e.g., poly- and oligonucleotides) with another that results in the formation of a double-stranded structure, a triplex structure or a quaternary structure. Annealing or hybridization can occur via Watson-Crick basepairing interactions, but may be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing. In some embodiments, the nucleobase polymer that anneals or hybridizes to another is a single nucleobase polymer while in other embodiments, the nucleobase polymers are separate nucleobase polymers.

[0127] “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and / or by manipulation using recombinant techniques.

[0128] “Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0129] “Coding sequence” and synonymously “encoding” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0130] “Percent (%) sequence identity” refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (see, e.g.. Altschul et al., J. Mol. BioL. 1990, 215: 403-410; and Altschul et al.. Nucleic Acids Res., 1977. 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T.” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wl). using default parameters provided.

[0131] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, the phrase “a reference sequence corresponding to SEQ ID NO: 2, having an asparagine at the residue corresponding to XU” (or “a reference sequence corresponding to SEQ ID NO: 2, having an asparagine at the residue corresponding to position 11”) refers to a reference sequence in which the corresponding residue at position XI1 in SEQ ID NO: 2 (e.g., an glycine), has been changed to asparagine.

[0132] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0133] “Corresponding to”, “reference to.” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered RNA ligase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0134] “Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0135] “Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The amino acid positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position XI1 as compared to SEQ ID NO: 2’’ (or a “residue difference at position 11 as compared to SEQ ID NO: 2”) refers to a difference of the amino acid residue at the polypeptide position corresponding to position 11 of SEQ ID NO: 2. Thus, if the reference polypeptide of SEQ ID NO: 2 has a glycine at position 11, then a “residue difference at position XI1 as compared to SEQ ID NO: 2” refers to an amino acid substitution of any residue other than glycine at the position of the polypeptide corresponding to position 11 of SEQ ID NO: 2. In some instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., in the Tables in the Examples), the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in the reference sequence. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a where appropriate. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino acid residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.

[0136] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “ / ” (e.g., X18A / X18H, X18A / H, or 18A / H). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either / or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0137] “Amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant RNA ligase polypeptides listed in any of the Tables in the Examples. In some embodiments, the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set. In the substitution sets, the individual substitutions are separated by a semicolon (“;”; e.g., S106G;K251F) or slash (“ / ”; e.g., S106G / K251F or 106G / 251F).

[0138] “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0139] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect; (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0140] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity and / or retaining the improved properties of an engineered RNA ligase. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated by and may be present in substitution sets.

[0141] “Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0142] “Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full length engineered RNA ligase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0143] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant RNA ligase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant RNA ligase polypeptides provided herein are isolated polypeptides.

[0144] “Substantially pure polypeptide” or “purified” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure RNA ligase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant RNA ligase polypeptides are substantially pure polypeptide compositions.

[0145] “Improved enzyme property” refers to an engineered RNA ligase polypeptide that exhibits an improvement in any enzyme property as compared to a reference RNA ligase polypeptide, such as a wild-type RNA ligase polypeptide or another engineered RNA ligase polypeptide. Improved properties include but are not limited to such properties as increased enzymatic activity, increased product yield, increased protein expression, increased thermoactivity, increased thermostability, increased stability, increased substrate specificity and / or affinity, increased substrate range, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved solvent stability, increased solubility, and increased inhibitor resistance or tolerance. Exemplary improved properties are provided in the Examples.

[0146] “Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered RNA ligase polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) and / or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of RNA ligase) as compared to the reference RNA ligase enzyme (e.g., wild-type RNA ligase and / or another engineered RNA ligase). Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km. or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to about 1.5 fold. 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold. 100-fold, 150-fold. 200-fold or more enzymatic activity than the naturally occurring RNA ligase or another engineered RNA ligase from which the RNA ligase polypeptides were derived.

[0147] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in O.lxSSPE, and 0.1% SDS at 65 °C. Another high stringency condition comprises hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in O.lx SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0148] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the RNA ligase enzymes are codon optimized for optimal production from the host organism selected for expression.

[0149] “Control sequence” refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present disclosure. Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals. In some embodiments, the control sequences are provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0150] “Operably linked” or “operatively linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide of interest, and where appropriate, expression of the encoded polypeptide of interest.

[0151] “Promoter” or “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0152] “Suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, cosolvents, etc.) under which an RNA ligase polypeptide of the present disclosure is capable of converting a polynucleotide(s) substrate to the desired ligated product polynucleotide. Exemplary “suitable reaction conditions” are provided herein (see, the Examples).

[0153] “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the RNA ligase polypeptide on the substrate.

[0154] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0155] “Vector” is a recombinant construct for introducing a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide or a polypeptide encoded in the polynucleotide. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0156] “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0157] “Produces” refers to the production of proteins and / or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to. transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0158] “Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

[0159] “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g.. a polynucleotide sequences encoding at least one RNA ligase variant). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques, and progeny thereof, as known in the art.

[0160] “Analogue” in the context of a polypeptide means a polypeptide more than 70 % sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, analogues include non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues. Engineered RNA ligase Polypeptides

[0161] In one aspect, the present disclosure provides RNA ligases engineered to have an improved property, particularly increased activity and increased product yield. In some embodiments, the increased activity and increased product yield is on ligation of modified polynucleotides, including polynucleotide substrates with 2’-modified sugar moieties and / or phosphorothioate intemucleoside linkages.

[0162] In some embodiments, the engineered RNA ligase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1470 and 14783058, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to a reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0163] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862,956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862,956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0164] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0165] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54. 244. 648, 862, 956. 1088, 1416, 1612, 1840. 1866. 1954. 2062. 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0166] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0167] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-218, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0168] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12. 13, 15, 17, 18. 21, 23, 25. 26, 28, 29. 30, 32, 33. 37, 38, 39, 40. 41, 42, 45, 52. 54, 56, 57. 58, 60, 61. 62, 64, 65, 67, 71, 73, 74. 75, 77, 80. 81, 83, 84. 86, 87, 89, 90, 91, 93, 95, 97, 99, 100, 101, 102. 103, 104, 105, 106. 107, 109, 113, 114. 116, 117, 119, 121. 122, 123, 125, 126. 127, 128, 129, 131, 135, 137, 140, 141. 143, 144, 148, 150. 154, 156, 157, 158. 159, 160, 161, 162. 165, 169, 170, 171, 173, 175, 177, 178, 179, 181, 183, 184, 185, 190, 191, 192, 193, 194, 197, 198, 204, 206. 208, 210, 212, 213, 214, 215, 218, 223, 224, 226, 228, 229, 230, 232, 236, 238, 241, 243, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 264, 265, 266, 267, 268, 270, 271, 272, 274, 276, 277, 280, 282, 283, 286, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301, 302, 305, 306, 307, 310, 311, 315, 316, 317, 318, 323, 324, 325, 326, 327, 330, 331, 332, 334, 335, 336, 337, 338, 341, 342, 344, 345, or 346, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0169] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F. 11G / N, 12A / F / L / M / P / R / T / V, 13W, 15M. 17P, 18A / H. 21S, 23K / T. 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 33A / I / L / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P. 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 56VL, 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 7 IT, 73Q / W, 74D, 75A / D, 77M, 80E, 81Y, 83A / E / G / L / T / V, 84V, 86D / G / L, 87E, 89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, 90E / F, 91A / E / V, 93K / V. 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / S / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / Q / S, 157F / L / Q / V, 158R / S, 159F, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L / V, 184L, 185C / F / G / K / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 21OG / V, 212E / G / I / K / L, 213D / N / R / T, 214A / C / H / K / L / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 23OT / V, 232G / M / N / R / S, 236L / V. 238K / S / V, 241E / G / Q / R, 243H, 247I / L / R / S, 248F / G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / K / L / N / Q / S / V, 255N / S / V / Y, 256A / E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I / L, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / N / W, 282A / M, 283C / V, 286D / E / F / M / V, 288E / L / R / W, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 292N / S, 294A / S / V / Y, 295C / F / G / K / L / M / P / Q / R / S / T / V / Y, 296E / G / L / Q / W, 297A / I / L, 298C / VK / R / S / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / VL / M / V, 3O2A / C / I / L / S. 305V, 306G / L / T, 307K / R, 310H / L / M / N / Q / R / S / T, 311C. 315E / S, 316I / L / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 33OA / D / E / G / K / R, 331D / G / R / S / W, 332E / H / W, 334E / R, 335H / Q, 336I / L / M, 337A / V, 338G / L / M, 341A, 342R / S / T, 344K, 345Q / S, or 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0170] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue S10F, GUN, M12A / F / L / P / R / T / V, F13W, Y15M, S17P, S18A / H, N21S, Y23K / T, N25A / D / F / G / L / M / S / T, K26L, I28A / L / M / V. E29D / S, K30A / L / T / V / W, R32E, G33A / I / L / T / V, D37A / F / K / L / M / R, M38A / E / L / R / S / T / V, H39P, T40D / I / P / S, V41E / I, E42T, A45S, T52C, F54L, V56I / L, I57V, I58M, P60A / C / D / R / S / V, T61A, E62A / V. V64A / Q / W, P65C, K67R, P71T, L73Q / W, E74D, G75A / D, S77M, G80E, H81Y, I83A / E / G / L / T / V, I84V, K86D / G / L, K87E, K89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, D90E / F, S91A / E / V, V93K, V95A / C / G / I. N97A / E, L99I, N100A / G / H / L / R / S / T / V, A101P / V / W, M102T, D103G / V, L104T, V105K / L / R, S106G / L / M / Q / R / V / W, V107M / S, I109F / L, F113L / M, A114E, G116L, G117A / Q, Q119L, G121N, V122C / F / G / L / Q / T / W, D123K / R, G125K / T, D126M, K127P, D128E, F129L / W, V131T, L135A / H / M / Q / V, D137N / R, N140I, E141D / G, S143A / C / E / F / H / M / T / V, Y144L, Y148F, V150D / L, C154S / V, T156G / H / Q / S, L157F / Q / V, G158R / S, L159F, K160R, L161M / V, A162C, L165M, S169G / N, F170H / Y, A171S, L173M, Q175A, P177K, N178V, D179E, K181E / N / P / R / S, I183L / V, V184L, N185C / F / G / K / M / P / S / T / V, L190EM, V191C, D192E / F / L / N, T193K / Q, T194A / G / V, E197W, H198M, F204W, P206L, A208H / VK / L / P / T, S210G / V, T212E / G / EK / L, G213D / N / R / T, L2I4A / C / H / K / L / Q / Y, A215G / T, Y218F, V223C, Y224A / Q / R, I226R, F228D / L, N229W, N23OT / V, T232G / M / N / R / S, I236L / V, C238K / S / V, S241E / G / Q / R, F243H, A247I / L / R / S, F248G / I / K / T, L249K / L / R / S, D250E / VS, K251C / E / F / G / L / R / S / T / Y, P252A / Q, I253L / W, K254E / L / N / Q / S / V, A255N / S / V / Y, K256A / E / G / M / S / W, V257D / E / G, F258D / M / P / V, V260G, E261D / P / T, K264A / E / G / S / V, R265A / F, V266C / I / L, L267I / M, E268F / G / M, F270L / M / V, S271A / E / G / T, E272M / S, V274A, W276A / G / M / V, N277A, S280L / N / W, V282A / M, L283C / V, I286D / E / F / M / V, S288E / L / R / W, V289A / D / E / I / P / T, T290D / H / N / P, A291H / L / Q / R / S / T / W, A292N / S, I;294A / S / V / Y, G295C / F / K / L / M / P / Q / R / S / T / V / Y, R296E / G / L / Q / W, V297A / I / L, M298C / I / K / R / S / V, G299E / I / K / Q / R / T / V, L300A, T301A / F / FL / M / V, M302A / C / I / L / S, I3O5V, I306G / L / T, N307K / R, A310H / L / M / N / Q / R / S / T, R31 IC, D315E / S, L316I / L / Y, L317E / F / G / Q / V / W, F318A / E / G / S, S323E / G, A324D / G / M, V325L, K326C / L, K327E / G / M / S, T330A / D / E / G / K / R, I331D / G / R / S / W, L332E / H / W, Q334E / R, N335H / Q, T336I / L / M, M337A / V, R338G / L / M, W341A, H342R / S / T, V344K, L345Q / S, or E346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0171] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 17, 21, 23, 25, 26, 28, 29, 30, 32, 37, 38, 40, 41, 42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 83, 84, 87, 90, 91, 93, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 148, 150, 157, 158, 160, 161, 162, 165, 169, 170, 173, 178, 179, 184, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 213, 215, 218, 223, 228, 229, 232, 236, 238, 241, 243, 248, 249, 250, 251, 252, 253, 254, 255, 258, 261, 264, 265, 267, 268, 270, 271, 272, 274, 276, 277, 282, 286, 289, 290, 292, 294, 295, 297, 298, 299, 300, 301, 302, 305, 311,318, 323, 324, 325, 327, 332, 336, 338, 341, 344, or 346; a substitution or amino acid residue 18A / H, 33A / I / T / V, 39P, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 95A / C / G / I, 116L, 117A / Q, 119L, 144L, 154S / V, 156G / H / S, 171S, 175A, 177K, 181E / N / P / R / S, 183L, 212E / G / K / L, 224A / Q / R, 226R, 230T / V, 247T / L / R / S, 256E / G / M / S / W, 257D / E / G, 260G, 280L / W, 283C / V, 288L / R / W, 291S, 296E / G / L / Q / W, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 315E, 316I / Y, 317E / F / G / Q / V / W, 326C / L, 330A / D / E / G / K, 331D / G / S, 334E, 335Q, 337A / V, 338G / L / M, 342R / S / T, or 345Q / S: or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0172] In some embodiments, the substitution at amino acid position 10, 11, 12, 13, 17, 21, 23, 25, 26, 28, 29, 30, 32, 37, 38, 40, 41, 42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 83, 84, 87, 90, 91, 93, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 148, 150, 157, 158, 160, 161, 162, 165, 169, 170, 173, 178, 179, 184, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 213, 215, 218, 223, 228, 229, 232, 236, 238, 241, 243, 248, 249, 250, 251, 252, 253, 254, 255, 258, 261, 264, 265, 267, 268, 270, 271, 272, 274, 276, 277, 282, 286, 289, 290, 292, 294, 295, 297, 298, 299, 300, 301, 302, 305, 311, 318, 323, 324, 325, 327, 332, 336, 338, 341, 344, or 346 is 10F, 1 IN, 12A / F / L / M / P / R / T / V, 13W, 17P, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 561 / L. 57V, 58M, 60A / C / D / R / S / V, 61 A, 62A / V, 64A / Q / W, 65C, 67R, 7 IT, 73Q / W, 74D, 75A / D, 77M, 80E, 83A / E / G / L / T / V. 84V, 87E, 90E / F, 91A / E / V. 93K, 97A / E, 991. 100A / G / H / L / R / S / T / V, 101P / V / W. 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E. 121N. 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P. 128E, 129L / W, 131T, 135A / H / M / Q / V. 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / T / V, 148F, 150D / L, 157F / Q / V, 158R / S, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 173M, 178V, 179E, 184L, 190I / M, 191C, 192E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 210G / V, 212E / G / K / L, 213D / N / R / T, 214A / C / H / K / Q / Y, 215G / T, 218F, 223C, 228D / L, 229W, 232G / M / N / R / S, 236L / V. 238K / S / V, 241E / G / Q / R, 243H, 248G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / L / N / Q / S / V, 255N / S / V / Y, 258D / M / P / V, 264A / E / G / S / V, 265A / F, 266C / I, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 282A / M, 286D / E / F / M / V, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 292N / S, 294A / S / V / Y, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 297A / I / L, 298I / S, 298C / K / R / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C / I / L / S, 305V, 31 IC, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 332E / H / W, 336I / L / M, 338G / L / M, 341 A, 344K, or 346S, respectively.

