Engineered RNA ligases

Abstract

The present disclosure provides engineered RNA ligases, recombinant polynucleotides encoding the engineered RNA ligases, and compositions of the engineered RNA ligases. The present disclosure also provides uses of the engineered RNA ligases for ligating polynucleotide substrates.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application 63 / 601,699, filed November 21, 2023, which is incorporated herein by reference.

[0002] References to sequence lists, tables, or computer programs Meanwhile, the sequence list, named CX9-256WO2_ST26.xml, created on November 13, 2024, and measuring 1,902,579 bytes, was submitted as part of the specification and is incorporated herein by reference. Background Technology

[0003] RNA ligases are a family of enzymes that catalyze the joining of adjacent RNA or DNA fragments. They are involved in RNA editing and repair, and are therefore essential proteins in biological processes. RNA ligases can be used for target sequence analysis, quantification of different RNA species, detection of specific RNA mutations, and polynucleotide synthesis. Based on their substrate preferences, known RNA ligases are divided into two groups. Single-stranded RNA ligases, also known as RNA ligase 1 or RNA ligase I, are capable of joining two RNA or DNA fragments without a hybridization template. Double-stranded RNA ligases, also known as RNA ligase 2 or RNA ligase II, preferentially join nicks in RNA duplexes. RNA ligase type 1 proteins have been identified in fungi, baculoviruses, archaea, and archaeoviruses (thermally stable). RNA ligase type 2 enzymes have been identified in vibrio phage KVP40, baculoviruses and insectpoxviruses, and some parasitic and archaeoviral species.

[0004] Prototypical RNA ligases are those derived from bacteriophage T4. T4 RNA ligase 1 ligates 5'-phosphoryl nucleic acid donors and 3'-hydroxy nucleic acid acceptors via a phosphodiester bond in an ATP-dependent reaction. Substrates for T4 RNA ligase 1 include ssRNA, ssDNA, and dinucleotide pyrophosphate. T4 RNA ligase 1 is used for ligation of ssRNA and DNA, RNA ligase-mediated rapid cDNA extension (RLM-RACE), ligation of oligonucleotide adaptors to cDNA or single-stranded primer extension products for PCR, oligonucleotide synthesis, and various 5' nucleotide modifications of nucleic acids. In the case of nicked dsRNA or RNA / DNA hybrids, T4 RNA ligase 2 attaches adjacent 5' phosphate esters at the ends of the RNA and DNA strands to the 3' OH group of the RNA strand. T4 RNA ligase 2 exhibits significant homology with DNA ligases and mRNA capping enzymes and is used to close nicks in dsRNA and dsRNA / DNA hybrids. Although T4 RNA ligase can act on both RNA and DNA, RNA is the preferred substrate, while DNA is a less effective acceptor in the ligation reaction.

[0005] While T4 RNA ligases have become useful tools for labeling, circularizing, and ligating RNA and RNA / DNA, there is a desire for ligases that provide easier and more efficient polynucleotide substrate ligation, including improved ligation of polynucleotides containing nucleotide analogs such as modified sugar residues and non-standard nucleotide internucleotide bonds. Summary of the Invention

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

[0007] In one aspect, this 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 2-220, 224-252 and 270-958. The reference sequences 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

[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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582. The reference sequences NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or with respect 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 343 of SEQ ID NO: 2 or with respect 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0011] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 4-220, 224-252, and 270-958, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 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 includes at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, 156, 167, 168, 170, 171, 17 Substitutions at 4, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least substitutions at amino acid positions 33, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least the substitutions or sets of substitutions at amino acid positions 179, 33 / 101 / 221 / 320 / 335 / 336 / 339, 38 / 71 / 114 / 184 / 191 / 196, 117, 292, 75 / 136 / 296, or 98, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least substitutions or sets of substitutions at amino acid positions 14, 32, 33, 38, 40, 57, 60, 71, 75, 82, 92, 96, 98, 101, 114, 141, 142, 145, 170, 171, 174, 179, 184, 191, 193, 196, 202, 205, 207, 209, 221, 255, 320, 327, 339, or 342, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least the substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the engineered RNA ligase comprises a reference sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 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 higher sequence identity.

[0019] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs in SEQ ID NOs: 4-220, 224-252, and 270-958.

[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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence is identical with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or ... The reference sequences 4, 106, 218, 286, 396, 436, 520, 552, or 582 contain one or more substitutions.