[0173] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F. UN, 12A / F / L / M / P / R / T / V, 13W, 17P, 18A / H, 21S, 23KZT, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 33A / VT / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P, 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 561 / L, 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 7 IT, 73Q / W, 74D, 75A / D, 77M, 80E, 83A / E / G / L / T / V, 84V, 86D / L, 87E, 89C / D / E / L / N / Q / R / S / T / V / Y, 90E / F, 91A / E / V, 93K, 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / S, 157F / Q / V, 158R / S, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L, 184L, 185C / F / M / P / S / T / V, 190I / M, 191C, 192E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 210G / V, 212E / G / K / L, 213D / N / R / T, 214A / C / H / K / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 230T / V, 232G / M / N / R / S, 236L / V. 238K / S / V, 241E / G / Q / R, 243H, 247I / L / R / S, 248G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / L / N / Q / S / V, 255N / S / V / Y, 256E / G / M / S / W, 257D / E / G, 258D / M / P / V. 260G, 261D / P / T. 264A / E / G / S / V, 265A / F, 266C / I. 267I / M, 268F / G / M, 270L / M / V. 271A / E / G / T. 272M / S, 274A, 276A / G / M / V, 277A, 280L / W, 282A / M, 283C / V, 286D / E / F / M / V, 288L / R / W, 289A / D / E / I / P / T. 290D / H / N / P, 291H / L / Q / R / S / T / W, 291S, 292N / S, 294A / S / V / Y, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 296E / G / L / Q / W, 297A / I / L, 298 l / S. 298C / K / R / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C / I / L / S, 305V, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 311C, 315E, 316I / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 330A / D / E / G / K, 331D / G / S, 332E / H / W, 334E, 335Q, 336FL / M, 337A / V, 338G / L / M, 341A, 342R / S / T, 344K, 345Q / S, or 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0174] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue S10F, G1 IN, M12A / F / L / P / R / T / V, F13W, Y15M, S17P, S18A / H, N21S, Y23K / T, N25A / D / F / G / L / M / S / T, K26L, I28A / L / M / V, E29D / S, K30A / L / T / V / W, R32E, G33A / I / L / T / V, D37A / F / K / L / M / R, M38A / E / L / R / S / T / V, H39P, T40D / EP / S, V41E / I, E42T, A45S, T52C, F54L, V56EL, I57V, I58M, P60A / C / D / R / S / V, T61A, E62A / V, V64A / Q / W, P65C, K67R, P71T, L73Q / W, E74D, G75A / D, S77M, G80E, H81Y, I83A / E / G / L / T / V, I84V, K86D / G / L, K87E, K89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, D90E / F, S91A / E / V, V93K, V95A / C / G / I, N97A / E, L99I, N100A / G / H / L / R / S / T / V, A101P / V / W, M102T, D103G / V, L104T, V105K / L / R, S106G / L / M / Q / R / V / W, V107M / S, I109F / L, F113L / M, A114E, G116L, G117A / Q, Q119L, G121N, V122C / F / G / L / Q / T / W, D123K / R, G125K / T, D126M, K127P, D128E, F129L / W, V131T, L135A / H / M / Q / V, D137N / R, N140I, E141D / G, S143A / C / E / F / H / M / T / V, Y144L, Y148F, V150D / L, C154S / V, T156G / H / Q / S, L157F / Q / V, G158R / S, L159F, K160R, L161M / V, A162C, L165M, S169G / N, F170H / Y, A171S, L173M, Q175A, P177K, N178V, D179E, K181E / N / P / R / S, I183L / V, V184L, N185C / F / G / K / M / P / S / T / V, L190I / M, V191C, D192E / F / L / N, T193K / Q, T194A / G / V, E197W, H198M, F204W, P206L, A208H / I / K / L / P / T, S210G / V, T212E / G / K / L, G213D / N / R / T, L214A / C / H / K / L / Q / Y, A215G / T, Y218F, V223C, Y224A / Q / R, I226R, F228D / L, N229W, N230T / V, T232G / M / N / R / S, I236L / V, C238K / S / V, S241E / G / Q / R, F243H, A247I / L / R / S, F248G / I / K / T, L249K / L / R / S, D250E / I / S, K251C / E / F / G / L / R / S / T / Y, P252A / Q, I253L / W, K254E / L / N / Q / S / V, A255N / S / V / Y, K256A / E / G / M / S / W, V257D / E / G, F258D / M / P / V, V260G, E261D / P / T, K264A / E / G / S / V, R265A / F, V266C / I / L, L267FM, E268F / G / M, F270L / M / V, S271A / E / G / T, E272M / S, V274A, W276A / G / M / V, N277A, S280L / N / W, V282A / M, L283C / V, I286D / E / F / M / V, S288E / L / R / W, V289A / D / E / PP / T, T290D / H / N / P, A291H / L / Q / R / S / T / W, A292N / S, F294A / S / V / Y, G295C / F / K / L / M / P / Q / R / S / T / V / Y, R296E / G / L / Q / W, V297A / I / L, M298C / I / K / R / S / V, G299E / I / K / Q / R / T / V, L300A, T301A / F / EL / M / V, M302A / C / EL / S, I3O5V, I306G / L / T, N307K / R, A310H / L / M / N / Q / R / S / T, R311C, D315E / S, L316VL / Y, L317E / F / G / Q / V / W, F318A / E / G / S, S323E / G, A324D / G / M, V325L, K326C / L, K327E / G / M / S, T330A / D / E / G / K / R, I331D / G / R / S / W, L332E / H / W, Q334E / R, N335H / Q, T336I / L / M, M337A / V, R338G / L / M, W341A, H342R / S / T, V344K, L345Q / S, E346S,, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0175] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 86, 89, 93, 101, 122, 143, 157, 183, 192, 194, 198, 212, 214, 230, 241, 248, 249, 250, 251, 254, 266, 268, 280, 291, 295, 298, 307, 316, 318, 326, 330, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0176] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 1 IN, 12A / F / L / P / R / T / V, 23K / T, 25A / D / F / G / L / M / S / T, 29D / S, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 75A / D, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 93K, 101P / V / W, 122C / F / G / L / Q / T / W, 143A / C / E / F / H / M / T / V, 157F / Q / V, I83L / V, 192E / F / L / N, 194A / G / V, 198M, 212E / G / I / K / L, 214A / C' / H / K / L / Q / Y, 23OT / V, 241E / G / Q / R, 248G / I / K / T, 249K / L / R / S, 250E / VS, 251C / E / F / G / L / R / S / T / Y, 254E / K / L / N / Q / S / V, 266C / VL, 268F / G / M, 280L / N / W, 291H / L / Q / R / S / T / W, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 298C / I / K / R / S / V, 307K / R, 316I / L / Y, 318A / E / G / S, 326C / L, 330A / D / E / G / K / R, or 331D / G / R / S / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0177] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 1 IN, 12T, 23T. 25A, 29S, 37L, 38A / S, 75D, 86D, 89M, 93K. 101P, 122F, 157V, 183L, 192E, 194V, 198M, 212E, 214L, 226R, 230T / V, 241G, 249L, 2501. 251E / S, 254S, 266L, 268G, 280L. 291Q, 295V, 298S / V, 307R, 316L, 318S. 326L. 330A, or 331G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0178] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 93, 101, 122, 143, 157, 192, 194, 198, 241, 248, 249, 250, 251, 254, 268, 295, 298, or 318; a substitution or amino acid residue 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 183L, 212E / G / K / L, 214A / C / H / K / L / Q / Y, 226R, 230T / V, 266C / I, 280L / W, 291H / L / Q / R / S / T / W, 307K / R, 326C / L, 33OA / D / E / G / K, or 331D / G / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0179] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 1 IN, 12A / F / L / P / R / T / V, 23K / T, 25A / D / F / G / L / M / S / T, 29D / S, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 75A / D, 86D / L, 89C / D / F / L / N / Q / R / S / T / V / Y, 93K, 101P / V / W, 122C / F / G / L / Q / T / W, 143A / C / E / F / H / M / T / V, 157F / Q / V, 183L, 192E / F / L / N, I94A / G / V, 198M, 212E / G / K / L, 2I4A / C / H / K / L / Q / Y, 226R, 23OT / V, 241E / G / Q / R, 248G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 254E / L / N / Q / S / V, 266C / I, 268F / G / M, 280L / W, 291H / L / Q / R / S / T / W, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 298C / K / R / V, 307K / R, 318A / E / G / S, 326C / L, 330A / D / E / G / K, or 331D / G / S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0180] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 1 IN, 12T, 23T, 25A, 29S, 37L, 38A / S, 75D, 86D, 93K, 101P. 122F, 157V, 183L. 192E. 194V, 198M, 212E, 214L, 226R, 230T / V, 241G, 249L, 2501, 251E / S, 254S, 266L, 268G, 280L, 291Q, 295V, 298S / V, 307R, 318S, 326L, 330A, or 331G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0181] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 37, 249, 266, or 316 or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 1 IN, 37L, 249L. 266L, or 316L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution G1 IN, D37L, S249L, V266L, or M316L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 37 / 249 / 266 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 11N / 37L / 249L / 266L / 316L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0182] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 12, 230, or 251, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 12T, 230T, or 25IS, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution M12T, N230T, or K251S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 12 / 230 / 251, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 12T / 230T / 251S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0183] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 198 or 326, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 198M or 326L, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution H198M or K326L, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 198 / 326, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 198M / 326L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0184] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 194, 298, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 194V, 298S, or 331G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution T194V, M298S, or 1331G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 194 / 298 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 194V / 298S / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0185] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 12 or 25, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 12M or 25A, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution T12M or N25A, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 12 / 25, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 12M / 25A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0186] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 89, 157, 183, or 214, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 89M, 157V, 183L, or 214L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution K89M, L157V, I183L, or V214L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 89 / 157 / 183 / 214, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 89M / 157V / 183L / 214L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0187] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 29, 226, or 230, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 29S, 226R, or 230V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution E29S, I226R, or T230V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 29 / 226 / 230, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 29S / 226R / 230V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0188] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 23 or 254, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 23T or 254K, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution Y23T or S254K, or combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 23 / 254, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 23T / 254K. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0189] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 75, 250, 268, or 307, or an combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 75D, 2501, 268G, or 307R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution G75D. D250I, E268G, or N307R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 75 / 250 / 268 / 307, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 75D / 250I / 268G / 307R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0190] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 38, 93, 192, 248, or 251, or an combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 38S, 93K, 192E, 248F, or 25 IE, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution M38S, V93K, D192E, K248F, or S251E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 38 / 93 / 192 / 248 / 251, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 38S / 93K / 192E / 248F / 251E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0191] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 38, 86, 101, 241, or 318, or an combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 38A, 86D, 101P, 241G, or 318S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution M38A, K86D, A101P, S241G, or F318S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 38 / 86 / 101 / 241 / 318, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 38A / 86D / 101P / 241G / 318S. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0192] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 122, 143, 212, or 295, or an combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 122F, 143S, 212E. or 295V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution V122F, V143S. T212E, or G295V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 122 / 143 / 212 / 295, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 122F / 143S / 212E / 295V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0193] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 291, 295, or 298, or an combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 291Q, 295G, or 298V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution A291Q, V295G, or S298V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 291 / 295 / 298, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set 291Q / 295G / 298V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0194] In some embodiments, the amino acid sequence of the engineered RNA ligase further comprises at least a substitution at amino acid position 2, 14, 15, 63, 69, 81, 85, 95, 138, 142, 149, 159, 171, 186, 195, 202, 266, 275, 283, 285, 303, 314, 333, or 339; a substitution or amino acid residue 181, 33L, 39Q / S, 86G, 89G / H / I / M / W, 116N, 117D, 119T, 144W, 154C, 156A / L / N / Q / T, 175K, 177P, 181D, 183V, 185G / K, 2121, 2141, 224Y, 2261, 230D, 247K, 256A, 257L, 260S, 280G / N, 288E, 291P, 296K, 306L, 307E / Q, 310K, 315S / T, 316L, 317A, 326R, 330M / R, 331L / R / W, 334R, 335D / H, 337L, 3421, or 345V; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0195] In some embodiments, the amino acid sequence of the engineered RNA ligase further comprises at least a substitution or amino acid residue 2E / G / V, 14K, 15E / L / M / P / Y, 181, 33L, 39Q / S, 63V, 69D, 81Y, 85L, 86G, 89G / H / I / M / W, 95V, 116N, 117D, 119T, 138R, 142R, 144W, 149R / T, 154C, 156A / L / N / Q / T, 159F, 171E. 175K, 177P, 181D, 183V, 185G / K, 186D / R, 195G, 202Y, 2121, 2141, 224Y, 2261, 230D, 247K, 256A, 257L. 260S, 266L, 275N, 280G / N, 2831, 285K, 288E, 291P, 296K, 303Q, 306L, 307E / Q, 310K, 314W, 315S / T, 316L, 317A, 326R, 330M / R, 331L / R / W, 333V, 334R, 335D / H, 337L, 339P, 3421, or 345V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0196] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 256, 258. 254, 251, 248, 37, 54, 232, 15. 71, 26, 116, 38, 25, 106 / 251, 52, 33, 247, 37 / 249 / 266 / 316 / 330 / 334 / 335, 21. 11 / 37 / 249 / 266 / 316. 261, 11 / 266 / 316. 11 / 248 / 249 / 266, 331, 11 / 248 / 249, 255. 37 / 249 / 266 / 316 / 334, 266. 11 / 37 / 156 / 249 / 335, 37 / 156 / 248 / 249 / 266 / 334,334, 156, 11 / 266 / 316 / 334, 11 / 39 / 156 / 248 / 316, 11 / 249, 37 / 156, 107 / 251, 156 / 249 / 316, 37 / 156 / 316, 11 / 37 / 156 / 249, 11 / 37 / 334, 194, 37 / 316, 175, 11 / 156 / 249 / 316, 11 / 37, 252, 12, 37 / 266, 11 / 37 / 156 / 266, 11 / 37 / 156 / 248 / 266, 37 / 236 / 248 / 249, 37 / 156 / 248 / 249, 11 / 266 / 330 / 335, 249, 11 / 156 / 248 / 249 / 316, 37 / 249 / 316, 11, 11 / 156, 11 / 37 / 249, 330, 316, 156 / 249 / 266 / 335, 249 / 266 / 316 / 330 / 335, 11 / 156 / 249, 266 / 316 / 330 / 334 / 335, 156 / 248 / 249 / 316 / 334, 37 / 156 / 248 / 249 / 316, 156 / 266, 248 / 249, 156 / 249 / 316 / 330 / 335, 218, 37 / 156 / 248 / 249 / 266, 11 / 37 / 156 / 248 / 249 / 316, 37 / 156 / 316 / 330, 11 / 37 / 248 / 249 / 316 / 330, 11 / 37 / 248 / 249, 249 / 330 / 335, 11 / 37 / 156 / 330 / 334, 40, 11 / 37 / 148 / 249 / 316, 83, 323, 193, 335, or 257, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0197] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 256A, 258P, 254L, 25IS, 248T, 37M. 54L, 258M, 232S. 15M, 71T, 26L, 232M, 116L, 38S. 25 IL. 25S, 106G / 251F. 52C, 33L, 251G. 256S, 247L, 37L / 249R / 266L / 316L / 330R / 334R / 335H. 21S, 11N / 37L / 249L / 266L / 316L. 261P. 11N / 266L / 316L, 11N / 248T / 249R / 266L, 33 ID, 11N / 248T / 249L, 37F. 255V, 37L / 249R / 266L / 316L / 334R, 38L, 266L, 232R, 11N / 37L / 156Q / 249R / 335H, 37L / 156Q / 248T / 249R / 266L / 334R, 334E, 156Q, 11N / 266L / 316L / 334R, 11N / 39P / 156Q / 248T / 316L, 11N / 249R, 37L / 156Q, 107M / 251R, 156Q / 249R / 316L, 37L / 156Q / 316L, 11N / 37L / 156Q / 249L, 11N / 37L / 334R, 194A, 37L / 316L, 175A, 11N / 156Q / 249L / 316L, 11N / 37L, 252Q, 37L, 12F, 11N / 249L, 37L / 266L, 11N / 37L / 156Q / 266L, 11N / 37L / 156Q / 248T / 266L, 37L / 236L / 248T / 249R, 37L / 156Q / 248T / 249L, 11N / 266L / 330R / 335H, 249L, 11N / 156Q / 248T / 249R / 316L, 37L / 249L / 316L, UN, 11N / 156Q, 11N / 37L / 249L, 33OR, 316L, 156Q / 249L / 266L / 335H, 249L / 266L / 316L / 330R / 335H, 11N / 156Q / 249L, 249R, 266L / 316L / 330R / 334R / 335H, 254V, 156Q / 248T / 249L / 316L / 334R, 33A, 37L / 156Q / 248T / 249R / 316L, 156Q / 266L, 248T / 249L, 25M, 156Q / 249R / 316L / 330R / 335H, 218F, 12V, 12T, 37L / 156Q / 248T / 249L / 266L, 12L, 11N / 37L / 156Q / 248T / 249L / 316L, 37L / 156Q / 316L / 330R, 11N / 37L / 248T / 249R / 316L / 330R, 11N / 37L / 248T / 249L, 249R / 330R / 335H, 11N / 37L / 156Q / 330R / 334R, 25L, 38R, 40D, 248T / 249R, 11N / 37L / 148F / 249L / 316L, 83V, 323E, 193Q, 335H, 257G, or 258V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0198] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) K256A. E258P, K254L, K251S, K248T, D37M, F54L, E258M, T232S, Y15M, P71T, K26L, T232M, G116L, M38S, K251L, N25S, S106G / K251F, T52C, G33L, K251G, K256S, A247L, D37L / S249R / V266L / M316L / T330R / Q334R / N335H, N21S, G11N / D37L / S249L / V266L / M316L, E261P, G11N / V266L / M316L, G11N / K248T / S249R / V266L, I331D, G11N / K248T / S249L, D37F, A255V, D37L / S249R / V266L / M316L / Q334R, M38L, V266L, T232R, G11N / D37L / T156Q / S249R / N335H, D37L / T156Q / K248T / S249R / V266L / Q334R, Q334E, T156Q, G11N / V266L / M316L / Q334R, G11N / H39P / T156Q / K248T / M316L, G11N / S249R, D37L / T156Q, V107M / K251R, T156Q / S249R / M316L, D37L / T156Q / M316L, G11N / D37L / T156Q / S249L, G11N / D37L / Q334R, T194A, D37L / M316L, Q175A, G11N / T156Q / S249L / M316L, G11N / D37L, P252Q, D37L, M12F, G11N / S249L, D37L / V266L, G11N / D37L / T156Q / V266L, G11N / D37L / T156Q / K248T / V266L, D37L / I236L / K248T / S249R, D37L / T156Q / K248T / S249L, G11N / V266L / T330R / N335H, S249L, G11N / T156Q / K248T / S249R / M316L, D37L / S249L / M316L, GUN, G11N / T156Q, G11N / D37L / S249L, T33OR, M316L, T156Q / S249L / V266L / N335H. S249L / V266L / M316L / T330R / N335H. G11N / T156Q / S249L. S249R, V266L / M316L / T330R / Q334R / N335H. K254V, T156Q / K248T / S249L / M316L / Q334R, G33A, D37L / T156Q / K248T / S249R / M316L, T156Q / V266L, K248T / S249L, N25M, T156Q / S249R / M316L / T330R / N335H. Y218F, M12V, M12T, D37L / T156Q / K248T / S249L / V266L, M12L. G11N / D37L / T156Q / K248T / S249L / M316L, D37L / T156Q / M316L / T330R, G11N / D37L / K248T / S249R / M316L / T330R, G11N / D37L / K248T / S249L, S249R / T330R / N335H, G11N / D37L / T156Q / T330R / Q334R, N25L, M38R, T40D, K248T / S249R, G11N / D37L / Y148F / S249L / M316L, I83V, S323E, T193Q, N335H, V257G, orE258V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0199] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 37 / 156 / 232 / 249 / 266 / 316 / 334 / 335, 11 / 12 / 37 / 249 / 254 / 256 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 251 / 254 / 256 / 266 / 316, 11 / 12 / 37 / 38 / 230 / 249 / 266 / 316, 11 / 37 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 38 / 249 / 254 / 266 / 316, 11 / 37 / 230 / 249 / 256 / 266 / 316, 11 / 37 / 249 / 254 / 266 / 316, 11 / 37 / 230 / 249 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 254 / 266 / 316, 11 / 12 / 37 / 38 / 230 / 249 / 251 / 256 / 266 / 316, 11 / 12 / 37 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 249 / 251 / 254 / 256 / 266 / 316, 11 / 37 / 38 / 249 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 256 / 266 / 316, 11 / 12 / 37 / 249 / 266 / 316, 11 / 12 / 25 / 37 / 38 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 38 / 102 / 230 / 249 / 251 / 256 / 266 / 316, 11 / 12 / 37 / 249 / 256 / 266 / 316, 11 / 37 / 38 / 230 / 249 / 256 / 266 / 316, 11 / 37 / 249 / 256 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 251 / 256 / 266 / 316, 11 / 37 / 230 / 249 / 251 / 254 / 266 / 316, 11 / 37 / 230 / 249 / 254 / 266 / 316, 11 / 37 / 249 / 251 / 254 / 266 / 316, 11 / 12 / 25 / 37 / 230 / 249 / 266 / 316, 11 / 37 / 38 / 230 / 249 / 266 / 316, 11 / 37 / 38 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 38 / 230 / 249 / 256 / 266 / 316, 11 / 37 / 102 / 249 / 266 / 316, 11 / 37 / 249 / 251 / 266 / 316, 11 / 12 / 25 / 37 / 249 / 251 / 256 / 266 / 316, 11 / 12 / 37 / 102 / 249 / 256 / 266 / 316, 11 / 12 / 37 / 38 / 230 / 249 / 251 / 254 / 256 / 266 / 316, 11 / 12 / 37 / 249 / 251 / 256 / 266 / 316, 11 / 37 / 102 / 230 / 249 / 251 / 254 / 256 / 266 / 316, 11 / 12 / 37 / 38 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 251 / 254 / 266 / 316, 11 / 37 / 156 / 248 / 249 / 266 / 316 / 334 / 335, 11 / 12 / 25 / 37 / 38 / 249 / 266 / 316, 11 / 12 / 37 / 102 / 230 / 249 / 251 / 254 / 266 / 316, 11 / 12 / 37 / 38 / 249 / 251 / 254 / 266 / 316, 11 / 12 / 37 / 249 / 251 / 254 / 266 / 316, 11 / 12 / 37 / 102 / 249 / 266 / 316, 11 / 37 / 38 / 230 / 249 / 251 / 254 / 256 / 266 / 316, 11 / 12 / 25 / 37 / 230 / 249 / 251 / 256 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 266 / 311 / 316,11 / 12 / 37 / 249 / 254 / 266 / 316, 11 / 37 / 38 / 230 / 249 / 251 / 254 / 266 / 316, 11 / 12 / 37 / 38 / 249 / 251 / 256 / 266 / 316, 11 / 37 / 156 / 249 / 266 / 316 / 334 / 335, 11 / 37 / 249 / 257 / 258 / 266 / 316 / 334, 11 / 12 / 25 / 37 / 38 / 249 / 251 / 256 / 266 / 316, 11 / 37 / 249 / 266 / 316 / 334, 11 / 12 / 25 / 37 / 230 / 249 / 254 / 256 / 266 / 316, 11 / 37 / 38 / 249 / 251 / 256 / 266 / 316, 11 / 12 / 37 / 38 / 230 / 249 / 251 / 254 / 266 / 316, 11 / 12 / 37 / 38 / 249 / 266 / 316, 11 / 37 / 249 / 257 / 258 / 266 / 316 / 334 / 335, 11 / 12 / 37 / 38 / 249 / 254 / 256 / 266 / 316, 11 / 37 / 232 / 248 / 249 / 257 / 258 / 266 / 316 / 334 / 335, 11 / 37 / 156 / 249 / 258 / 266 / 316 / 334 / 335, 11 / 12 / 37 / 102 / 230 / 249 / 254 / 266 / 316, 11 / 25 / 37 / 249 / 266 / 316, 11 / 37 / 156 / 249 / 257 / 258 / 266 / 316 / 334 / 335, 11 / 12 / 37 / 102 / 230 / 249 / 266 / 316, 11 / 37 / 156 / 248 / 249 / 257 / 258 / 266 / 316 / 334, 11 / 37 / 248 / 249 / 266 / 316 / 334 / 335, 11 / 37 / 232 / 249 / 257 / 258 / 266 / 316 / 334 / 335, 11 / 37 / 249 / 266 / 316 / 334 / 335, 11 / 37 / 156 / 249 / 266 / 316 / 334, 11 / 12 / 37 / 38 / 102 / 249 / 256 / 266 / 316, 11 / 37 / 156 / 249 / 257 / 266 / 316 / 334 / 335, 11 / 12 / 37 / 140 / 249 / 251 / 254 / 266 / 316, 11 / 37 / 249 / 251 / 254 / 256 / 266 / 316, 11 / 37 / 156 / 249 / 258 / 266 / 316 / 334, 11 / 37 / 156 / 248 / 249 / 266 / 316 / 330 / 335, 11 / 12 / 37 / 38 / 249 / 256 / 266 / 316, 11 / 37 / 232 / 249 / 266 / 316 / 334 / 335, 11 / 37 / 249 / 266 / 316 / 330 / 334 / 335, 11 / 37 / 156 / 248 / 249 / 266 / 316 / 334, 11 / 37 / 156 / 249 / 266 / 316 / 335, 11 / 37 / 249 / 257 / 266 / 316 / 334, 11 / 37 / 248 / 249 / 266 / 316 / 334, 11 / 37 / 232 / 248 / 249 / 266 / 316 / 334 / 335, 11 / 12 / 25 / 37 / 38 / 230 / 249 / 251 / 256 / 266 / 316. 11 / 37 / 249 / 266 / 316 / 330 / 335, 11 / 37 / 248 / 249 / 257 / 266 / 316 / 334 / 335, 11 / 37 / 232 / 248 / 249 / 266 / 316 / 334, 11 / 37 / 156 / 232 / 248 / 249 / 266 / 316 / 334 / 335, 11 / 37 / 232 / 249 / 257 / 266 / 316 / 330 / 334, 11 / 37 / 156 / 248 / 249 / 266 / 291 / 316 / 334 / 335, 11 / 37 / 156 / 232 / 248 / 249 / 257 / 258 / 266 / 316 / 334, 11 / 12 / 25 / 37 / 249 / 266 / 316, 11 / 37 / 156 / 248 / 249 / 266 / 316 / 330 / 334 / 335, 11 / 37 / 232 / 248 / 249 / 266 / 316 / 330 / 335, 11 / 37 / 156 / 232 / 249 / 266 / 316 / 330 / 334 / 335, 11 / 37 / 197 / 249 / 266 / 316, 11 / 37 / 249 / 266 / 316 / 338, 11 / 37 / 249 / 266 / 316 / 327, 11 / 37 / 192 / 249 / 266 / 316, 11 / 37 / 249 / 266 / 316 / 331, 11 / 37 / 249 / 266 / 316 / 326, 11 / 37 / 249 / 266 / 316 / 345, 11 / 37 / 101 / 249 / 266 / 316, 11 / 37 / 100 / 249 / 266 / 316, 11 / 37 / 141 / 249 / 266 / 316, 11 / 37 / 198 / 249 / 266 / 316, 11 / 37 / 64 / 249 / 266 / 316, 11 / 37 / 249 / 266 / 306 / 316, 11 / 37 / 249 / 266 / 316 / 317, 11 / 37 / 249 / 265 / 266 / 316, 11 / 37 / 249 / 266 / 296 / 316, 11 / 37 / 194 / 249 / 266 / 316, 11 / 37 / 62 / 249 / 266 / 316, 11 / 37 / 90 / 249 / 266 / 316, 11 / 37 / 249 / 266 / 307 / 316, 11 / 37 / 103 / 249 / 266 / 316, 11 / 37 / 210 / 249 / 266 / 316, 11 / 37 / 105 / 249 / 266 / 316, or 11 / 37 / 61 / 249 / 266 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0200] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 37L / 156Q / 232G / 249L / 266L / 316L / 334R / 335H, 11N / 12V / 37L / 249L / 254V / 256A / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 266L / 316L, 11N / 12L / 37L / 230T / 249L / 251T / 266L / 316L, 11N / 12L / 37L / 230T / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 12A / 37L / 38R / 230T / 249L / 266L / 316L, 11N / 37L / 230T / 249L / 251S / 266L / 316L, 11N / 12P / 37L / 38R / 249L / 254V / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 251T / 266L / 316L, 11N / 37L / 230T / 249L / 256A / 266L / 316L, 11N / 37L / 249L / 254V / 266L / 316L, 11N / 37L / 230T / 249L / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 254V / 266L / 316L, 11N / 12V / 37L / 38R / 230T / 249L / 251T / 256A / 266L / 316L, 11N / 12L / 37L / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 249L / 251T / 254V / 256A / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 251S / 266L / 316L, 11N / 37L / 38R / 249L / 266L / 316L. 11N / 12T / 37L / 230T / 249L / 256A / 266L / 316L, 11N / 12L / 37L / 249L / 266L / 316L, 11N / 12V / 37L / 249L / 266L / 316L, 11N / 12L / 25M / 37L / 38R / 230T / 249L / 251T / 266L / 316L, 11N / 12L / 37L / 38R / 102T / 230T / 249L / 251T / 256A / 266L / 316L. 11N / 12L / 37L / 230T / 249L / 266L / 316L, 11N / 12V / 37L / 249L / 256A / 266L / 316L, 11N / 37L / 38R / 230T / 249L / 256A / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 37L / 249L / 256A / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 251S / 256A / 266L / 316L, 11N / 12T / 37L / 249L / 251T / 266L / 316L, 11N / 37L / 230T / 249L / 251S / 254L / 266L / 316L, 11N / 37L / 230T / 249L / 254V / 266L / 316L, 11N / 37L / 249L / 251S / 254L / 266L / 316L, 11N / 12T / 25L / 37L / 230T / 249L / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 251S / 254L / 256A / 266L / 316L, 11N / 37L / 38R / 230T / 249L / 266L / 316L, 11N / 12T / 37L / 249L / 251S / 266L / 316L, 11N / 37L / 38R / 249L / 251T / 266L / 316L, 11 N / l 2V / 37L / 230T / 249L / 251S / 256A / 266L / 316L, 11N / 12L / 37L / 230T / 249L / 251T / 254V / 256A / 266L / 316L, 11N / 12T / 37L / 38R / 230T / 249L / 256A / 266L / 316L, 11N / 12L / 37L / 230T / 249L / 256A / 266L / 316L, 11N / 37L / 102T / 249L / 266L / 316L, 11N / 37L / 249L / 251T / 266L / 316L, 11N / 12V / 25M / 37L / 249L / 251T / 256A / 266L / 316L. 11N / 12T / 37L / 102T / 249L / 256A / 266L / 316L, 11N / 37L / 230T / 249L / 251T / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 251S / 254L / 256A / 266L / 316L. 11N / 12V / 37L / 38R / 230T / 249L / 251T / 254V / 256A / 266L / 316L, 11N / 12T / 37L / 38R / 230T / 249L / 266L / 316L. 11N / 12L / 37L / 38R / 249L / 254V / 266L / 316L. 11N / 12L / 37L / 249L / 251T / 256A / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 266L / 316L, 11N / 37L / 102T / 230T / 249L / 251S / 254V / 256A / 266L / 316L. 11N / 37L / 230T / 249L / 251S / 254V / 266L / 316L. 11N / 12T / 37L / 230T / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 12T / 37L / 38R / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 251S / 254V / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 251T / 266L / 316L, 11N / 37L / 156Q / 248T / 249L / 266L / 316L / 334R / 335H, 11N / 12V / 37L / 38R / 230T / 249L / 266L / 316L, 11N / 12T / 25M / 37L / 38R / 249L / 266L / 316L, 11N / 12V / 37L / 102T / 230T / 249L / 251 T / 254V / 266L / 316L, 11N / 12L / 37L / 249L / 256A / 266L / 316L, 11N / 12T / 37L / 249L / 266L / 316L, 11N / 37L / 230T / 249L / 251T / 254L / 266L / 316L, 11N / 12V / 25L / 37L / 38R / 249L / 266L / 316L, 11N / 12T / 37L / 38R / 249L / 251S / 254V / 266L / 316L, 11N / 12V / 37L / 249L / 251S / 254L / 256A / 266L / 316L, 11N / 12T / 37L / 249L / 251 T / 254V / 266L / 316L, 11N / 12T / 37L / 102T / 249L / 266L / 316L, 11 N / l 2V / 37L / 38R / 249L / 251S / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 251S / 254L / 266L / 316L, 11N / 12T / 37L / 249L / 251T / 256A / 266L / 316L, 11N / 37L / 38R / 230T / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 12V / 37L / 230T / 249L / 251T / 256A / 266L / 316L, 11 N / l 2L / 25M / 37L / 230T / 249L / 251S / 256A / 266L / 316L, 11 N / l 2T / 37L / 230T / 249L / 266L / 311C / 316L, 11N / 12L / 37L / 38R / 230T / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 12L / 37L / 230T / 249L / 251T / 254L / 266L / 316L, 11N / 12L / 37L / 249L / 254V / 266L / 316L, 11N / 37L / 38R / 230T / 249L / 251S / 254V / 266L / 316L, 11N / 12L / 37L / 38R / 249L / 251S / 256A / 266L / 316L. 11N / 12L / 37L / 38R / 249L / 251S / 266L / 316L. 11N / 37L / 156Q / 249L / 266L / 316L / 334R / 335H. 11N / 12A / 37L / 230T / 249L / 266L / 316L, 11N / 12P / 37L / 230T / 249L / 266L / 316L, 11N / 12T / 37L / 38R / 249L / 251T / 266L / 316L, 11N / 37L / 249L / 257G / 258P / 266L / 316L / 334R, 11N / 12L / 25L / 37L / 38R / 249L / 251T / 256A / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 334R, 11N / 12T / 25L / 37L / 230T / 249L / 254V / 256A / 266L / 316L, 11N / 12T / 37L / 249L / 251S / 256A / 266L / 316L, 11N / 37L / 38R / 249L / 251T / 256A / 266L / 316L, 11N / 12V / 37L / 249L / 251S / 266L / 316L, 11N / 12V / 37L / 249L / 251T / 254L / 266L / 316L, 11N / 12V / 37L / 38R / 230T / 249L / 251T / 254V / 266L / 316L, 11N / 12L / 37L / 38R / 249L / 266L / 316L, 11N / 12L / 37L / 249L / 251S / 254L / 256A / 266L / 316L, 11N / 37L / 249L / 257G / 258P / 266L / 316L / 334R / 335H, 11N / 12L / 37L / 38R / 249L / 254L / 256A / 266L / 316L, 11N / 12V / 37L / 249L / 254L / 266L / 316L, 11N / 12T / 37L / 249L / 254L / 266L / 316L, 11N / 12T / 37L / 38R / 249L / 266L / 316L, 11N / 37L / 232G / 248T / 249L / 257G / 258P / 266L / 316L / 334R / 335H, 11N / 12L / 37L / 102T / 249L / 266L / 316L, 11N / 37L / 156Q / 249L / 258P / 266L / 316L / 334R / 335H, 11N / 37L / 156Q / 249R / 266L / 316L / 334R / 335H, 11N / 12T / 37L / 102T / 230T / 249L / 254L / 266L / 316L, 11N / 25M / 37L / 249L / 266L / 316L, 11N / 12V / 37L / 249L / 254L / 256A / 266L / 316L, 11N / 37L / 156H / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 156Q / 249L / 257G / 258P / 266L / 316L / 334R / 335H, 11N / 12T / 37L / 102T / 230T / 249L / 266L / 316L, 11N / 37L / 156Q / 248T / 249L / 257G / 258P / 266L / 316L / 334R, 11N / 12T / 37L / 249L / 256A / 266L / 316L, 11N / 37L / 248T / 249R / 266L / 316L / 334R / 335H, 11N / 37L / 232G / 249L / 257G / 258P / 266L / 316L / 334R / 335H, 11N / 12T / 37L / 38R / 249L / 251T / 254V / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 156Q / 249L / 266L / 316L / 334R, 11N / 12V / 37L / 38R / 249L / 254V / 266L / 316L, 11N / 37L / 249L / 251T / 254V / 266L / 316L, 11N / 12T / 37L / 38R / 102T / 249L / 256A / 266L / 316L, 11N / 37L / 156Q / 249L / 257G / 266L / 316L / 334R / 335H, 11N / 37L / 38R / 249L / 251S / 256A / 266L / 316L, 11N / 12T / 37L / 140I / 249L / 251S / 254L / 266L / 316L. 11N / 37L / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 12V / 37L / 249L / 251T / 266L / 316L, 11N / 37L / 248T / 249L / 266L / 316L / 334R / 335H, 11N / 12T / 37L / 249L / 254V / 266L / 316L, 11N / 12V / 37L / 38R / 249L / 266L / 316L, 11N / 12V / 37L / 249L / 251T / 256A / 266L / 316L, 11N / 37L / 156Q / 249L / 258P / 266L / 316L / 334R. 11N / 37L / 156Q / 248T / 249L / 266L / 316L / 330R / 335H, 11N / 12T / 37L / 38R / 249L / 256A / 266L / 316L, 11N / 37L / 232G / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 156Q / 249R / 266L / 316L / 334R, 11N / 37L / 249L / 266L / 316L / 330R / 334R / 335H, 11N / 37L / 156Q / 248T / 249L / 266L / 316L / 334R, 11N / 37L / 156Q / 249L / 266L / 316L / 335H, 11N / 37L / 249L / 257G / 266L / 316L / 334R, 11N / 37L / 156H / 248T / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 248T / 249L / 266L / 316L / 334R, 11N / 37L / 232G / 248T / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 156H / 248T / 249L / 266L / 316L / 334R, 11N / 37L / 156H / 249L / 266L / 316L / 334R, 11N / 12T / 25M / 37L / 38R / 230T / 249L / 251S / 256A / 266L / 316L, 11N / 12V / 37L / 249L / 251S / 254V / 256A / 266L / 316L, 11N / 37L / 156Q / 248T / 249R / 266L / 316L / 334R / 335H, 11N / 37L / 249L / 266L / 316L / 330R / 335H, 11N / 37L / 248T / 249L / 257G / 266L / 316L / 334R / 335H, 11N / 37L / 232G / 248T / 249L / 266L / 316L / 334R, 11N / 37L / 156Q / 232G / 248T / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 232G / 249L / 257G / 266L / 316L / 330R / 334R, 11N / 37L / 156Q / 248T / 249L / 266L / 291T / 316L / 334R / 335H, 11N / 37L / 156Q / 232G / 248T / 249L / 257G / 258P / 266L / 316L / 334R, 11N / 12V / 37L / 38R / 249L / 251T / 254L / 266L / 316L, 11N / 12T / 25L / 37L / 38R / 249L / 266L / 316L. 11N / 37L / 156Q / 232G / 249R / 266L / 316L / 334R / 335H, 11N / 12T / 25M / 37L / 249L / 266L / 316L, 11N / 37L / 156Q / 248T / 249L / 266L / 316L / 330R / 334R / 335H, 11N / 37L / 156Q / 248T / 249R / 266L / 316L / 334R, 11N / 37L / 156H / 232G / 248T / 249L / 266L / 316L / 334R / 335H, 11N / 37L / 232G / 248T / 249L / 266L / 316L / 330R / 335H, 11N / 37L / 156Q / 232G / 249L / 266L / 316L / 330R / 334R / 335H, 11N / 37L / 197 W / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 338L, 11N / 37L / 249L / 266L / 316L / 327S, 11N / 37L / 192F / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 331S, 11N / 37L / 249L / 266L / 316L / 326C, 11N / 37L / 249L / 266L / 316L / 338G, 11N / 37L / 249L / 266L / 316L / 345S, 11N / 37L / 249L / 266L / 316L / 327G, 11N / 37L / 101V / 249L / 266L / 316L, 11N / 37L / 100V / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 331G, 11N / 37L / 141G / 249L / 266L / 316L, 11N / 37L / 198M / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 326L, 11N / 37L / 64 W / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 306G / 316L, 11N / 37L / 249L / 266L / 316L / 345Q, 11N / 37L / 101W / 249L / 266L / 316L, 11N / 37L / 100R / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 331W, 11N / 37L / 249L / 266L / 316L / 317F, 11N / 37L / 249L / 266L / 316L / 338M, 11N / 37L / 249L / 265F / 266L / 316L, 11N / 37L / 249L / 266L / 296W / 316L, 11N / 37L / 194V / 249L / 266L / 316L, 11N / 37L / 62V / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 316L / 317W, 11N / 37L / 90F / 249L / 266L / 316L, 11N / 37L / 249L / 266L / 307R / 316L, 11N / 37L / 249L / 266L / 316L / 317G, 11N / 37L / 100T / 249L / 266L / 316L, 11N / 37L / 100L / 249L / 266L / 316L, 11N / 37L / 194G / 249L / 266L / 316L, 11N / 37L / 103G / 249L / 266L / 316L, 11N / 37L / 62A / 249L / 266L / 316L, 11N / 37L / 210G / 249L / 266L / 316L, 11N / 37L / 105R / 249L / 266L / 316L, or 11N / 37L / 61A / 249L / 266L / 316L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0201] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 12 / 37 / 141 / 197 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 100 / 101 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 141 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 64 / 100 / 101 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 100 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 101 / 141 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 101 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 64 / 100 / 101 / 230 / 249 / 251 / 266 / 316 / 338 / 345, 11 / 12 / 37 / 197 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 141 / 198 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 197 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 64 / 100 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 64 / 100 / 101 / 156 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 64 / 100 / 101 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 100 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 101 / 197 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 64 / 100 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 101 / 198 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 141 / 197 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 64 / 100 / 101 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 101 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 101 / 197 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 197 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 197 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 141 / 197 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 64 / 197 / 198 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 141 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 141 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 100 / 101 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 141 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 197 / 198 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 64 / 101 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 64 / 197 / 198 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 197 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 100 / 101 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 100 / 109 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 101 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 197 / 198 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 141 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 100 / 197 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 64 / 100 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 64 / 197 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 100 / 197 / 198 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 141 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 101 / 230 / 249 / 251 / 266 / 306 / 316, 11 / 12 / 37 / 64 / 100 / 101 / 197 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 100 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 316 / 326 / 345, 11 / 12 / 37 / 64 / 100 / 101 / 197 / 198 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 64 / 101 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 100 / 101 / 197 / 230 / 249 / 251 / 266 / 316, 11 / 12 / 37 / 101 / 230 / 249 / 251 / 266 / 316 / 327, 11 / 12 / 37 / 64 / 100 / 197 / 198 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 230 / 249 / 251 / 266 / 306 / 316, 11 / 12 / 37 / 64 / 230 / 249 / 251 / 266 / 316 / 338, 11 / 12 / 37 / 101 / 141 / 230 / 249 / 251 / 266 / 316 / 326 / 327, 11 / 12 / 37 / 64 / 101 / 197 / 230 / 249 / 251 / 266 / 316 / 338, or 11 / 12 / 37 / 101 / 198 / 230 / 249 / 251 / 266 / 306 / 316 / 327, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0202] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 12T / 37L / 141G / 197W / 230T / 249L / 251S / 266L / 316L / 326L / 327S, 11N / 12T / 37L / 100V / 101V / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 141G / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 64W / 100V / 101V / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 100V / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 101V / 141G / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11 N / l 2T / 37L / 101 V / l 98M / 230T / 249L / 251S / 266L / 316L / 326L / 327G, 11N / 12T / 37L / 100V / 101V / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 64W / 100V / 101V / 230T / 249L / 251S / 266L / 316L / 338G / 345Q. 11N / 12T / 37L / 64W / 100V / 101V / 230T / 249L / 251S / 266L / 316L / 338L / 345Q, 11N / 12T / 37L / 197W / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 327S, 11N / 12T / 37L / 141G / 198M / 230T / 249L / 251S / 266L / 316L / 327S, 11N / 12T / 37L / 100V / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 327G, 11N / 12T / 37L / 197 W / 230T / 249L / 251S / 266L / 316L / 326L / 327G, 11N / 12T / 37L / 64W / 100V / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 64W / 100V / 101V / 156S / 197W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 64W / 100V / 101V / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 100V / 197 W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 327S, 11N / 12T / 37L / 197W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 101 V / l 97 W / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 64W / 100V / 197W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 101 V / l 98M / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 141G / 197 W / 230T / 249L / 251S / 266L / 316L / 327S, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 327G, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326C / 327S, 11N / 12T / 37L / 64W / 100V / 101V / 197W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 101 V / l 97 W / 230T / 249L / 251S / 266L / 316L / 326L / 327G, 11N / 12T / 37L / 141G / 197 W / 230T / 249L / 251S / 266L / 316L / 326L / 327G, 11 N / l 2T / 37L / 100V / 101W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 197W / 230T / 249L / 251S / 266L / 316L / 327S, 11N / 12T / 37L / 197W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 141G / 197W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101 V / l 98M / 230T / 249L / 251S / 266L / 316L / 326C. 11N / 12T / 37L / 64W / 197W / 198M / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 141G / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 64W / 100V / 101W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 101V / 141G / 230T / 249L / 251S / 266L / 316L. 11N / 12T / 37L / 100V / 101V / 197W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 141G / 230T / 249L / 251S / 266L / 316L / 326C, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 197W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 197 W / l 98M / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 197 W / l 98M / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 64 W / 101W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 64W / 197W / 198M / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101 V / l 97 W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 100V / 101V / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 141G / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 100V / 109F / 197 W / 230T / 249L / 251S / 266L / 316L / 338L, 11 N / l 2T / 37L / 101 W / l 97 W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 64W / 100V / 101W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 197 W / l 98M / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 100V / 101W / 230T / 249L / 251S / 266L / 316L / 338L. 11N / 12T / 37L / 141G / 230T / 249L / 251S / 266L / 316L / 326C / 327S, 11N / 12T / 37L / 100V / 197 W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 197W / 230T / 249L / 251S / 266L / 316L / 326C / 327G. 11N / 12T / 37L / 64W / 100V / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 316L / 326C, 11N / 12T / 37L / 100V / 101W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 64W / 197W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 100V / 197W / 198M / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 141G / 230T / 249L / 251S / 266L / 316L / 327S, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 306G / 316L, 11N / 12T / 37L / 64W / 100V / 101W / 197W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 316L / 326L / 327G, 11N / 12T / 37L / 100V / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 316L / 327G, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 316L / 326C / 345S, 11N / 12T / 37L / 64W / 100V / 101V / 197W / 198M / 230T / 249L / 251S / 266L / 316L, 11 N / l 2T / 37L / 230T / 249L / 251S / 266L / 316L / 326C / 327S, 11N / 12T / 37L / 64 W / 101W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 100V / 101V / 197 W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 316L / 327S, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 100V / 101V / 197W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 100V / 101W / 197W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 64W / 100V / 197W / 198M / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 230T / 249L / 251S / 266L / 306G / 316L, 11N / 12T / 37L / 64W / 230T / 249L / 251S / 266L / 316L / 338G, 11N / 12T / 37L / 64W / 100V / 101V / 197W / 230T / 249L / 251S / 266L / 316L, 11N / 12T / 37L / 64W / 197W / 198M / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 101V / 230T / 249L / 251S / 266L / 316L / 327G, 11N / 12T / 37L / 101V / 141G / 230T / 249L / 251S / 266L / 316L / 326C / 327S, 11N / 12T / 37L / 64W / 101W / 197W / 230T / 249L / 251S / 266L / 316L / 338L, 11N / 12T / 37L / 101W / 230T / 249L / 251S / 266L / 316L / 338G, or 11N / 12T / 37L / 101V / 198M / 230T / 249L / 251S / 266L / 306G / 316L / 327S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. [0203J In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 331, 11 / 12 / 37 / 192 / 193 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330, 11 / 12 / 37 / 192 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 192 / 193 / 194 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 331, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 192 / 193 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 192 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 192 / 193 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330, 11 / 12 / 37 / 60 / 100 / 156 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 193 / 198 / 230 / 249 / 251 / 266 / 296 / 316 / 326 / 331, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 193 / 194 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 193 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326, 11 / 12 / 37 / 193 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 330, 11 / 12 / 37 / 192 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 192 / 193 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 192 / 193 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 330, 11 / 12 / 37 / 192 / 193 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 100 / 156 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 62 / 100 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 330, 11 / 12 / 37 / 60 / 100 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 192 / 193 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 330 / 331, 11 / 12 / 37 / 100 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 193 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326 / 338, 11 / 12 / 37 / 62 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 247 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 18 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 254 / 266 / 316 / 326, 11 / 12 / 33 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 261 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 260 / 266 / 316 / 326, 11 / 12 / 37 / 117 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 256 / 266 / 316 / 326, 11 / 12 / 37 / 198 / 230 / 249 / 251 / 255 / 266 / 316 / 326, 11 / 12 / 37 / 38 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 29 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, 11 / 12 / 25 / 37 / 198 / 230 / 249 / 251 / 266 / 316 / 326, or 11 / 12 / 37 / 198 / 230 / 249 / 251 / 252 / 266 / 316 / 326. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0204] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11 N / l 2T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D / 331W, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 331W, 11N / 12T / 37L / 192L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D. 11N / 12T / 37L / 192L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D / 331G, 11N / 12T / 37L / 192L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 331S, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 298I / 316L / 326L / 330D / 331S, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 330D / 331G, 11N / 12T / 37L / 192L / 193K / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D / 331S, 11N / 12T / 37L / 192L / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331S, 11N / 12T / 37L / 192L / 193K / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 331G, 11N / 12T / 37L / 192L / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331W, 11N / 12T / 37L / 60D / 100R / 156G / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 193K / 198M / 230T / 249L / 251S / 266L / 296G / 316L / 326L / 331W, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D / 331W, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 193K / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L, 11N / 12T / 37L / 193K / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D, 11N / 12T / 37L / 192L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 192L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331S, 11N / 12T / 37L / 192L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 330D, 11N / 12T / 37L / 192L / 193K / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331S, 11 N / l 2T / 37L / 100R / 156G / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331S, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 298I / 316L / 326L / 331G, 11N / 12T / 37L / 62V / 100V / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 330D, 11N / 12T / 37L / 193K / 198M / 230T / 249L / 251S / 266L / 298I / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 331S, 11N / 12T / 37L / 60D / 100R / 198M / 230T / 249L / 251S / 266L / 316L / 326L. 11N / 12T / 37L / 192L / 193K / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 330D / 331S, 11N / 12T / 37L / 192L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331W. 11N / 12T / 37L / 192L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 330D / 331S, 11N / 12T / 37L / 1 OOR / l 98M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 193K / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331S, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L / 338L, 11N / 12T / 37L / 62V / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 247S / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 18H / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37 A / l 98M / 230T / 249L / 251S / 266L / 316L / 326L, 11 N / l 2T / 37L / 198M / 230T / 249L / 251S / 254S / 266L / 316L / 326L, 11N / 12T / 33L / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 261T / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251Y / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 260G / 266L / 316L / 326L. 11N / 12T / 37L / 198M / 230T / 249L / 251C / 266L / 316L / 326L, 11N / 12R / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L. 11N / 12T / 37L / 117 A / l 98M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 256S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 255Y / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 255S / 266L / 316L / 326L, 11N / 12T / 37L / 38E / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251T / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 255V / 266L / 316L / 326L, 11N / 12T / 29D / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 247R / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 25A / 37L / 198M / 230T / 249L / 251S / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 256E / 266L / 316L / 326L, 11N / 12T / 37L / 198M / 230T / 249L / 251S / 252A / 266L / 316L / 326L, or 11N / 12T / 37L / 198M / 230T / 249L / 251S / 261D / 266L / 316L / 326L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0205] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331 / 342, 11 / 12 / 33 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331 / 338, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 12 / 33 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 265 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 265 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 33 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331 / 338, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331 / 338, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 247 / 249 / 251 / 265 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 296 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 296 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 265 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 33 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 296 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 18 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331 / 338, 11 / 12 / 33 / 37 / 156 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 296 / 298 / 316 / 326 / 331, 11 / 12 / 33 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 296 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 296 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 296 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 266 / 296 / 298 / 316 / 317 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 247 / 249 / 251 / 266 / 296 / 298 / 316 / 326 / 331, 11 / 12 / 33 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 266 / 298 / 307 / 316 / 326 / 331, or 11 / 12 / 33 / 37 / 194 / 198 / 230 / 249 / 251 / 265 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0206] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11 N / l 21718H / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37A / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G / 342R, 11N / 12T / 33L / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 18H / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 18H / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G / 338L, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 307R / 316L / 326L / 331G, 11N / 12T / 33L / 37A / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37A / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37A / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 317 W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 33L / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37 A / l 94 V / l 98M / 230T / 249L / 251S / 266L / 298S / 316L / 317 W / 326L / 331G, 11N / 12T / 18H / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G / 338M, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G / 338M, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 18H / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G / 338L, 11N / 12T / 33L / 37L / 194V / 198M / 230I7249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11 N / l 2T / 37A / 194 V / 198M / 230T / 247S / 249L / 251S / 265F / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37 A / l 94 V / l 98M / 230T / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 33L / 37A / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 296W / 298S / 316L / 317W / 326L / 331G. 11N / 12T / 18H / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 307R / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37A / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 18H / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 307R / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 307R / 316L / 326L / 331G. 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 33L / 37A / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 18H / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 33L / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 18H / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G / 338L, 11N / 12T / 37 A / l 94 V / l 98M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 33L / 37A / 156G / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 317W / 326L / 331G, 11 N / l 2T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 326L / 331G, 11N / 12T / 33L / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G / 338L. 11N / 12T / 37A / 194V / 198M / 230T / 249L / 251S / 265F7266L / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 296W / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 33L / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37A / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 317F / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 247S / 249L / 251S / 266L / 296W / 298S / 316L / 326L / 331G, 11N / 12T / 33L / 37A / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 266L / 296W / 298S / 316L / 317W / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 307R / 316L / 326L / 331G, 11N / 12T / 18H / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 266L / 298S / 307R / 316L / 326L / 331G, or 11N / 12T / 33L / 37A / 194V / 198M / 230T / 249L / 251S / 265F / 266L / 298S / 316L / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0207] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331. 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 247 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 247 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 38 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 38 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 38 / 194 / 198 / 230 / 247 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 327 / 331, 11 / 12 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 38 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 192 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 18 / 37 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 38 / 194 / 198 / 230 / 249 / 250 / 251 / 252 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 60 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 29 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 256 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 25 / 29 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 257 / 261 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 256 / 261 / 266 / 298 / 316 / 326 / 331, or 11 / 37 / 156 / 194 / 198 / 230 / 249 / 251 / 254 / 255 / 261 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0208] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / T194V / 198M / 230T / 247R / 249L / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331 G, 11N / 12T / 18H / 37L / 194V / 198M / 230T / 247R / 249L / 251T / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 194 V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 256S / 257D / 266L / 298S / 316L / 326L / 331G. 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 18H / 37L / 38E / 194V / 198M / 230T / 249L / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331G. 11N / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 38E / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251T / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249R / 251T / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 266L / 298S / 316L / 326L / 331G, 11N / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 18A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 257D / 266L / 298S / 316L / 326L / 331G. 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 18H / 37L / 38E / 194V / 198M / 230T / 247R / 249L / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 327M / 331G, 11N / 12T / 18H / 37L / 194V / 198M / 230T / 247R / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / H198M / N230T / S249L / K251S / K254S / A255N / V266L / M298S / M316L / K326L / I331G, 11N / 12T / 18H / 37L / 156G / 194V / 198M / 230T / 249L / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11 N / l 2T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 261D / 266L / 298S / 316L / 326L / 331G. 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 38E / 194V / 198M / 230T / 249L / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249R / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 29D / 37L / 194 V / l 98M / 230T / 249L / 251S / 254S / 255N / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 192N / 194V / 198M / 230T / 249R / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 25 A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 261D / 266L / 298S / 316L / 326L / 331G. 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 18A / 37L / 194V / 198M / 230T / 249L / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 12T / 37L / 194V / 198M / 230T / 249R / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 250I / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 38E / 194V / 198M / 230T / 249L / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11 N / l 2T / 37L / 194V / 198M / 230T / 249L / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 257D / 266L / 298S / 316L / 326L / 331G. 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249R / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 266L / 298S / 316L / 326L / 331G, 11N / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 38E / 194V / 198M / 230T / 249L / 250I / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 247R / 249R / 251S / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 60S / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255Y / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194 V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 247R / 249L / 251T / 252A / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 38E / 194V / 198M / 230T / 249L / 251T / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255N / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 261D / 266L / 298S / 316L / 326L / 331G , 11N / 29D / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 256S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 257D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 25A / 29D / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 257D / 261D / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 256S / 261D / 266L / 298S / 316L / 326L / 331G, or 11N / 37L / 156G / 194V / 198M / 230T / 249L / 251S / 254S / 255V / 261D / 266L / 298S / 316L / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0209] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 12 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 28 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 89 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 95 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 143 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 181 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 185 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 190 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 12 / 37 / 194 / 198 / 213 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, or 11 / 12 / 37 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0210] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 12T / 37L / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11 N / l 2T / 37L / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 95A / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 183V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 185P / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 181E / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 213D / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 89M / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 28M / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 190M / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 143A / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 12T / 37L / 89Q / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, or 11N / 12T / 37L / 95G / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0211] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 25 / 37 / 95 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 95 / 183 / 185 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 28 / 37 / 89 / 143 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 28 / 143 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 181 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 181 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 181 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 143 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 28 / 37 / 143 / 181 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 95 / 181 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 143 / 157 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 95 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 95 / 143 / 181 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 95 / 143 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 181 / 183 / 185 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 143 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 28 / 37 / 95 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 143 / 157 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 28 / 37 / 95 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 95 / 143 / 183 / 185 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, or 11 / 25 / 37 / 95 / 143 / 157 / 183 / 194 / 198 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0212] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 25A / 37L / 95A / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 95G / 183V / 185P / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 183V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25 A / 28M / 37L / 89G / 143M / 183L / 194 V / l 98M / 230T7249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 28M / 143M / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 181E / 183V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143A / 181E / 183V / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143A / 181E / 183V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89G / 143M / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 28M / 37L / 143M / 181E / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143M / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 95A / 181E / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143A / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143A / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89G / 143A / 157V / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 95 A / l 57V / 194 V / l 98M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89G / 95A / 143A / 181E / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25 A / 37L / 95 A / l 43 A / l 94 V / l 98M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143M / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 25A / 37L / 143M / 181E / 183V / 185P / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 143M / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 28M / 37L / 95A / 183V / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 143A / 157V / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 28M / 37L / 95A / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 95A / 143M / 183V / 185P / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, or 11N / 25A / 37L / 95A / 143A / 157V / 183L / 194V / 198M / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0213] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 25 / 37 / 83 / 89 / 137 / 150 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 213 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 137 