[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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4-220, 224-252 and 270-958. The reference sequences 4, 106, 218, 286, 396, 436, 520, 552, or 582 contain one or more substitutions.

[0022] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, 156, 167, 168, 170, 171, 17 Substitutions at 4, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ Reference sequences of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to reference sequences corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0023] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 33, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4.

[0025] 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0026] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 33 / 101 / 221 / 320 / 335 / 336 / 339, 71 / 92 / 171 / 184 / 202, 40 / 92 / 171 / 202 / 327, 196 / 300 / 335 / 339, 33 / 184 / 221 / 300 / 320, 335 / 336 / 339, 184 / 196 / 221 / 336, 33 / 184 / 320 / 335 / 336, 207 / 209 / 221 / 300 / 320 / 336 / 339, 101 / 196 / 221 / 300 / 335, 207 / 209 / 300 / 320, 40 / 1 70 / 171 / 202 / 327, 33 / 101 / 184 / 196 / 209 / 336 / 339, 40 / 71 / 92 / 171 / 184 / 327, 207 / 335 / 336 / 339, 33 / 101 / 196 / 300 / 320 / 335 / 336 / 339, 57 / 171 / 179 / 184 / 202 / 327, 57 / 92 / 202 / 327, 33 / 101 / 207 / 221 / 300 / 335 / 336 / 339, 33 / 221 / 336, 196 / 221 / 300 / 335 / 336 / 339, 33 / 184 / 221 / 336 / 339, 33 / 184 / 196 / 221 / 3 00, 101 / 196 / 207 / 209 / 221 / 300 / 336 / 339, 101 / 184 / 335 / 336 / 339, 33 / 184 / 196 / 320 / 336 / 339, 33 / 184 / 196 / 300 / 320 / 335 / 336 / 339, 184 / 320 / 335 / 33 6 / 339, 184 / 196 / 207 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 320 / 335 / 336 / 339, 33 / 196 / 209 / 221 / 339, 184 / 320 / 335 / 336 / 339, 101 / 209 / 335 / 336 / 339, 101 / 196 / 209 / 339, 40 / 71 / 82 / 170 / 171 / 179 / 202 / 327, 320 / 335 / 336 / 339, 101 / 184 / 300 / 335 / 336 / 339, 33 / 101 / 196 / 209 / 335 / 336 / 339, 33 / 196 / 300 / 320, 221 / 335, 33 / 184 / 196 / 207 / 221, 221 / 335 / 336 / 339, 196 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 300 / 320, 57 / 171 / 202 / 327, 40 / 71 / 171 / 184, 184 / 196 / 221,The substitution set at positions 33 / 196 / 207 / 335 / 336, 32 / 57 / 71 / 202 / 327 / 342, 33 / 196 / 221 / 336, 101 / 221 / 300 / 320 / 335 / 336 / 339, 33 / 101 / 207 / 221, 101 / 184 / 196 / 209 / 221, 33 / 207 / 209 / 300 / 335 / 336 / 339, or 33 / 83 / 184 / 196 / 300 / 336, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0027] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO: 106, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO: 106.

[0028] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 218-220 and 224-252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0029] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 38 / 71 / 114 / 184 / 191 / 196, 141 / 170 / 171 / 174, 114, 57 / 60 / 170 / 171 / 174 / 192 / 196 / 320, 60 / 92 / 170 / 171, 141 / 174 / 196, 57 / 170 / 171 / 174, 141 / 184, 57 / The substitutions or sets of substitutions at positions 60 / 141 / 170 / 171 / 174 / 192, 60 / 170 / 174, 141 / 170 / 171, 69 / 71 / 114 / 184, 38 / 40, 40 / 71 / 98 / 184 / 259, 32 / 60 / 141 / 170 / 171 / 174 / 196 / 320, 32 / 170 / 207 / 320, or 196, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO: 106.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO: 218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO: 218.

[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%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 270-312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 218 or relative to the reference sequence corresponding to SEQ ID NO: 218.

[0032] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a set of substitutions or substitutions at amino acid positions 102, 17, 190, 16, 68, 23, 270, 117 / 229, or 117, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to a reference sequence corresponding to SEQ ID NO: 218.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO: 286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO: 286.

[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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 286 or the reference sequence corresponding to SEQ ID NO: 286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or relative to the reference sequence corresponding to SEQ ID NO: 286.