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 150 / 157 / 183 / 190 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 143 / 150 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 60 / 89 / 143 / 150 / 157 / 183 / 190 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 60 / 89 / 150 / 157 / 183 / 190 / 194 / 198 / 213 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 104 / 137 / 154 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 123 / 137 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 137 / 150 / 154 / 157 / 183 / 190 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 123 / 143 / 150 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 60 / 89 / 104 / 143 / 150 / 157 / 183 / 194 / 198 / 213 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 60 / 89 / 95 / 143 / 157 / 183 / 190 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 123 / 137 / 157 / 183 / 190 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 60 / 89 / 123 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 104 / 123 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, or 11 / 25 / 37 / 60 / 89 / 123 / 143 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0214] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residue 11N / 25A / 37L / 83L / 89M / 137R / 150D / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37R / 89M / 157V / 183L / 194V / 198M / 213R / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G 11N / 25A / 37L / 89M / 137R / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G 11N / 25A / 37L / 89M / 150D / 157V / 183L / 190M / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 143A / 150D / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 143T / 150D / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 60A / 89M / 143A / 150D / 157V / 183L / 190M / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 60A / 89M / 150D / 157V / 183L / 190M / 194V / 198M / 213R / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 104T / 137R / 154V / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 123K / 137R / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 137R / 150D / 154V / 157V / 183L / 190M / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298 S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 123K / 143T / 150D / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 60A / 89M / 104T / 143A / 150D / 157V / I183L / 194V / 198M / 213R / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 60A / 89M / 95G / 143T / 157V / 183L / 190M / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 3 16L / 326L / 331G, 11N / 25A / 37L / 89M / 123R / 137R / 157V / 183L / 190M / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 60A / 89M / 123R / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 104T / 123R / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, or 11N / 25A / 37L / 60A / 89M / 123R / 143A / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0215] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 33 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 81 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 268 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 65 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 232 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331 / 332, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 330 / 331, 11 / 25 / 37 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 137 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 223 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 280 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 289 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 288 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331 / 342, 11 / 25 / 37 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 241 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 126 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 277 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 87 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 247 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 243 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 84 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 83 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 105 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 250 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 250 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 33 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 33 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 33 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 33 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 251 / 254 / 266 / 291 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 33 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 33 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 33 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 33 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 33 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 33 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 228 / 230 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 77 / 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, or 11 / 23 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0216] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residue 11N / 25A / 37L / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 23K / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 33L / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25G / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 10F / 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194 V / 198M / 214L / 230T / 249L / 250S / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251E / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 23T / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 25A / 33V / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 81Y / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G. 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 266L / 298S / 316L / 326L / 331G. 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 268F / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 65C / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 232N / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G / 332E, 11N / 25A / 37K / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331R, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 330K / 331G, 11N / 25A / 37L / 89M / 101 P / l 57V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 268G / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 307R / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224Q / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 137N / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 223C / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 280N / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 289I / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 288E / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G / 342T, 11N / 25A / 37L / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 268M / 298S / 316L / 326L / 331G , 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 241Q / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 126M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G 11N / 25 A / 37L / 89M / 157 V / l 83L / 194 V / l 98M / 214L / 230T / 249L / 251S / 254S / 266L / 277A / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 87E / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 247I / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194 V / 198M / 214L / 230T / 243H / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 84V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 83A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 105K / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25G / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25G / / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 250S / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25G / 29S / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250S / 251S / 254S / 255S / 266L / 298 S / 316L / 326L / 331G, 11N / 25G / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25G / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25G / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 250S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 29S / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326 L / 331G, 11N / 25A / 29S / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 29S / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / I 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / I3 31G, 11N / 25A / 33V / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 33V / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 33V / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 33V / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 33V / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 33V / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 33V / 37L / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250S / 251S / 254S / 255S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 250S / 251S / 254S / 255S / 266L / 298S / 3 16L / 326L / 331G. 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250S / 251S / 254S / 255S / 266L / 298S / 3 16L / 326L / 331G, 11N / 25A / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 32 6L / 331G, 11N / 25A / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250S / 251S / 254S / 255S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 250S / 251S / 254S / 255S / 266L / 2 98S / 316L / 326L / 331G, 11N / 25A / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 250S / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 250S / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G , 11N / 25A / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37M / 38V / 89M / 157 V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 251S / 254S / 266L / 291T / 298S / 316L / 326L / 331G. 10F / 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 10F / 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 10F / 11N / 25A / 33L / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G. 10F / 11N / 25A / 33L / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G. 10F / 11N / 25A / 33L / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G, 10F / 11N / 25A / 33L / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 331 G, 10F / 11N / 25A / 33L / 37L / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326 L / 331G, 10F / 11N / 25A / 33V / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251E / 254S / 266L / 298S / 316L / 326L / 33 1G, 10F / 11N / 25A / 33V / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G, 10F / 11N / 25A / 33V / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G. 10F / 11N / 25A / 33V / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 10F / 11N / 25A / 33V / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 10F / 11N / 25A / 33V / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 10F / 11N / 25A / 33V / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G, 10F / 11N / 25A / 33V / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 10F / 11N / 25A / 33V / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G, 10F / 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 10F / 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 10F / 11N / 25A / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 1OF / 11N / 25 A / 37M / 89M / 157 V / l 83L / 194 V / l 98M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 10F / 11N / 25A / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 10F / 11N / 25A / 37M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 10F / 11N / 25A / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G, 10F / 11N / 25A / 37M / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 10F / 11N / 25A / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230I7249L / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G. 10F / 11N / 25A / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G, 10F / 11N / 25A / 37M / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G, 10F / 11N / 25A / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249K / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G, 10F / 11N / 25A / 37L / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G, 10F / 11N / 25A / 37L / 38V / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 33 1G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 228L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250S / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250S / 251S / 254S / 255S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, 77M / 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 3 31G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 250S / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 250S / 251S / 254S / 255S / 266L / 298S / 316L / 326L / 331G, 11N / 23T / 25G / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230T / 249L / 251S / 254S / 255S / 266L / 298S / 316L / 3 26L / 331G, 11N / 23T / 25G / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31G, or 11N / 23T / 25G / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0217] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 25 / 37 / 86 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 255 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 306 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 318 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331 / 335, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 241 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 248 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331 / 344, 11 / 25 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 272 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 83 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 257 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 258 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 323 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0218] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residue 11N / 25A / 37L / 86D / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 255V / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 306T / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250E251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 75A / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 318S / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G / 335Q, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 241G / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 250E / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 248F / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G / 344K, 11N / 25A / 37L / 38S / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194 V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 272S / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 83G / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 306L / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 272M / 298S / 316L / 326L / 331G 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 248G / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T7249L / 251S / 254S / 257E / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 258M / 266L / 298S / 316L / 326L / 331G 11N / 25A / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 230T / 249L / 251S / 254S / 266L / 298S / 316L / 323G / 326L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0219] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 25 / 29 / 37 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 291 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 291 / 298 / 307 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 33 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 33 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 291 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 33 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 33 / 37 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 291 / 298 / 307 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 38 / 75 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 271 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 38 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 291 / 298 / 316 / 326 / 331, 10 / 11 / 25 / 29 / 37 / 38 / 75 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 268 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 64 / 81 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 254 / 266 / 268 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 33 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 298 / 316 / 326 / 331, 11 / 25 / 29 / 37 / 38 / 75 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 254 / 266 / 291 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0220] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residues 11N / 25A / 29S / 37L / 89M / 101 P / l 57V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 291T / 298S / 316L / 3 26L / 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 291T / 298S / 307R / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 3 31R, 11N / 25A / 29S / 33V / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 29S / 33V / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 254S / 266L / 291T / 29 8S / 316L / 326L / 331R, 11N / 25A / 29S / 33V / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 29S / 33V / 37L / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 11N / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 291T / 29 8S / 307R / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 38V / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 250S / 251S / 254S / 266L / 29 8S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 38V / 75D / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 271 G / 298S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 38V / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 10F / 11N / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 31 6L / 326L / 331G, 10F / 11N / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298 S / 316L / 326L / 331G, 10F / 11N / 25A / 29S / 37L / 38V / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 291 T / 298S / 316L / 326L / 331G, 10F / 11N / 25A / 29S / 37L / 38V / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 254S / 266L / 298 S / 316L / 326L / 331G, 10F / 11N / 25A / 29S / 37L / 38V / 75D / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 3 16L / 326L / 331G. 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224Q / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 101P / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 30 7K / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 268F / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37K / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 266L / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 64Q / 81Y / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298 S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 254S / 266L / 268F / 2 98S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 3 26L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 32 6L / 331G, 11N / 23T / 25A / 29S / 37L / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 254S / 266L / 29 8S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 254S / 266L / 298S / 307R / 3 16L / 326L / 331G, 11N / 25A / 29S / 37L / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25A / 29S / 33V / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 254S / 266L / 298 S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 25S / 29S / 37L / 38V / 75D / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 291 T / 298S / 316L / 326L / 331G, 11N / 25A / 29S / 33V / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298 S / 316L / 326L / 331G, 11N / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 254S / 266L / 298S / 316L / 3 26L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 326L / 3 31G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0221] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 23 / 25 / 29 / 37 / 38 / 65 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 65 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 65 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 65 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331 / 342, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 65 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 268 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 64 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 291 / 298 / 316 / 326 / 331 / 342, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 307 / 316 / 326 / 331 / 342. 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 291 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 224 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 86 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331 / / 342, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 77 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331 / 342, 11 / 23 / 25 / 29 / 37 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 268 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 251 / 266 / 298 / 316 / 326 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0222] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residue 11N / 23T / 25A / 29S / 37L / 38A / 65C / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298 S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38T / 65C / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316 L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 65C / 89M / 157V / 183L / 194V / 198M / 214L / 224R / 226R / 230V / 249L / 251S / 266L / 298 S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38 A / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316 L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 307R / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 32 6L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 266L / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 224R / 226R / 230V / 249L / 251S / 266L / 29 8S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 65C / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 32 6L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 266L / 29 8S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 224A / 226R / 230V / 249L / 251S / 266L / 298S / 3 16L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 307R / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268G / 29 8S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268G / 298S / 30 7R / 316L / 326L / 331G / 342T, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 224R / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 3 26L / 331G, 11N / 23T / 25A / 29S / 37L / 65C / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157 V / l 83L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 268G / 298S / 316L / 3 26L / 331G, 11N / 23T / 25A / 29S / 37L / 64Q / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268G / 291T / 29 8S / 316L / 326L / 331G / 342T. 11N / 23T / 25A / 29S / 37L / 75D / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 266L / 298S / 307 R / 316L / 326L / 331G / 342T, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 291T / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 64Q / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298 S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 224R / 226R / 230V / 249L / 251S / 266L / 298 S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249K / 251S / 266L / 298S / 307R / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 64Q / 86G / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 241R / 249L / 251 S / 266L / 298S / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 32 6L / 331G / 342T, 11N / 23T / 25 A / 29S / 37L / 89M / 157 V / l 83L / 192E / 194 V / l 98M / 214L / 224R / 226R / 230V / 249L / 251S / 266L / 298S / 3 16L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248G / 249L / 250I / 251S / 266L / 298S / 30 7R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 77M / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G / 342T, 11N / 23T / 25A / 29S / 37L / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 268G / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 3 26L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 31 6L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 251S / 266L / 298S / 316L / 32 6L / 331G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0223] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set at amino acid positions 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331. 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / / 250 / 250 / 251 / 266 / 268 / 277 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 93 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 3 31, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331 / 344, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 248 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 277 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 86 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / / 241 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 3 18 / 326 / 331 / 335, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 3875 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331 / 335, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331 / 344, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331 / 335, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331 / 335, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 277 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 93 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 192 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 250 / 251 / 255 / 266 / 268 / 298 / 307 / 316 / 318 / 3 26 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 64 / 75 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 318 / 326 / 331 / 335, 11 / 23 / 25 / 29 / 37 / 64 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 298 / 307 / 316 / / 318 / 326 / 3 31, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 318 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 86 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 3 26 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 331 / 335, 11 / 23 / 25 / 29 / 37 / 38 / 64 / 75 / 89 / 101 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 318 / 326 / 3 31, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 64 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 249 / 250 / 251 / 255 / 266 / 268 / 280 / 298 / 307 / 316 / 318 / 3 26 / 331, 11 / 23 / 25 / 29 / 37 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 250 / 251 / 255 / 266 / 268 / 298 / 307 / 316 / / 318 / 326 / 33 1, 11 / 23 / 25 / 29 / 37 / 38 / 75 / 89 / 157 / 183 / 194 / 198 / 214 / 226 / 230 / 241 / 249 / 250 / 251 / 266 / 268 / 298 / 307 / 316 / 326 / 331 / 335, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0224] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution set or amino acid residue 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250I / 251E / 266L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / / 250E / 250I / 251 E / 266L / 268G / 277A / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250E / 251E / 266L / 26 8G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 93K / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250I / 251E / 266L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250I / 251E / 266L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250E / 250I / 251E / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250E / 251E / 266 L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250E251E / 266L / 268 G / 298S / 307R / 316L / 326L / 331G / 344K, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250E / 251R / 26 6L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250E / 251S / 266 L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25 A / 29S / 37L / 38S / 75D / 89M / 93K / 157 V / l 83L / 194 V / l 98M / 214L / 226R / 230V / 249L / 250E / 251E / 266L / 268G / 277A / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255V / 266 L / 268G / 280N / 298S / 307R / 316L / 318S / 326L / 331G. 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248F / 249L / 250V251S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38 A / 75D / 86D / 89M / 157V / 183L / 194 V / 198M / 214L / 226R / 230V / 241G / 249L / 250T / 251S / 255V / 266L / 268G / 280N / 298S / 307R / 316L / 318S / 326L / 331G / 335Q, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250V251S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250F251S / 266L / 268G / 29 8S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 101P / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255 S / 266L / 268G / 280N / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268 G / 298S / 307R / 316L / 318S / 326L / 331G / 335Q, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250E251S / 255V / 266 L / 268G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 250E / 251E / 266L / 26 8G / 298S / 307R / 316L / 326L / 331G / 344K, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250E251S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G / 335Q, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250F251S / 266L / 268G / 29 8S / 307R / 316L / 318S / 326L / 331G / 335Q, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268 G / 277A / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 93K / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251E / 266L / 268G / 298S / 307R / 316L / 326L / 331G. 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 192E / 194V / 198M / 214L / 226R / 230V / 249L / 2501 / 251E / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250P251S / 255S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 64Q / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 248I / 249L / 250I / 251S / 266L / 268 G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 64Q / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 241G / 249L / 250I / 251S / 255V / 266L / 268G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250V251E / 266L / 268G / 29 8S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 64Q / 75D / 89M / 101P / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255 V / 266L / 268G / 280N / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 101P / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255 V / 266L / 268G / 280N / 298S / 307R / 316L / 318S / 326L / 331G / 335Q, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268G / 29 8S / 307R / 316L / 326L331G. 11N / 23T / 25A / 29S / 37L / 38S / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251R / 266L / 268 G / 277A / 298S / 307R / 316L / 326L / 331G. 11N / 23T / 25A / 29S / 37L / 64Q / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268 G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 64Q / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255V / 266L / 268G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 241G / 249L / 250I / 251S / 255S / 266 L / 268G / 280N / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255V / 266L / 26 8G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 86D / 89M / 101P / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 241G / 249L / 250I / 251S / 266L / 268G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 241G / 249L / 250I / 251S / 266 L / 268G / 298S / 307R / 316L / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255S / 266 L / 268G / 298S / 307R / 316L / 318S / 326L / 331G / 335Q, 11N / 23T / 25A / 29S / 37L / 38 A / 64Q / 75D / 89M / 101 P / l 57V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 266L / 268G / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 64Q / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 249L / 250I / 251S / 255V / 266L / 268G / 280N / 298S / 307R / 316L / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 241G / 249L / 250I / 251S / 255S / 266 L / 268G / 298S / 307R / 316L / / 318S / 326L / 331G, 11N / 23T / 25A / 29S / 37L / 38A / 75D / 89M / 157V / 183L / 194V / 198M / 214L / 226R / 230V / 241G / 249L / 250I / 251S / 266 L / 268G / 298S / 307R / 316L / 326L / 331G / 335Q, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0225] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at an amino acid position provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1. 10.1, 11.1, 12.1,13.1, 14.1, 15.1, 16.1, 17.1, 18.1,19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0226] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least one substitution provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1,10.1,11.1,12.1,13.1,14.1,15.1,16.1,17.1. 18.1, 19.1, 20.1, 21.1, 22.1. 23.1, 24.1. 25.1, and 26.1. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0227] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at the amino acid position(s) provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1, 10.1,11.1, 12.1,13.1,14.1,15.1, 16.1,17.1,18.1,19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0228] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set of an RNA ligase variant provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1, 10.1,11.1, 12.1,13.1,14.1,15.1, 16.1,17.1,18.1,19.1, 20.1. 21.1, 22.1, 23.1, 24.1, 25.1, and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0229] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence comprising a substitution or substitution set of an RNA ligase variant provided in Tables 5.1, 6.1, 6.2, 7.1, 8.1, 8.2, 9.1,10.1,11.1,12.1, 13.1,14.1, 15.1, 16.1, 17.1. 18.1,19.1. 20.1, 21.1. 22.1, 23.1. 24.1, 25.1. and 26.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0230] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0231] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058.

[0232] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54. 244. 648, 862, 956. 1088, 1416, 1612, 1840. 1866. 1954. 2062. 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54. 244, 648, 862, 956. 1088. 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244. 648, 862, 956, 1088, 1416, 1612, 1840, 1866. 1954, 2062, 2674, 2754, or 2786.

[0233] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54. 244. 648, 862, 956. 1088, 1416, 1612, 1840. 1866. 1954, 2062, 2674, 2754. or 2786. or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648. 862. 956, 1088. 1416, 1612, 1840, 1866, 1954. 2062. 2674, 2754, or 2786.

[0234] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 15, 17, 18, 21, 23, 25, 26, 28, 29, 30, 32, 33, 37, 38, 39, 40, 41, 42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 81, 83, 84, 86, 87, 89, 90, 91, 93, 95,97,99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 116, 117, 119, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 144, 148, 150, 154, 156, 157, 158, 159, 160, 161, 162, 165, 169, 170, 171, 173, 175, 177, 178, 179, 181, 183, 184, 185, 190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 212, 213, 214, 215, 218, 223, 224, 226, 228, 229, 230, 232, 236, 238, 241, 243, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 264, 265, 266, 267, 268, 270, 271, 272, 274, 276, 277, 280, 282, 283, 286, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301, 302, 305, 306, 307, 310, 311, 315, 316, 317, 318, 323, 324. 325, 326, 327, 330. 331, 332, 334. 335. 336, 337, 338. 341. 342, 344, 345. or 346, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244. 648, 862, 956, 1088, 1416, 1612, 1840. 1866. 1954, 2062, 2674, 2754. or 2786. or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754, or 2786.

[0235] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or an amino acid residue 10F, 11G / N, 12A / F / L / M / P / R / T / V, 13W, 15M, 17P, 18A / H, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 33A / I / L / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P. 40D / FP / S, 41E / I, 42T, 45S, 52C, 54L, 561 / L, 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 7 IT, 73Q / W, 74D, 75A / D, 77M, 80E, 81Y, 83A / E / G / L / T / V, 84V, 86D / G / L, 87E, 89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, 90E / F, 91A / E / V, 93K / V. 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / S / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / Q / S, 157F / L / Q / V, 158R / S, 159F, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L / V, 184L, 185C / F / G / K / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, I94A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 2I0G / V, 212E / G / I / K / L, 213D / N / R / T, 214A / C / H / K / L / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 230T / V, 232G / M / N / R / S, 2361, / V, 238K / S / V, 241E / G / Q / R, 243H, 247T / L / R / S, 248F / G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / L / N / Q / S / V, 255N / S / V / Y. 256A / E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I / L, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / N / W, 282A / M, 283C / V, 286D / E / F / M / V, 288E / L / RAV, 289A / D / E / FP / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 292N / S, 294A / S / V / Y, 295C / F / K / L / M / P / Q / R / S / T / V / Y. 296E / G / L / Q / W, 297A / I / L, 298C / VK / R / S / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / VL / M / V, 302A / C / FL / S. 305V, 306G / L / T, 307K / R, 310H / L / M / N / Q / R / S / T. 311C. 315E / S, 316I / L / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 330A / D / E / G / K / R, 331D / G / R / S / W, 332E / H / W, 334E / R, 335H / Q, 336I / L / M, 337A / V, 338G / L / M, 341A. 342R / S / T, 344K. 345Q / S, or 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0236] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 17, 21, 23, 25, 26, 28, 29, 30, 32, 37, 38, 40, 41,42, 45, 52, 54, 56, 57, 58,60, 61, 62,64, 65, 67, 71,73, 74, 75, 77, 80, 83, 84, 87, 90,91, 93, 97. 99, 100.101, 102, 103, 104, 105, 106, 107, 109, 113, 114. 121, 122, 123, 125. 126, 127, 128, 129. 131, 135, 137, 140. 141, 143, 148. 150, 157, 158. 160, 161, 162, 165. 169, 170, 173, 178. 179, 184, 190, 191. 192, 193, 194, 197. 198, 204, 206, 208, 210, 213. 215, 218, 223, 228. 229, 232, 236, 238. 241, 243, 248, 249. 250, 251, 252, 253. 254, 255, 258, 261, 264, 265, 267, 268, 270, 271, 272, 274, 276, 277. 282, 286, 289, 290. 292, 294, 295, 297. 298, 299, 300, 301, 302, 305, 311, 318, 323, 324, 325, 327, 332, 336, 338, 341, 344, or 346; a substitution or amino acid residue 18A / H, 33A / ET / V, 39P, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 95A / C / G / I, 116L, 117A / Q, 119L. 144L, 154S / V, 156G / H / S, 171S, 175A, 177K, 181E / N / P / R / S, 183L, 212E / G / K / L, 224A / Q / R, 226R, 230T / V, 247FL / R / S, 256E / G / M / S / W, 257D / E / G, 260G, 280L / W, 283C / V, 288L / R / W, 291S, 296E / G / L / Q / W. 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 315E, 316I / Y, 3I7E / I7G / Q / V / W, 326C / L, 330A / D / E / G / K, 331D / G / S, 334E, 335Q, 337A / V, 338G / L / M, 342R / S / T, or 345Q / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062. 2674, 2754, or 2786.

[0237] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F, 1 1G / N, 12A / F / L / M / P / R / T / V, 13W, 17P, 18A / H, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 33A / I / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P, 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 561 / L. 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V. 64A / Q / W. 65C, 67R, 7 IT. 73Q / W. 74D, 75A / D. 77M. 80E, 83A / E / G / L / T / V. 84V, 86D / L. 87E, 89C / D / E / L / N / Q / R / S / T / V / Y, 90E / F, 91A / E / V, 93K / V. 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W. 107M / S. 109F / L. 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W. 123K / R. 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R. 1401, 141D / G, I43A / C / E / F / H / M / S / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / S, 157F / L / Q / V, 158R / S, 160R, 161M / V, 162C, 165M, 169G / N. 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E. 181E / N / P / R / S, 183L. 184L, 185C / F / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 210G / V, 212E / G / K / L, 213D / N / R / T, 214A / C / H / K / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 23OT / V, 232G / M / N / R / S, 236L / V, 238K / S / V, 241E / G / Q / R, 243H, 247I / L / R / S, 248F / G / EK / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / K / L / N / Q / S / V, 255N / S / V / Y, 256E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I, 267VM, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / W, 282A / M, 283C / V, 286D / E / F / M / V, 288L / R / W, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 291S, 292N / S, 294A / S / V / Y, 295C / F / KZL / M / P / Q / R / S / T / V / Y, 295G, 296E / G / L / Q / W, 297A / VL, 298l / S, 298C / K / R / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C / VL / S, 305V, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 311C, 315E, 316I / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 330A / D / E / G / K, 331D / G / S, 332E / H / W, 334E, 335Q, 336I / L / M, 337A / V, 338G / L / M, 341 A, 342R / S / T, 344K, 345Q / S, 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416. 1612, 1840, 1866, 1954, 2062. 2674. 2754, or 2786.

[0238] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 86, 89, 93, 101, 122, 143, 157, 183, 192, 194, 198, 212, 214, 230, 241, 248, 249, 250, 251, 254, 266, 268, 280, 291, 295, 298, 307, 316, 318, 326, 330, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0239] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or an amino acid residue 11G / N, 12A / F / L / M / P / R / T / V, 23K / T, 25A / D / F / G / L / M / S / T. 29D / S, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 75A / D, 86D / G / L, 89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, 93K / V, 101P / V / W, 122C / F / G / L / Q / T / W, 143A / C / E / F / H / M / S / T / V, 157F / L / Q / V, 183L / V, 192D / E / F / L / N. I94A / G / V, 198M, 212E / G / I / K / L, 214A / C / H / K / L / Q / Y, 230T / V, 241E / G / Q / R, 248F / G / EK / T, 249K / L / R / S, 250E / VS, 251C / E / F / G / L / R / S / T / Y, 254E / L / N / Q / S / V, 266C / I / L, 268F / G / M, 280L / N / W, 291H / L / Q / R / S / T / W, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 298C / I / K / R / S / V, 307K / R, 316I / L / Y, 318A / E / G / S, 326C / L, 330A / D / E / G / K / R, or 331D / G / R / S / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0240] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or an amino acid residue 1 IN, 12M / T, 23T, 25A, 29S, 37L, 38A / S, 75D, 86D, 89M. 93K, 101P, 122F. 143S, 157V. 183L, 192D / E. 194V. 198M, 212E, 214L, 226R, 230T / V, 241G, 248F, 249L, 2501, 251E / S. 254K / S. 266L. 268G. 280L. 29IQ. 295G / V, 298S / V, 307R, 316L, 318S, 326L. 330A, or 331G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612. 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0241] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 93, 101, 122, 143, 157, 192, 194, 198, 241, 248, 249. 250. 251, 254, 268. 295. 298, or 318: a substitution 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 183L, 212E / G / K / L, 214A / C / H / K / L / Q / Y. 226R. 230T / V. 266C / I. 280L / W, 291H / L / Q / R / S / T / W, 307K / R. 326C / L. 330A / D / E / G / K, or 331D / G / S: or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416. 1612, 1840, 1866, 1954, 2062. 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54. 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754, or 2786.

[0242] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 11G / N, 12A / F / L / M / P / R / T / V, 23K / T, 25A / D / F / G / L / M / S / T, 29D / S, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 75A / D, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 93K / V, 101P / V / W, 122C / F / G / L / Q / T / W, 143A / C / E / F / H / M / S / T / V, 157F / L / Q / V, 183L, 192D / E / F / L / N, 194A / G / V, 198M, 212E / G / K / L, 241E / G / Q / R, 214A / C / H / K / L / Q / Y, 226R, 230T / V, 248F / G / VK / T, 249K / L / R / S, 250E / VS, 251C / E / F / G / L / R / S / T / Y. 254E / L / N / Q / S / V, 266C / I, 268F / G / M, 280L / W, 291H / L / Q / R / S / T / W, 295C / F / K. / L / M / P / Q / R / S / T / V / Y. 298C / I / K / R / S / V. 299E / I / K / Q / R / T / V. 307K / R. 318A / E / G / S, 326C / L, 330A / D / E / G / K, or 331D / G / S. or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416. 1612, 1840, 1866, 1954, 2062. 2674. 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54. 244, 648, 862, 956, 1088. 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754, or 2786.

[0243] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 1 IN, 12T, 23T, 25A, 29S, 37L, 38A / S, 75D, 86D, 93K, 101P, 122F, 157V, 183L, 192E, 194V, 198M, 212E, 214L, 226R, 230T / V, 241G, 249L, 2501, 251E / S, 254S, 266L, 268G, 280L, 291Q, 295V, 298S / V, 307R, 318S, 326L, 330A, or 331G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0244] In some embodiments, the amino acid sequence of the engineered RNA ligase further comprises at least a substitution at amino acid position 2, 14. 15, 63, 69, 81, 85, 95, 138, 142, 149, 159, 171, 186, 195, 202, 266, 275, 283,285, 303, 314, 333, or 339; a substitution 181, 33L, 39Q / S, 86G, 89G / H / I / M / W, 116N, 117D, 119T. 144W, 154C, 156A / L / N / Q / T, 175K, 177P, 181D, 183V. 185G / K. 2121, 2141, 224Y, 2261, 230D. 247K, 256A, 257L, 260S, 280G / N, 288E, 291P. 296K, 306L. 307E / Q, 310K, 315S / T, 316L. 317A, 326R, 330M / R, 331L / R / W, 334R, 335D / H, 337L, 3421, or 345V; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088. 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

[0245] In some embodiments, the amino acid sequence of the engineered RNA ligase further comprises at least a substitution or amino acid residue 2E / G / V, 14K, 15E / L / M / P / Y, 181, 33L, 39Q / S, 63V, 69D, 81Y, 85L, 86G, 89G / H / VM / W, 95V, 116N, 117D, 119T, 138R, 14...

Claims

1. An engineered RNA ligase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1470 and 14783058, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

2. The engineered RNA ligase of claim 1. comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54. 244, 648, 862,956, 1088, 1416, 1612, 1840. 1866. 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648. 862, 956, 1088, 1416, 1612. 1840, 1866. 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862,956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

3. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

4. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862,956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

5. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

6. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-218, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

7. The engineered RNA ligase of any one of claims 1-6, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 15, 17, 18, 21, 23, 25, 26, 28, 29, 30, 32, 33, 37, 38, 39,40, 41,42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 81, 83, 84, 86, 87, 89, 90, 91, 93, 95, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 116, 117, 119, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 144, 148, 150, 154, 156, 157, 158, 159, 160, 161, 162, 165, 169, 170, 171, 173, 175, 177, 178,179,181, 183, 184, 185,190, 191, 192, 193, 194, 197. 198, 204, 206.

208. 210, 212, 213.

214. 215, 218, 223.

224. 226, 228, 229.

230. 232, 236, 238.241, 243, 247. 248, 249, 250, 251. 252, 253, 254, 255. 256, 257, 258.

260. 261, 264, 265.

266. 267, 268, 270.271, 272, 274. 276, 277, 280, 282. 283, 286, 288, 289. 290, 291, 292, 294. 295, 296, 297, 298. 299, 300, 301,302, 305, 306. 307, 310, 311, 315. 316, 317, 318, 323. 324, 325, 326, 327. 330, 331, 332, 334. 335, 336, 337,338, 341, 342, 344, 345, or 346, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

8. The engineered RNA ligase of any one of claims 1-7, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F, 11G / N, 12A / F / L / M / P / R / T / V, 13W, 15M, 17P, 18A / H, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W. 32E, 33A / I / L / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P, 40D / I / P / S, 41E / 1,42T, 45S, 52C, 54L, 56I / L, 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 71T, 73Q / W, 74D, 75A / D, 77M, 80E, 81Y. 83A / E / G / L / T / V. 84V, 86D / G / L. 87E, 89C / D / E / G / L / M / N / Q / R / SN7V / W / Y, 90E / F, 91A / E / V, 93K / V. 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R. 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / S / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / Q / S. 157F / L / Q / V. 158R / S. 159F, 160R. 161M / V, 162C, 165M, 169G / N. 170H / Y, 171S. 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L / V, 184L. 185C / F / G / K / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L. 208H / I / K / L / P / T, 210G / V, 212E / G / I / K / L, 213D / N / R / T, 2I4A / C / H / K / L / Q / Y, 215G / T, 218F, 223C,224A / Q / R, 226R, 228D / L, 229W, 230T / V, 232G / M / N / R / S, 236L / V, 238K / S / V, 241E / G / Q / R, 243H, 247I / L / R / S, 248F / G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / K / L / N / Q / S / V, 255N / S / V / Y, 256A / E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I / L, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / N / W, 282A / M, 283C / V, 286D / E / F / M / V, 288E / L / R / W, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 292N / S, 294A / S / V / Y, 295C / F / G / K / L / M / P / Q / R / S / T / V / Y, 296E / G / L / Q / W, 297A / I / L, 298C / I / K / R / S / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C / VL / S, 305V, 306G / L / T, 307K / R, 310H / L / M / N / Q / R / S / T, 311C, 315E / S, 316I / L / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 33OA / D / E / G / K / R, 331D / G / R / S / W, 332E / H / W, 334E / R, 335H / Q, 336I / L / M, 337A / V, 338G / L / M, 341 A, 342R / S / T, 344K, 345Q / S, or 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

9. The engineered RNA ligase of any one of claims 1-7, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 17, 21, 23, 25, 26, 28, 29, 30, 32, 37, 38,40, 41, 42, 45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 83, 84, 87, 90, 91, 93, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 113, 114, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137, 140, 141, 143, 148, 150, 157, 158, 160, 161, 162, 165, 169, 170, 173, 178, 179, 184,190, 191, 192, 193, 194, 197, 198, 204, 206, 208, 210, 213, 215, 218, 223, 228, 229, 232, 236, 238, 241, 243,248, 249, 250, 251, 252, 253, 254, 255, 258, 261, 264, 265, 267, 268, 270, 271, 272, 274, 276, 277, 282, 286,289, 290, 292, 294, 295, 297, 298, 299, 300, 301, 302, 305, 311, 318, 323, 324, 325, 327, 332, 336, 338, 341,344, or 346; a substitution or amino acid residue 18A / H, 33A / I / T / V, 39P, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 95A / C / G / I, 116L, 117A / Q, 119L, 144L, 154S / V, 156G / H / S, 171S, 175A, 177K, 181E / N / P / R / S, 183L, 212E / G / K / L, 224A / Q / R, 226R, 230T / V, 247I / L / R / S, 256E / G / M / S / W, 257D / E / G, 260G, 280L / W, 283C / V, 288L / R / W, 291S, 296E / G / L / Q / W, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 315E, 316I / Y, 317E / F / G / Q / V / W, 326C / L, 330A / D / E / G / K, 331D / G / S, 334E, 335Q, 337A / V, 338G / L / M, 342R / S / T, or 345Q / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

10. The engineered RNA ligase of any one of claims 1-9, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F, UN, 12A / F / L / M / P / R / T / V, 13W, 17P, 18A / H, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 33A / I / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P, 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 56VL, 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 71T, 73Q / W, 74D, 75A / D, 77M, 80E, 83A / E / G / L / T / V, 84V, 86D / L, 87E, 89C / D / E / L / N / Q / R / S / T / V / Y, 90E / F, 91A / E / V, 93K, 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, I0IP / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401,141D / G, 143A / C / E / F / H / M / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / S, 157F / Q / V, 158R / S, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L, 184L, 185C / F / M / P / S / T / V, 190I / M, 191C, 192E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M. 204W, 206L, 208H / I / K / L / P / T, 210G / V, 212E / G / K / L, 213D / N / R / T.214A / C / H / K / Q / Y, 215G / T, 218F, 223C, 224A / Q / R. 226R. 228D / L, 229W, 230T / V, 232G / M / N / R / S. 236L / V, 238K / S / V, 241E / G / Q / R, 243H, 247I / L / R / S, 248G / I / K / T, 249K / L / R / S, 250E / VS, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / L / N / Q / S / V, 255N / S / V / Y, 256E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / W, 282A / M, 283C7V, 286D / E / F / M / V, 288L / R / W, 289A / D / E / VP / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 291S, 292N / S, 294A / S / V / Y, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 296E / G / L / Q / W, 297A / I / L, 298I / S, 298C7K / IW, 299E / VK / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C7I / L / S, 305V, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 31 IC, 315E, 316I / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 330A / D / E / G / K, 331D / G / S, 332E / H / W, 334E, 335Q, 336I / L / M, 337A / V, 338G / L / M, 341 A, 342R / S / T, 344K, 345Q / S, or 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

11. The engineered RNA ligase of any one of claims 1-7, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29, 37, 38, 75, 86, 89, 93, 101, 122, 143, 157, 183, 192, 194, 198, 212, 214, 230, 241, 248, 249, 250, 251, 254, 266, 268, 280, 291, 295, 298, 307, 316, 318, 326, 330, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

12. The engineered RNA ligase of any one of claims 1-7 and 11, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11. 12, 23, 25.

29. 37, 38, 75. 93, 101. 122, 143, 157, 192. 194, 198, 241, 248, 249, 250, 251, 254. 268, 295, 298, or 318; a substitution 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 183L, 212E / G / K / L, 214A / C / H / K / L / Q / Y, 226R, 230T / V, 266C / I, 280L / W, 291H / L / Q / R / S / T / W, 307K / R, 326C / L, 33OA / D / E / G / K, or 331D / G / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

13. The engineered RNA ligase of any one of claims 7-12, wherein the amino acid sequence of the engineered RNA ligase further comprises at least a substitution at amino acid position 2, 14, 15, 63, 69, 81, 85, 95, 138, 142, 149, 159, 171, 186, 195, 202, 266, 275, 283, 285, 303, 314, 333, or 339; a substitution or amino acid residue 181, 33L, 39Q / S, 86G, 89G / H / I / M / W, 116N. 117D, 119T. 144W, 154C, 156A / L / N / Q / T, 175K, 177P, 181D, 183V, 185G / K, 2121, 2141, 224Y, 2261, 230D, 247K, 256A, 257L, 260S, 280G / N, 288E, 291P, 296K, 306L. 307E / Q, 310K, 315S / T. 316L. 317A. 326R, 330M / R, 331L / R / W. 334R. 335D / H. 337L. 3421. or 345V; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

14. The engineered RNA ligase of any one of claims 1-7, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position!s) 256, 258, 254, 251, 248, 37, 54, 232, 15, 71, 26, 116, 38, 25, 106 / 251, 52, 33, 247, 37 / 249 / 266 / 316 / 330 / 334 / 335, 21, 11 / 37 / 249 / 266 / 316, 261, 11 / 266 / 316, 11 / 248 / 249 / 266, 331, 11 / 248 / 249, 255, 37 / 249 / 266 / 316 / 334, 266,11 / 37 / 156 / 249 / 335, 37 / 156 / 248 / 249 / 266 / 334, 334, 156, 11 / 266 / 316 / 334, 11 / 39 / 156 / 248 / 316, 11 / 249, 37 / 156, 107 / 251, 156 / 249 / 316, 37 / 156 / 316, 11 / 37 / 156 / 249, 11 / 37 / 334, 194, 37 / 316, 175, 11 / 156 / 249 / 316, 11 / 37,252, 12, 37 / 266, 11 / 37 / 156 / 266, 11 / 37 / 156 / 248 / 266, 37 / 236 / 248 / 249, 37 / 156 / 248 / 249, 11 / 266 / 330 / 335,249, 11 / 156 / 248 / 249 / 316, 37 / 249 / 316, 11, 11 / 156, 11 / 37 / 249, 330,316, 156 / 249 / 266 / 335,249 / 266 / 316 / 330 / 335, 11 / 156 / 249, 266 / 316 / 330 / 334 / 335, 156 / 248 / 249 / 316 / 334, 37 / 156 / 248 / 249 / 316, 156 / 266, 248 / 249, 156 / 249 / 316 / 330 / 335, 218, 37 / 156 / 248 / 249 / 266, 11 / 37 / 156 / 248 / 249 / 316, 37 / 156 / 316 / 330, 11 / 37 / 248 / 249 / 316 / 330, 11 / 37 / 248 / 249, 249 / 330 / 335, 11 / 37 / 156 / 330 / 334, 40, 11 / 37 / 148 / 249 / 316, 83, 323, 193, 335, or 257, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

15. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866. 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

16. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058.

17. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

18. The engineered RNA ligase of claim 1, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674,2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862. 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

19. The engineered RNA ligase of claim 17 or 18, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 15, 17, 18,21, 23, 25, 26, 28, 29, 30, 32, 33, 37, 38, 39,40, 41, 42,45, 52, 54, 56, 57, 58, 60, 61, 62, 64, 65, 67, 71, 73, 74, 75, 77, 80, 81, 83, 84, 86, 87, 89, 90, 91,93, 95, 97,99, 100,101, 102, 103, 104,105, 106, 107, 109,113, 114, 116, 117, 119, 121, 122, 123, 125, 126, 127, 128, 129, 131, 135, 137.

140. 141, 143, 144. 148, 150, 154, 156, 157, 158, 159, 160, 161, 162, 165, 169, 170, 171, 173, 175, 177, 178, 179,181, 183, 184, 185,190, 191, 192, 193, 194, 197. 198, 204, 206, 208. 210, 212, 213, 214. 215, 218, 223, 224. 226, 228, 229, 230, 232, 236, 238,241, 243, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 264, 265, 266, 267, 268, 270,271, 272, 274, 276, 277, 280, 282, 283, 286, 288, 289, 290, 291, 292, 294. 295, 296, 297, 298, 299, 300, 301,302, 305, 306, 307, 310, 311, 315, 316, 317, 318, 323, 324, 325, 326, 327, 330, 331, 332, 334, 335, 336, 337,338, 341, 342, 344, 345, or 346, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

20. The engineered RNA ligase of any one of claims 17-19, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F, 11G / N, 12A / F / L / M / P / R / T / V. 13W. 15M, 17P. 18A / H, 21S. 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S. 30A / L / T / V / W, 32E, 33A / I / L / T / V. 37A / F / K / L / M / R. 38A / E / L / R / S / T / V, 39P. 40D / I / P / S. 41E / 1.42T, 45S, 52C, 54L, 56I / L, 57V. 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 71T. 73Q / W. 74D, 75A / D. 77M, 80E, 81Y. 83A / E / G / L / T / V, 84V, 86D / G / L. 87E, 89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, 90E / F, 91A / E / V, 93K / V, 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / S / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / Q / S, 157F / L / Q / V. 158R / S, 159F, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L / V, 184L, 185C / F / G / K / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 210G / V, 212E / G / I / K / L, 213D / N / R / T, 2I4A / C / H / K / L / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 230T / V, 232G / M / N / R / S, 236L / V, 238K / S / V, 241E / G / Q / R, 243H, 247VL / R / S, 248F / G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / L / N / Q / S / V, 255N / S / V / Y, 256A / E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I / L, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / N / W, 282A / M, 283C / V, 286D / E / F / M / V, 288E / L / R / W, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 292N / S, 294A / S / V / Y, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 296E / G / L / Q / W, 297A / VL, 298C / I / K / R / S / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C / VL / S, 305V, 306G / L / T, 307K / R, 310H / L / M / N / Q / R / S / T, 31 IC, 315E / S, 316I / L / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S. 33OA / D / E / G / K / R, 331D / G / R / S / W. 332E / H / W, 334E / R, 335H / Q. 3361 / L / M. 337A / V, 338G / L / M, 341 A, 342R / S / T, 344K. 345Q / S. or 346S. or any combinations thereof,wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

21. The engineered RNA ligase of any one of claims 17-20, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 10, 11, 12, 13, 17, 21, 23, 25, 26, 28, 29, 30, 32, 37, 38, 40, 41. 42.45, 52, 54. 56, 57, 58. 60, 61, 62. 64, 65, 67.

71. 73, 74, 75. 77, 80, 83. 84, 87, 90.91, 93, 97.99, 100.101, 102, 103, 104.105, 106, 107, 109.113, 114, 121, 122. 123, 125, 126, 127, 128, 129. 131, 135, 137, 140. 141, 143, 148, 150. 157, 158, 160, 161. 162, 165, 169, 170. 173, 178, 179,184, 190, 191, 192, 193, 194, 197. 198, 204, 206, 208. 210, 213, 215, 218. 223, 228, 229, 232. 236, 238, 241,243, 248, 249, 250, 251, 252, 253, 254, 255, 258, 261. 264, 265, 267, 268. 270, 271, 272, 274. 276, 277, 282,286, 289, 290, 292, 294, 295, 297, 298, 299, 300, 301, 302, 305, 311, 318, 323, 324, 325, 327. 332, 336, 338,341, 344, or 346; a substitution or amino acid residue 18A / H, 33A / I / T / V, 39P, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 95A / C / G / I, 116L, 117A / Q, 119L, 144L, 154S / V, 156G / H / S, 171S, 175A, 177K, 181E / N / P / R / S, 183L, 212E / G / K / L, 224A / Q / R, 226R, 230'17V, 247I / L / R / S, 256E / G / M / S / W, 257D / E / G, 260G, 280L / W, 283C / V, 288L / R / W, 291S, 296E / G / L / Q / W, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 315E, 3I6I / Y, 317E / F / G / Q / V / W, 326C / L, 33OA / D / E / G / K, 331D / G / S, 334E, 335Q, 337A / V, 338G / L / M, 342R / S / T, or 345Q / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

22. The engineered RNA ligase of claim 21, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or amino acid residue 10F, 11G / N, 12A / F / L / M / P / R / T / V, 13W. 17P, 18A / H, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E. 33A / I / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P. 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 56I / L, 57V, 58M. 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 71T, 73Q / W, 74D, 75A / D, 77M, 80E, 83A / E / G / L / T / V, 84V, 86D / L, 87E, 89C / D / E / L / N / Q / R / S / T / V / Y, 90E / F, 91A / E / V, 93K / V, 95A / C / G / I, 97A / E, 991, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L, 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / S / T / V, 144L, 148F, 150D / L, 154S / V, 156G / H / S, 157F / L / Q / V, 158R / S, 160R, 161M / V, 162C, 165M, 169G / N, 170H / Y, 17 IS, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L, 184L, 185C / F / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, I94A / G / V, 197W, 198M, 204W, 206L, 208H / I / K / L / P / T, 2I0G / V, 212E / G / I / K / L, 213D / N / R / T, 2I4A / C / H / K / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 230T / V, 232G / M / N / R / S, 236L / V, 238K / S / V, 241E / G / Q / R, 243H, 247I / L / R / S, 248F / G / I / K / T, 249K / L / R / S, 250E / I / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / K / L / N / Q / S / V. 255N / S / V / Y, 256E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / F / G / S / V, 265A / F, 266C / I, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V. 277A. 280L / W, 282A / M, 283C / V. 286D / E / F / M / V, 288L / R / W, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 291S, 292N / S, 294A / S / V / Y. 295C / F / K / L / M / P / Q / R / S / T / V / Y, 295G, 296E / G / L / Q / W,297A / I / L, 298I / S, 298C / K / R / V, 299E / VK / Q / R / T / V, 300A, 301A / F / I / L / M / V, 302A / C / I / L / S, 305V, 306G / T, 307K / R, 310H / L / M / N / Q / R / S / T, 311C, 315E, 316I / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 330A / D / E / G / K, 331D / G / S, 332E / H / W, 334E, 335Q, 336I / L / M, 337A / V, 338G / L / M, 341 A, 342R / S / T, 344K, 345Q / S, or 346S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

23. The engineered RNA ligase of any one of claims 17-20, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12. 23, 25, 29, 37, 38, 75, 86, 89, 93, 101, 122, 143, 157, 183, 192, 194, 198, 212, 214, 230, 241. 248, 249, 250, 251. 254, 266, 268, 280, 291, 295, 298, 307, 316, 318, 326, 330, or 331, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

24. The engineered RNA ligase of any one of claims 17-20 and 23, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 11, 12, 23, 25, 29. 37, 38, 75. 93, 101. 122, 143, 157, 192. 194, 198, 241, 248. 249, 250, 251.

254. 268, 295, 298. or 318; a substitution 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 183L, 212E / G / K / L, 214A / C / H / K / L / Q / Y, 226R, 23017V, 266C / I. 280L / W, 291H / L / Q / R / S / T / W, 307K / R. 326C / L. 33OA / D / E / G / K, or 331D / G / S; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244, 648, 862. 956, 1088, 1416. 1612, 1840, 1866, 1954, 2062. 2674, 2754, or 2786, or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416, 1612, 1840, 1866, 1954, 2062, 2674. 2754, or 2786.

25. The engineered RNA ligase of any one of claims 19-24, wherein the amino acid sequence of the engineered RNA ligase further comprises at least a substitution at amino acid position 2, 14, 15, 63, 69, 81, 85, 95, 138, 142, 149, 159, 171, 186, 195, 202, 266, 275, 283, 285, 303, 314, 333, or 339; a substitution 181, 33L, 39Q / S, 86G, 89G / H / I / M / W, 116N, 117D, 119T, 144W, 154C, 156A / L / N / Q / T, 175K, 177P, 181D, 183V, 185G / K, 2121, 2141, 224Y, 2261, 230D, 247K, 256A, 257L, 260S, 280G / N, 288E, 291P, 296K, 306L, 307E / Q, 310K, 315S / T, 316L, 317 A, 326R, 330M / R, 331L / R / W. 334R. 335D / H, 337L. 3421. or 345V; or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, 244. 648, 862, 956, 1088, 1416, 1612, 1840. 1866. 1954, 2062, 2674, 2754. or 2786. or relative to the reference sequence corresponding to SEQ ID NO: 54, 244, 648, 862. 956, 1088. 1416, 1612, 1840, 1866, 1954. 2062, 2674, 2754, or 2786.

26. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 ofSEQ ID NO: 54, or to the reference sequence corresponding to SEQ ID NO: 54, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or relative to the reference sequence corresponding to SEQ ID NO: 54.

27. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 220-630, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 220-630, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or relative to the reference sequence corresponding to SEQ ID NO: 54.

28. The engineered RNA ligase of claim 26 or 27, wherein the amino acid sequence of the engineered RNA ligase comprises a substitution or substitution set at amino acid positions(s) 156 / 232 / 334 / 335, 12 / 254 / 256, 12 / 230, 12 / 230 / 251, 12 / 230 / 251 / 254 / 256, 12 / 38 / 230, 230 / 251, 12 / 38 / 254, 230 / 256, 254, 230, 12 / 230 / 254, 12 / 38 / 230 / 251 / 256, 12 / 251, 12 / 251 / 254 / 256, 38, 12 / 230 / 256, 12, 12 / 25 / 38 / 230 / 251, 12 / 38 / 102 / 230 / 251 / 256, 12 / 256, 38 / 230 / 256, 256, 12 / 230 / 251 / 256, 230 / 251 / 254, 230 / 254, 251 / 254, 12 / 25 / 230, 38 / 230, 38 / 251, 12 / 38 / 230 / 256, 102, 251, 12 / 25 / 251 / 256, 12 / 102 / 256, 12 / 38 / 230 / 251 / 254 / 256, 12 / 251 / 256, 102 / 230 / 251 / 254 / 256, 12 / 38 / 251, 12 / 230 / 251 / 254, 156 / 248 / 334 / 335, 12 / 25 / 38, 12 / 102 / 230 / 251 / 254, 12 / 38 / 251 / 254, 12 / 251 / 254, 12 / 102,38 / 230 / 251 / 254 / 256, 12 / 25 / 230 / 251 / 256, 12 / 230 / 311,12 / 254, 38 / 230 / 251 / 254, 12 / 38 / 251 / 256, 156 / 334 / 335, 257 / 258 / 334.12 / 25 / 38 / 251 / 256, 334. 12 / 25 / 230 / 254 / 256, 38 / 251 / 256. 12 / 38 / 230 / 251 / 254, 12 / 38, 257 / 258 / 334 / 335, 12 / 38 / 254 / 256, 232 / 248 / 257 / 258 / 334 / 335, 156 / 258 / 334 / 335, 156 / 249 / 334 / 335. 12 / 102 / 230 / 254, 25, 156 / 257 / 258 / 334 / 335, 12 / 102 / 230, 156 / 248 / 257 / 258 / 334, 248 / 249 / 334 / 335, 232 / 257 / 258 / 334 / 335, 334 / 335, 156 / 334, 12 / 38 / 102 / 256, 156 / 257 / 334 / 335, 12 / 140 / 251 / 254, 251 / 254 / 256, 248 / 334 / 335, 156 / 258 / 334. 156 / 248 / 330 / 335, 12 / 38 / 256, 232 / 334 / 335, 156 / 249 / 334, 330 / 334 / 335, 156 / 248 / 334, 156 / 335, 257 / 334, 248 / 334. 232 / 248 / 334 / 335, 12 / 25 / 38 / 230 / 251 / 256, 156 / 248 / 249 / 334 / 335, 330 / 335, 248 / 257 / 334 / 335, 232 / 248 / 334, 156 / 232 / 248 / 334 / 335, 232 / 257 / 330 / 334, 156 / 248 / 291 / 334 / 335, 156 / 232 / 248 / 257 / 258 / 334, 156 / 232 / 249 / 334 / 335, 12 / 25, 156 / 248 / 330 / 334 / 335, 156 / 248 / 249 / 334, 232 / 248 / 330 / 335, 156 / 232 / 330 / 334 / 335, 197, 338, 327, 192, 331, 326, 345, 101, 100, 141, 198, 64, 306, 317, 265, 296, 194, 62, 90, 307, 103, 210, 105, or 61, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 54, or relative to the reference sequence corresponding to SEQ ID NO: 54.

29. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%. 87%, 88%. 89%. 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or to the reference sequence corresponding to SEQ ID NO: 244, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or relative to the reference sequence corresponding to SEQ ID NO: 244.

30. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%.97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 632-820, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 632-820, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or relative to the reference sequence corresponding to SEQ ID NO: 244.

31. The engineered RNA ligase of claim 29 or 30, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 141 / 197 / 326 / 327, 100 / 101 / 338, 141 / 198 / 326,64 / 100 / 101, 100 / 338, 101 / 141 / 198 / 326, 101 / 198 / 326 / 327, 198 / 326, 64 / 100 / 101 / 338 / 345, 197 / 326, 198 / 327, 141 / 198 / 327, 198 / 326 / 327, 197 / 326 / 327, 64 / 100 / 338, 64 / 100 / 101 / 156 / 197 / 338,64 / 100 / 101 / 338, 100 / 197 / 338, 197 / 338, 101 / 197 / 326,64 / 100 / 197 / 338. 101 / 198, 141 / 197 / 327,64 / 100 / 101 / 197 / 338, 101 / 326, 101 / 197 / 326 / 327, 197 / 327, 197, 141 / 197, 101, 101 / 198 / 326, 64 / 197 / 198 / 338, 141, 101 / 141, 100 / 101 / 197 / 338, 141 / 326,338, 326, 197 / 198 / 338,64 / 101 / 338, 64 / 197 / 198, 101 / 197, 100 / 101, 100 / 109 / 197 / 338, 101 / 197 / 338, 197 / 198, 141 / 326 / 327, 100 / 197, 64 / 100, 64 / 197 / 338, 100 / 197 / 198, 101 / 338, 141 / 327, 101 / 306, 64 / 100 / 101 / 197, 101 / 326 / 327, 100, 327, 326 / 345, 64 / 100 / 101 / 197 / 198, 326 / 327, 64 / 101, 100 / 101 / 197, 101 / 327, 64 / 100 / 197 / 198 / 338, 306, 64 / 338, 101 / 141 / 326 / 327, 64 / 101 / 197 / 338, or 101 / 198 / 306 / 327, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 244, or relative to the reference sequence corresponding to SEQ ID NO: 244.

32. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or to the reference sequence corresponding to SEQ ID NO: 648, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or relative to the reference sequence corresponding to SEQ ID NO: 648.

33. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 822-954, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 822-954, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or relative to the reference sequence corresponding to SEQ ID NO: 648.

34. The engineered RNA ligase of claim 32 or 33, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 194 / 330 / 331, 298, 331, 192 / 193 / 194 / 298, 330, 192 / 330 / 331, 192 / 193 / 194 / 331, 298 / 330 / 331, 194 / 298 / 330 / 331, 192 / 193 / 330 / 331, 192 / 298 / 331, 192 / 193, 194 / 330, 60 / 100 / 156, 193 / 296 / 331,298 / 331, 193 / 194 / 330 / 331, 194, 194 / 298 / 331, 193 / 298, 193 / 330, 192, 192 / 193 / 194 / 298 / 331, 192 / 193 / 194 / 298 / 330, 192 / 193 / 298 / 331, 100 / 156, 62 / 100, 298 / 330, 60 / 100, 192 / 193 / 298 / 330 / 331, 100, 193 / 194 / 298, 338, 62, 247, 18, 37, 254, 33, 261, 251, 260, 12, 117, 256, 255, 38, 29, 25, or 252, wherein the amino acid positions arerelative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 648, or relative to the reference sequence corresponding to SEQ ID NO: 648.

35. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or to the reference sequence corresponding to SEQ ID NO: 862, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO:

862. or relative to the reference sequence corresponding to SEQ ID NO: 862.

36. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 956-1078, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 956-1078, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or relative to the reference sequence corresponding to SEQ ID NO: 862.

37. The engineered RNA ligase of claim 35 or 36. wherein the amino acid sequence of the engineered RNA ligase comprises a substitution or substitution set at amino acid positions(s) 254, 18 / 254, 247 / 317, 37 / 317 / 342, 33, 18 / 156 / 254, 18, 156, 254 / 338, 254 / 307, 33 / 37 / 317, 37, 37 / 156 / 317, 156 / 265 / 317, 265 / 317, 156 / 254, 33 / 156 / 317, 317, 156 / 317, 37 / 317, 18 / 338, 338, 156 / 247 / 317, 33 / 317, 37 / 247 / 265 / 317, 33 / 37 / 156 / 317, 156 / 247 / 296 / 317, 18 / 307, 156 / 296 / 317, 156 / 247, 37 / 247 / 317, 18 / 254 / 307, 307, 265, 33 / 37, 18 / 156, 33 / 156 / 296 / 317, 18 / 156 / 254 / 338, 37 / 265 / 317, 33 / 37 / 156 / 247 / 317, 296, 33 / 296 / 317, 156 / 296, 247 / 296 / 317, 37 / 296 / 317, 247 / 296, 33 / 37 / 156, 296 / 317, 156 / 254 / 307, 18 / 156 / 307, or 33 / 37 / 265, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 862, or relative to the reference sequence corresponding to SEQ ID NO: 862.

38. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%. 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or to the reference sequence corresponding to SEQ ID NO: 956, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

39. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1080-1374, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1080-1374, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

40. The engineered RNA ligase of claim 38 or 39. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 12 / 25 / 29 / 156, 12 / 255 / 256 / 257, 12 / 156 / 255, 247 / 252, 12 / 25, 12, 12 / 25 / 29 / 156 / 255, 256, 12 / 156, 12 / 25 / 156 / 255 / 256 / 257 / 261, 18 / 247 / 251, 25 / 255 / 256 / 257, 12 / 25 / 257, 255 / 257, 12 / 25 / 29 / 255 / 257, 256 / 257, 12 / 255 / 257, 12 / 156 / 255 / 256 / 257, 25, 18 / 38 / 251 / 252, 12 / 256 / 257 / 261, 12 / 29, 12 / 25 / 255 / 256 / 257 / 261, 38, 12 / 25 / 29 / 256 / 257, 29 / 257, 12 / 261, 251, 249 / 251, 12 / 255, 12 / 29 / 156 / 256 / 257, 252, 255 / 257 / 261, 18, 29 / 255 / 257, 255 / 256 / 257, 12 / 25 / 29 / 156 / 256 / 257, 25 / 255 / 256 / 257 / 261, 29 / 255 / 256 / 257 / 261, 25 / 256, 18 / 38 / 247 / 252 / 327, 18 / 247,25 / 29, 12 / 25 / 156 / 255 / 257, 18 / 156 / 251 / 252, 12 / 255 / 261,25 / 255, 12 / 255 / 256 / 261, 156 / 255 / 257, 156 / 257, 12 / 156 / 256 / 257,25 / 156 / 256 / 257, 12 / 25 / 29 / 156 / 257 / 261,25 / 261, 12 / 156 / 255 / 256 / 261, 12 / 25 / 29 / 255 / 256 / 257,38 / 252,255,249 / 252, 12 / 156 / 255 / 256, 12 / 25 / 29 / 261,257, 12 / 255 / 256, 12 / 29 / 255, 12 / 255 / 257 / 261, 12 / 156 / 257, 192 / 249 / 251 / 252, 12 / 25 / 29 / 255 / 261, 12 / 156 / 255 / 257, 12 / 29 / 255 / 257, 12 / 29 / 256 / 257, 18 / 252,249, 156 / 250, 38 / 251 / 252, 156 / 255 / 256 / 257,25 / 257, 12 / 255 / 256 / 257 / 261, 25 / 156 / 261, 156 / 252, 12 / 25 / 255 / 256 / 257, 12 / 25 / 29 / 156 / 255 / 257,29 / 156,251 / 252,256 / 257 / 261, 25 / 156 / 255 / 256 / 257. 156 / 249 / 251 / 252, 25 / 29 / 255, 12 / 29 / 156, 255 / 256, 38 / 250 / 252, 247 / 249 / 252, 12 / 256 / 257, 60, 12 / 256, 12 / 156 / 256, 25 / 255 / 257 / 261, 12 / 25 / 29 / 255 / 256 / 261, 12 / 29 / 156 / 255 / 256, 12 / 25 / 156 / 255 / 256, 247 / 251 / 252, 256 / 261, 38 / 251, 12 / 25 / 29 / 156 / 261, 12 / 257, 156 / 255 / 256 / 261, 12 / 25 / 156 / 255. 12 / 25 / 29 / 156 / 255 / 257 / 261, 12 / 29 / 256 / 261, 29 / 156 / 261, 25 / 29 / 156 / 257 / 261, 12 / 25 / 255 / 256 / 261. or 12 / 156 / 255 / 261, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

41. The engineered RNA ligase of claim 38 or 39, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid position 157, 183, 95, 185, 181, 213, 89, 28, 190, 214, or 143, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 956, or relative to the reference sequence corresponding to SEQ ID NO: 956.

42. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or to the reference sequence corresponding to SEQ ID NO: 1088, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or relative to the reference sequence corresponding to SEQ ID NO: 1088.

43. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%. 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1376-1434. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1376-1434, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or relative to the reference sequence corresponding to SEQ ID NO: 1088.

44. The engineered RNA ligase of claim 42 or 43. wherein the amino acid sequence of theengineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 95 / 157. 95 / 183 / 185 / 214, 89 / 183, 28 / 89 / 143 / 183, 28 / 143 / 157, 181 / 183, 143 / 181 / 183 / 214, 143 / 181 / 183, 89 / 143 / 183, 28 / 143 / 181 / 183, 143 / 157, 95 / 181 / 183, 157, 143 / 183, 89 / 143 / 157 / 183, 89 / 95 / 157, 89 / 157, 89 / 95 / 143 / 181, 95 / 143, 89 / 157 / 183 / 214, 143 / 214, 143 / 181 / 183 / 185, 28 / 95 / 143, 143 / 183 / 214, 28 / 95 / 183 / 214, 89 / 143 / 157, 28 / 95, 95 / 143 / 183 / 185, or 95 / 143 / 157 / 183, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1088, or relative to the reference sequence corresponding to SEQ ID NO: 1088.

45. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416. or to the reference sequence corresponding to SEQ ID NO: 1416, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

46. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1436-1470, 1478-1766, and 1908-1946, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1436-1470, 1478-1766, and 1908-1946, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

47. The engineered RNA ligase of claim 45 or 46, wherein the amino acid sequence of theengineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 83 / 137 / 150, 137 / 213, 137, 150 / 190, 143 / 150, 104 / 150, 60 / 143 / 150 / 190, 60 / 150 / 190 / 213, 104 / 137 / 154, 123 / 137, 137 / 150 / 154 / 190, 123 / 143 / 150, 60 / 104 / 143 / 150 / 213,60 / 95 / 143 / 190, 123 / 137 / 190, 60 / 123, 104 / 123, or 60 / 123 / 143, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

48. The engineered RNA ligase of claim 45 or 46. wherein the amino acid sequence of theengineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38. 249, 255, 23, 33, 25, 226, 29, 37, 10, 250, 75, 230, 77, 251, 228, 81, 224, 64, 254, 268, 65, 232, 332, 331, 330, 101, 307, 137, 223, 280, 289, 288, 342, 93, 296, 86, 241, 126, 277, 87, 247, 243, 84, 83, 192, 105, 25 / 29, 25 / 29 / 250, 25 / 29 / 75 / 77 / 250 / 255, 25 / 75 / 230, 25 / 77 / 226 / 255, 25 / 230, 250 / 255, 29 / 75 / 226 / 230 / 255, 29 / 75 / 77, 29 / 75 / 77 / 226, 29 / 75 / 77 / 230 / 255, 29 / 226, 29 / 226 / 230, 29 / 230 / 255, 33 / 228, 33 / 249, 33 / 37 / 228, 33 / 37 / 38 / 228, 33 / 37 / 38 / 228 / 249, 33 / 38, 75 / 250 / 255, 75 / 226, 75 / 226 / 250 / 255, 75 / 226 / 230 / 250 / 255, 75 / 77, 75 / 77 / 255, 75 / 77 / 250 / 255, 75 / 77 / 226 / 255, 75 / 77 / 226 / 250 / 255, 75 / 77 / 230, 75 / 77 / 230 / 250, 75 / 230, 75 / 230 / 255, 75 / 230 / 250, 226 / 255, 226 / 250, 226 / 230, 226 / 230 / 255, 37 / 228, 37 / 228 / 249, 37 / 38, 37 / 38 / 228, 37 / 38 / 228 / 249,37 / 38 / 249, 38 / 249 / 291, 10 / 228, 10 / 228 / 249, 10 / 33 / 249, 10 / 33 / 37, 10 / 33 / 37 / 38 / 249, 10 / 33 / 38 / 249, 10 / 33 / 38, 10 / 33 / 249 / 251, 10 / 33 / 37 / 228, 10 / 33 / 37 / 249, 10 / 33 / 37 / 38 / 228 / 249, 10 / 33 / 37 / 38, 10 / 249, 10 / 37, 10 / 37 / 228, 10 / 37 / 228 / 249, 10 / 37 / 249, 10 / 37 / 38 / 228, 10 / 37 / 38 / 228 / 249, 10 / 37 / 38, 10 / 38 / 249, 10 / 38, 10 / 38 / 228, 77 / 255, 77 / 250, 77 / 250 / 255, 77 / 226, 77 / 226 / 255, 77 / 230, 77 / 230 / 255, 230 / 255, 230 / 250, 230 / 250 / 255, 23 / 25 / 226 / 255, 23 / 25 / 226 / 230, or 23 / 25 / 230, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding toSEQIDNO: 1416.

49. The engineered RNA ligase of claim 45 or 46. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 86, 255, 306, 250, 75, 318, 335, 241, 248, 344, 38, 272, 83, 257, 258, or 323, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1416, or relative to the reference sequence corresponding to SEQ ID NO: 1416.

50. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or to the reference sequence corresponding to SEQ ID NO: 1612, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or relative to the reference sequence corresponding to SEQ ID NO: 1612.

51. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1768-1840, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 768-1840, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or relative to the reference sequence corresponding to SEQ ID NO: 1612.

52. The engineered RNA ligase of claim 50 or 51. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 101, 291, 291 / 307, 331, 33 / 75, 33 / 75 / 249 / 291 / 331, 33 / 38. 33 / 93 / 249, 38 / 75 / 291 / 307, 38 / 75 / 249 / 250, 38 / 75 / 93 / 271, 38 / 93, 10 / 75, 10 / 38 / 75, 10 / 38 / 75 / 291, 10 / 38 / 75 / 249, 10 / 38 / 75 / 93, 64 / 224, 224, 23 / 101 / 307, 23 / 268, 23 / 37 / 64 / 224 / 254, 23 / 64 / 81, 23 / 224 / 268, 23 / 224 / 254, 23, 23 / 64 / 224, 64, 75 / 249 / 307, 93, 33 / 38 / 75 / 249, 75 / 249, 25 / 38 / 75 / 93 / 291, 33 / 38 / 75, 75, or 23 / 254. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1612, or relative to die reference sequence corresponding to SEQ ID NO: 1612.

53. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840. or to the reference sequence corresponding to SEQ ID NO: 1840. wherein the amino acidsequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or relative to the reference sequence corresponding to SEQ ID NO: 1840.

54. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1842-1906, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1842-1906, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or relative to the reference sequence corresponding to SEQ ID NO: 1840.

55. The engineered RNA ligase of claim 53 or 54, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 65 / 192, 38 / 65, 38 / 65 / 224, 38 / 64, 93 / 307, 38, 38 / 224, 38 / 192 / 224, 65, 192 / 224, 75 / 307, 75 / 250 / 268 / 307, 250 / 268 / 307 / 342, 224, 65 / 192, 268, 64 / 192, 250 / 268 / 291 / 342, 75 / 93 / 249 / 307 / 342, 250 / 291, 38 / 64 / 192, 38 / 64 / 224, 93 / 249 / 307, 38 / 64 / 86 / 192 / 241, 38 / 342, 248 / 250 / 307, 75 / 77 / 250 / 268 / 307 / 342, 93 / 268, 192, 38 / 192, or 64, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1840, or relative to the reference sequence corresponding to SEQ ID NO: 1840.

56. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or to the reference sequence corresponding to SEQ ID NO: 1866, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

57. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1948-2038 and 2130-2620, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1948-2038 and 2130-2620. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

58. The engineered RNA ligase of claim 56 or 57, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 248 / 251,38 / 248 / 250 / 251 / 277, 248 / 250 / 251, 38 / 93 / 192 / 248 / 251,38 / 192 / 248 / 251,250 / 251, 38 / 248 / 250 / 251, 248 / 251 / 344, 192 / 248 / 250 / 251, 38 / 248 / 250, 38 / 93 / 250 / 251 / 277, 38 / 255 / 280 / 318, 248, 38 / 86 / 241 / 255 / 280 / 318 / 335, 38 / 255, 38, 318, 38 / 101 / 255 / 280, 38 / 318 / 335, 38 / 255 / 318, 192 / 250 / 251 / 344, 38 / 335, 318 / 335, 38 / 277. 38 / 93 / 251.192 / 251, 255, 38 / 64 / 318, 38 / 64 / 241 / 255 / 318.

251. 64 / 101 / 255 / 280 / 318. 38 / 101 / 255 / 280 / 318 / 335. 38 / 255 / 280, 38 / 251 / 277, 64 / 318. 38 / 64 / 255 / 318, 241 / 255 / 280 / 318,38 / 86 / 101 / 241 / 318, 38 / 241, 38 / 255 / 318 / 335, 38 / 64 / 101 / 318, 38 / 64 / 255 / 280 / 318, 241 / 255 / 318, or 38 / 241 / 335, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

59. The engineered RNA ligase of claim 56 or 57, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 89, 122, 181, 28, 125, 157, 238, 330, 212, 143, 208, 290, 161, 214, 84, 270, 91, 295, 316, 286, 301, 170, 264, 289, 190, 183, 128, 60, 310, 131, 302, 267, 288, 185, 291, 129, 280, 298, 158 / 215, 28 / 122. 276, 274, 271, 215, 294, 106, 58, 135,45, 299. 315, 204, 332.

169. 305, 154, 158, 297. 95, 337. 324, 150, 179, 283. 206, 236, 266, 213. 336, 178, 99, 184, 282, 57, 177, 109, 191. 107, 160, 144, 56, 159, 341. 325, or 91, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1866, or relative to the reference sequence corresponding to SEQ ID NO: 1866.

60. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954, or to the reference sequence corresponding to SEQ ID NO: 1954, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

61. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2040-2128, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2040-2128, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

62. The engineered RNA ligase of claim 60 or 61. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 93 / 232 / 291 / 306, 93 / 232 / 280 / 306, 93 / 241 / 257 / 306 / 335, 232 / 280, 93 / 291 / 306, 93 / 257. 93 / 306, 93 / 280, 192 / 318, 93 / 280 / 291, 93 / 241 / 291 / 306, 192. 257 / 291, 241 / 257 / 306, 280 / 335, 93 / 291 / 306 / 335, 93 / 335, 93 / 232 / 335, 64 / 318, 93 / 241, 93 / 232 / 241 / 280, 64 / 192 / 318 / 323, 232 / 280 / 306, 93 / 232, 291 / 335, 257 / 258, 93 / 232 / 241 / 257 / 258 / 280 / 335, 318 / 323, 93 / 241 / 291 / 335, 83 / 86 / 323, 257 / 258 / 291, 101 / 224 / 318, 83 / 192 / 255 / 296, 171 / 232 / 257 / 258 / 280, 83 / 224 / 255 / 318, 64 / 192 / 250 / 318, 101 / 296, 232 / 241 / 280, 64 / 255 / 318, 86 / 250 / 255, 83 / 86 / 101, 83 / 86, 93 / 241 / 258 / 335, 250 / 296 / 323, or 257 / 258 / 280, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

63. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062. or to the reference sequence corresponding to SEQ ID NO: 2062. wherein the amino acidsequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062, or relative to the reference sequence corresponding to SEQ ID NO: 2062.

64. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2622-2746, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2622-2746, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062. or relative to the reference sequence corresponding to SEQ ID NO: 2062.

65. The engineered RNA ligase of claim 63 or 64, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 83 / 84 / 330, 91 / 270 / 290, 83 / 181, 161 / 290 / 330, 181 / 330, 161 / 270 / 330, 270 / 290, 83 / 84 / 270 / 318 / 330, 64 / 83 / 84 / 161 / 192 / 290 / 318,91 / 161 / 290 / 318 / 330, 161 / 330, 161 / 214 / 270 / 290 / 330, 192 / 270 / 318, 91 / 161 / 192 / 330, 161 / 270, 181 / 270 / 330, 91 / 181 / 192 / 290, 161 / 290, 83 / 161 / 270, 83 / 192 / 318,214 / 330, 83 / 161 / 290 / 330, 64 / 83 / 161 / 290 / 330, 83 / 161 / 192 / 214 / 318, 122 / 143 / 212 / 295, 161 / 192 / 270 / 290, 181 / 290, 91 / 161 / 214 / 270 / 290 / 318, 91 / 192 / 214 / 270, 214 / 290, 64 / 83 / 161 / 270 / 318 / 330, 91 / 161 / 181 / 330, 161 / 318 / 330, 91 / 270 / 290 / 330, 64 / 181 / 290 / 318, 181 / 270 / 290 / 318, 64 / 83 / 161 / 192 / 270 / 290 / 318 / 330, 64 / 83 / 161 / 214 / 290 / 318, 122 / 192 / 212, 64 / 83 / 91 / 192 / 214 / 330, 83 / 91 / 161 / 181 / 290 / 318, 91 / 161 / 192 / 214 / 270 / 290, 91 / 161 / 181 / 290 / 330, 83 / 91 / 125 / 161 / 270, 28 / 212. 122 / 212, 28 / 64 / 83 / 122 / 143 / 192 / 212 / 295 / 318, 122 / 295, 91 / 161 / 192 / 318, 28 / 122 / 143 / 212. 64 / 161 / 290, 64 / 122 / 208 / 318, 64 / 83 / 192 / 290 / 318, 64 / 83 / 161 / 290, 181 / 192 / 318. 83 / 161 / 192 / 318. 214 / 270, 83 / 122, or 192 / 212, 28 / 122. wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2062, or relative to the reference sequence corresponding to SEQ ID NO: 2062.

66. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or to the reference sequence corresponding to SEQ ID NO: 2674, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

67. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2748-2782, 2822-2914, and 2916-3012, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2748-2782, 2822-2914, and 2916-3012, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

68. The engineered RNA ligase of claim 66 or 67. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 28 / 291 / 295 / 298 / 315,28 / 95 / 291 / 295 / 298 / 315, 143 / 295 / 298 / 315, 291 / 295 / 298, 28 / 143 / 295 / 298, 28 / 291 / 295 / 298, 143 / 185 / 291 / 295 / 298, 28 / 143 / 185 / 291 / 298 / 336, 28 / 298, 280 / 295, 143 / 298 / 336, 143 / 291 / 295 / 298, 291 / 295 / 298 / 315 / 336, 143 / 295 / 298, 28 / 143 / 295 / 298 / 336, 95 / 143 / 295 / 298, 143 / 280 / 290, or 143 / 291 / 298, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

69. The engineered RNA ligase of claim 66 or 67. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 208, 25, 90, 141. 258, 248, 318, 117, 299, 33. 80, 253, 83, 254, 256, 30, 327, 73, 100, 97, 119. 265, 296, 32, 156, 40, 105, 107, 41 / 114, 41. or 64, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

70. The engineered RNA ligase of claim 66 or 67, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162, 208, 25, 103, 346, 42, 74, 113, 13, 228, 318, 121, 299, 33, 342, 253, 127, 254, 292, 30, 67, 86, 165, 173, 300, 317, 326, 11, 229, 97, 71, 17, 210, 241, 271, 40, 56, or 95, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2674, or relative to the reference sequence corresponding to SEQ ID NO: 2674.

71. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or to the reference sequence corresponding to SEQ ID NO: 2754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or relative to the reference sequence corresponding to SEQ ID NO: 2754.

72. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%. 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%. 89%, 90%, 91%. 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2784-2820. or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2784-2820, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or relative to the reference sequence corresponding to SEQ ID NO: 2754.

73. The engineered RNA ligase of claim 71 or 72, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 106 / 264 / 280, 280 / 330, 28 / 106 / 264 / 291, 28 / 264 / 280 / 330, 57 / 60 / 135 / 264 / 291, 28 / 135 / 264 / 280, 106 / 264 / 280 / 291, 28 / 106 / 264 / 280, 280 / 299, 28 / 57 / 89 / 106 / 264 / 280 / 291. 264, 185 / 291, 28 / 106 / 299, 28 / 214 / 264 / 299. 264 / 330, 28 / 89 / 214 / 291, 28 / 185 / 271 / 310. 122 / 271, or 89 / 106 / 135 / 264 / 280 / 330, wherein theamino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2754, or relative to the reference sequence corresponding to SEQ ID NO: 2754.

74. The engineered RNA ligase of claim 17, comprising an amino acid sequence having at least 70%. 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or to the reference sequence corresponding to SEQ ID NO: 2786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2786.

75. The engineered RNA ligase of claim 18, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to 346 of an even-numbered SEQ ID NO. of SEQ ID NOs: 3014-3058, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 3014-3058, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2786.

76. The engineered RNA ligase of claim 74 or 75. wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 25 / 228 / 248 / 264, 32 / 113 / 228 / 248, 25 / 32 / 113 / 241 / 248 / 264 / 299, 25 / 113 / 248 / 264 / 318, 25 / 127 / 228 / 264 / 299 / 318, 25 / 113 / 228 / 248 / 327, 228 / 241 / 264, 264 / 299 / 318 / 327, 25 / 113 / 241 / 248 / 264 / 318, 25 / 127 / 248 / 299 / 318, 113 / 127 / 228 / 264 / 299, 25 / 127, 25 / 228 / 248 / 264 / 327, 25 / 113 / 264 / 299, 32 / 113 / 264 / 318, 25 / 32 / 241 / 264 / 327, 113 / 264 / 327, 25 / 127 / 228 / 248 / 318, 327, 32 / 113 / 254 / 264, 25 / 228 / 241 / 264, 25 / 113 / 228 / 241 / 318, or 113 / 327, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 2786, or relative to the reference sequence corresponding to SEQ ID NO: 2786.

77. The engineered RNA ligase of claim 1, wherein the RNA ligase comprises an amino acid sequence comprising residues 12 to 346 of an even numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 14783058. or an amino acid sequence comprising an even numbered SEQ ID NO. of SEQ ID NOs: 4-1470 and 1478-3058.

78. An engineered RNA ligase comprising an amino acid sequence comprising(a) in the N-terminal domain one or more amino acid residues 12A / F / L / M / P / R / T / V, 13W, 15M, 17P, 18A / H, 21S, 23K / T, 25A / D / F / G / L / M / S / T, 26L, 28A / L / M / V, 29D / S, 30A / L / T / V / W, 32E, 33A / VL / T / V, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 39P, 40D / I / P / S, 41E / I, 42T, 45S, 52C, 54L, 56I / L, 57V, 58M, 60A / C / D / R / S / V, 61A, 62A / V, 64A / Q / W, 65C, 67R, 71T, 73Q / W, 74D, 75A / D, 77M, 80E, 81Y, 83A / E / G / L / T / V, 84V, 86D / G / L, 87E, 89C / D / E / G / L / M / N / Q / R / S / T / V / W / Y, 90E / F, 91A / E / V, 93K / V, 95A / C / G / I, 97A / E,99I, 100A / G / H / L / R / S / T / V, 101P / V / W, 102T, 103G / V, 104T, 105K / L / R, 106G / L / M / Q / R / V / W, 107M / S, 109F / L, 113L / M, 114E, 116L. 117A / Q, 119L, 121N, 122C / F / G / L / Q / T / W, 123K / R, 125K / T, 126M, 127P, 128E, 129L / W, 131T, 135A / H / M / Q / V, 137N / R, 1401, 141D / G, 143A / C / E / F / H / M / S / T / V. 144L, 148F, 150D / L, 154S / V, 156G / H / Q / S. 157F / L / Q / V. 158R / S. 159F. 160R. 161M / V, 162C, 165M. 169G / N. 170H / Y, 171S, 173M, 175A, 177K, 178V, 179E, 181E / N / P / R / S, 183L / V,184L. 185C / F / G / K / M / P / S / T / V, 190I / M, 191C, 192D / E / F / L / N, 193K / Q, 194A / G / V, 197W, 198M, 204W, 206L. 208H / I / K / L / P / T, 210G / V, 212E / G / I / K / L, 213D / N / R / T, 214A / C / H / K / L / Q / Y, 215G / T, 218F, 223C, 224A / Q / R, 226R, 228D / L, 229W, 230T / V, 232G / M / N / R / S, 236L / V, 238K / S / V, 241E / G / Q / R, and 243H, or at equivalent positions thereof;(b) in the C-terminal domain one or more amino acid residues 256A / E / G / M / S / W, 257D / E / G, 258D / M / P / V, 260G, 261D / P / T, 264A / E / G / S / V, 265A / F, 266C / I / L, 267I / M, 268F / G / M, 270L / M / V, 271A / E / G / T, 272M / S, 274A, 276A / G / M / V, 277A, 280L / N / W, 282A / M, 283C / V, 286D / E / F / M / V, 288E / L / R / W, 289A / D / E / I / P / T, 290D / H / N / P, 291H / L / Q / R / S / T / W, 292N / S, 294A / S / V / Y, 295C / F / G / K / L / M / P / Q / R / S / T / V / Y, 296E / G / L / Q / W, 297A / I / L, 298C / FK / R / S / V, 299E / I / K / Q / R / T / V, 300A, 301A / F / VL / M / V, 302A / C / I / L / S, 305V, 306G / L / T, 307K / R, 310H / L / M / N / Q / R / S / T, 311C, 315E / S, 316I / L / Y, 317E / F / G / Q / V / W, 318A / E / G / S, 323E / G, 324D / G / M, 325L, 326C / L, 327E / G / M / S, 33OA / D / E / G / K / R, 331D / G / R / S / W, 332E / H / W, 334E / R, 335H / Q, 336I / L / M, 337A / V, 338G / L / M, 341A, 342R / S / T, 344K, 345Q / S, and 346S; or at equivalent positions thereof; and / or(c) in the linker region connecting the N-terminal and C-terminal domain one or more amino acid residues 2471 / L / R / S, 248F / G / I / K / T, 249K / L / R / S, 250E / 1 / S, 251C / E / F / G / L / R / S / T / Y, 252A / Q, 253L / W, 254E / K / L / N / Q / S / V, and 255N / S / V / Y, or at equivalent positions thereof.

79. The engineered ds RNA ligase of claim 78, wherein the N-terminal domain comprises one or more amino acid residues 12A / F / L / P / R / T / V, 23K / T, 25A / D / F / G / L / M / S / T, 29D / S, 37A / F / K / L / M / R, 38A / E / L / R / S / T / V, 75A / D, 86D / L, 89C / D / E / L / N / Q / R / S / T / V / Y, 93K, 101P / V / W, 122C / F / G / L / Q / T / W, 143A / C / E / F / H / M / T / V, 157F / Q / V, 183L / V, 192E / F / L / N, 194A / G / V, 198M, 212E / G / I / K / L, 214A / C / H / K / L / Q / Y, 230T / V, and 241E / G / Q / R, or at equivalent positions thereof.

80. The engineered ds RNA ligase of claim 79, wherein the N-terminal domain comprises one or more amino acid residues 12T, 23T, 25A, 29S, 37L, 38A / S, 75D, 86D, 89M, 93K, 101P, 122F, 157V, 183L, 192E. 194V, 198M, 212E, 214L, 226R, 230T / V, and 241G, or at equivalent positions thereof.

81. The engineered ds RNA ligase of claim 78, wherein the C-terminal domain comprises one or more amino acid residues 266C / I / L, 268F / G / M, 280L / N / W, 291H / L / Q / R / S / T / W, 295C / F / K / L / M / P / Q / R / S / T / V / Y, 298C / I / K / R / S / V, 307K / R, 316FL / Y, 318A / E / G / S, 326C / L, 330A / D / E / G / K / R, and 331D / G / R / S / W, or at equivalent positions thereof.

82. The engineered ds RNA ligase of claim 81, wherein the C-terminal domain comprises one or more amino acid residues 266L, 268G, 280L, 291Q, 295V. 298S / V, 307R, 316L. 318S, 326L, 33OA, and 331G, or at equivalent positions thereof.

83. The engineered ds RNA ligase of any one of claims 78-82, wherein the linker region connecting the N-terminal and C-terminal domains comprises one or more amino acid residues 248G / FK / T, 249K / L / R / S, 250E / VS, 251C / E / F / G / L / R / S / T / Y, and 254E / K / L / N / Q / S / V, or at equivalent positions thereof.

84. The engineered ds RNA ligase of claim 83, wherein the linker region connecting the N-terminal and C-terminal domains comprises one or more amino acid residues 249L, 2501, 251E / S, and 254S, or at equivalent positions thereof.

85. The engineered RNA ligase of any one of claims 1-84, wherein the engineered RNA ligase comprises a fusion polypeptide.

86. The engineered RNA ligase of any one of claims 1-85, wherein the engineered RNA ligase has RNA ligase 2 activity.

87. The engineered RNA ligase of any one of claims 1-86, having at least one improved property as compared to a reference RNA ligase.

88. The engineered RNA ligase of claim 87, wherein the improved property is selected from i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased activity on polynucleotides ligase substrates with phosphorothioate internucleoside linkages, vi) increased activity on oligonucleotides with 2’-modifications, vii) increased substrate tolerance, or any combination of i), ii), iii), iv), v), vi), and vii), compared to a reference RNA ligase.

89. The engineered RNA ligase of claim 87 or 88. wherein the reference RNA ligase has the sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786, or the sequence corresponding to SEQ ID NO: 2, 54, 244, 648, 862, 956, 1088, 1416, 1612, 1840, 1866, 1954, 2062, 2674, 2754, or 2786.

90. The engineered RNA ligase of claim 87 or 88, wherein the reference RNA ligase has the sequence corresponding to residues 12 to 346 of SEQ ID NO: 2, or the sequence corresponding to SEQ ID NO: 2.

91. The engineered RNA ligase of any one of claims 1-90, wherein the engineered RNA ligase is purified.

92. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered RNA ligase of any one of claims 1-90.

93. The recombinant polynucleotide of claim 92, wherein the polynucleotide sequence has least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 53, 243,647, 861,955, 1087, 1415, 1611, 1839, 1865, 1953, 2061,2673, 2753, or 2785, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 53, 243, 647, 861, 955, 1087, 1415, 1611, 1839, 1865, 1953, 2061, 2673, 2753, or 2785, wherein the recombinant polynucleotide encodes an RNA ligase.

94. The recombinant polynucleotide of claim 92, wherein the polynucleotide sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 14773057, wherein the recombinant polynucleotide encodes an RNA ligase.

95. The recombinant polynucleotide of any one of claims 92-94, wherein the polynucleotide sequence is codon-optimized for expression of the encoded engineered RNA ligase.

96. The recombinant polynucleotide of claim 92, comprising(a) a polynucleotide sequence comprising nucleotide residues 34 to 1038 of SEQ ID NO. 53, 243, 647, 861, 955, 1087, 1415, 1611, 1839, 1865, 1953, 2061, 2673, 2753, or 2785, or a polynucleotide sequence comprising SEQ ID NOs: 53, 243, 647, 861, 955, 1087, 1415, 1611, 1839, 1865, 1953, 2061, 2673, 2753, or 2785, or(b) a polynucleotide sequence comprising nucleotide residues 34 to 1038 of an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057. or a polynucleotide sequence comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 3-1469 and 1477-3057.

97. An expression vector comprising a recombinant polynucleotide of any one of claims 92-96.

98. The expression vector of claim 97. wherein the polynucleotide is operably linked to a control sequence.

99. The expression vector of claim 98, wherein the control sequence comprises at least a promoter.

100. A host cell comprising an expression vector of any one of claims 97-99.

101. The host cell of claim 100, comprising a prokaryotic cell or eukaryotic cell.

102. The host cell of claim 101, comprising a bacterial cell, fungal cell, insect cell, or mammaliancell.

103. A method of producing an engineered RNA ligase polypeptide in a host cell comprising culturing a host cell of any one of claims 100-102, under suitable culture conditions such that the engineered RNA ligase is produced.

104. The method of claim 103, further comprising recovering the engineered RNA ligase from the culture and / or host cells.

105. The method of claim 103 or 104. further comprising purifying the engineered RNA ligase.

106. A composition comprising an RNA ligase of any one of claims 1-91.

107. The composition of claim 106, further comprising one or more of a buffer, ATP or dATP, ligation enhancer, and one or more polynucleotide substrates for the RNA ligase.

108. A method of ligating at least a first polynucleotide strand and a second polynucleotide strand, comprising contacting a first polynucleotide strand and a second polynucleotide strand with an engineered RNA ligase of any one of claims 1-91 in presence of a nucleotide substrate under conditions suitable for ligation of the first polynucleotide strand to the second polynucleotide strand, wherein the first polynucleotide strand comprises a ligatable 5’-end and the second polynucleotide strand comprises a 3’-end ligatable to the 5’-end of the first polynucleotide.

109. The method of claim 108, further comprising a third polynucleotide strand, wherein the first polynucleotide strand and second polynucleotide strand hybridize adjacent to one another on the third polynucleotide strand to position the 5’-end of the first polynucleotide strand adjacent to the 3’-end of the second polynucleotide strand.

110. The method of claim 109, wherein the third polynucleotide strand is continuous with the first polynucleotide strand or second polynucleotide strand.

111. The method of claim 109, wherein the third polynucleotide strand is continuous with the first polynucleotide strand and second polynucleotide strand to form a single continuous polynucleotide substrate.

112. The method of claim 111, wherein the third polynucleotide strand comprises a splint or bridging polynucleotide, wherein the 5’-terminal sequence of the first polynucleotide strand and the 3’-terminal sequence of the second polynucleotide strand hybridize adjacent to one another on the splint or bridging polynucleotide to position the 5’-end of the first polynucleotide strand adjacent to the 3’-end of the second polynucleotide strand.

113. The method of claim 109, wherein the first and third polynucleotide strands hybridize to each other to form a first double stranded polynucleotide fragment, and the second polynucleotide strand hybridizes to another polynucleotide strand to form a second double stranded fragment, wherein the first and second double stranded fragments have complementary ends that can base pair to form substrates for the engineered RNA ligase.

114. The method of any one of claims 108-113, wherein the first polynucleotide strand and / or second polynucleotide strand comprises RNA or a mixture of RNA and DNA.

115. The method of any one of claims 108-114, wherein the 3’-end of the second polynucleotide strand is a 3’-OH.

116. The method of any one of claims 108-115, wherein the 5’-end of the first polynucleotide strand is a 5’-phosphate.

117. The method of any one of claims 108-116, wherein the intemucleoside linkage comprises a phosphate linkage.

118. The method of any one of claims 108-114, wherein the first polynucleotide strand and / or second polynucleotide strand comprise one or more nucleotide analogs, wherein the nucleotide analog comprises a modified nucleobase, modified nucleoside sugar residue, modified nucleobase or nucleobase analog, modified intemucleoside linkage, modified 5’-end phosphate group, or any combinations thereof.

119. The method of claim 118, wherein the modified 5’-end phosphate group is a phosphate analog.

120. The method of claim 119, wherein the phosphate analog is a phosphorothioate or monomethy Ipho sphate.

121. The method of claim 118, wherein the modified nucleoside sugar residue is a 2’-0-alkyl, a 2’-fluoro, a p-D-ribo LNA, or a a-L-ribo-LNA (locked nucleic acids).

122. The method of claim 118, wherein the modified nucleobase or nucleobase analog is xanthine, hypoxanthine, inosine, 7-methylguanine, 2,6-diaminopurine, 5-methylcytosine, 5-hydroxycytosine, 5-bromocytosine, 5-iodocytosine, 2-thiothymine, 5-fluorouracil, 5-bromouracil, 8-bromoguanine, 8-aminoguanine, or 8-aza-7-deazaguanine.

123. The method of claim 118, wherein the internucleoside linkage is a phosphorothioate, phosphorodithioate, phosphoacetate, phosphoramidate, methylphosphonate, or phosphonocarboxylate.

124. The method of any one of claims 108-123, wherein the nucleotide substrate is ATP or dATP.

125. A kit comprising at least an RNA ligase of any one of claims 1-91.

126. The kit of claim 125, further comprising one or more of a buffer, nucleotide substrate, a ligation enhance, and a polynucleotide substrate.