[0035] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions or sets of substitutions at amino acid positions 1296, 114 / 117, 26, 30, 117 / 118, 117 / 119, 14, 183, 117, 185, 307, 12, 93 / 135, 135, 92, 288, 117 / 120, 79, 100, 287, 292, 25, 75, 327, or 111, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286, or relative to the reference sequence corresponding to SEQ ID NO: 286.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396.

[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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396.

[0038] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 102 / 119, 75 / 102, 75 / 102 / 117, 75 / 119, 75 / 136 / 296, 75 / 327, 75 / 117 / 119, 119, 119 / 296, 14 / 102 / 119, 14 / 102 / 296, 14 / 75, 14 / 75 / 119, 14 / 75 / 119 / 327, and 14 / 75 / 117 / 119. The substitutions or sets of substitutions at 14 / 75 / 117 / 119 / 296, 14 / 119, 14 / 296, 14 / 30 / 270, 14 / 25, 14 / 117, 14 / 117 / 119, 14 / 117 / 119 / 296, 14 / 117 / 296 / 327, 26 / 75 / 327, 25 / 75, 25 / 30 / 102, 117, 117 / 119, or 117 / 119 / 296, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436.

[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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 436 or the reference sequence corresponding to SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or relative to the reference sequence corresponding to SEQ ID NO: 436.

[0041] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 332, 156, 177, 333, 96, 11, 189, 327, 25, 73, 98, 335, 95, 188, or 52, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to the reference sequence corresponding to SEQ ID NO: 436.

[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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520.

[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%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520.

[0044] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the set of substitutions at amino acid positions 96 / 98 / 156 / 189 / 335, 73 / 96 / 98 / 189 / 335, 73 / 177 / 189, 73 / 98 / 177 / 189 / 335, 25 / 73 / 95 / 96 / 98 / 189 / 327, 25 / 98 / 189, 73 / 95 / 96 / 156 / 177, 73 / 335, 73 / 96 / 98 / 156 / 335, or 73 / 177 / 189 / 333, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0045] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552.

[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%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552.

[0047] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 292, 147, 255, 295, 75, 300, 333, 200, 289, 95, or 168, or combinations thereof, wherein said amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to a reference sequence corresponding to SEQ ID NO: 552.

[0048] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582.

[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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0050] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 95 / 156 / 337, 189 / 200 / 271, 200 / 271 / 289 / 337, 200 / 271 / 289, 189 / 200, 156 / 200 / 289, 156 / 168 / 189 / 271 / 300, 156 / 189 / 200 / 289, 156 / 168 / 289, 95 / 156 / 271 / 289, 156 / 333, 95 / 200 / 289 / 337, 168 / 200 / 289, 189 / 200 / 289, 333 / 337, 103 / 156 / 271, 95 / 156 / 200 / 30 0 / 333, 156 / 271 / 289 / 333, 95 / 156 / 271, 95 / 189 / 289, 95 / 156 / 168 / 189 / 271 / 289 / 300, 189 / 271 / 289, 95 / 168 / 271 / 300, 156 / 189 / 289 / 337, 200 / 271, 15 6 / 189 / 333, 189 / 289, 156 / 189 / 200 / 300 / 333 / 337, 156 / 189 / 200 / 271 / 289 / 337, 156 / 200 / 271 / 289 / 333 / 337, 168 / 271 / 300, 289 / 337, 156 / 168 / 337, 15 6 / 168 / 189 / 289, 156 / 271 / 289, 189 / 300, 156 / 289 / 300 / 337, 168 / 189 / 289, 95 / 156 / 168 / 300, 156 / 200 / 300 / 337, 289 / 333 / 337, 95 / 189 / 200 / 300 / 337 95 / 156 / 189 / 200 / 271 / 333, 200 / 271 / 289 / 300, 289 / 333, 189 / 271 / 300, 95 / 300, 95 / 189 / 333 / 337, 95 / 189 / 271 / 300, 95 / 189 / 200 / 289, 168, 156 / 200 / 2 71 / 289 / 337, 95 / 168 / 289, 156 / 289, 95 / 156 / 189 / 271 / 289, 156 / 168 / 189 / 200 / 289 / 333, 156 / 300 / 333 / 337, 95 / 156 / 189 / 200 / 271 / 337, 271, 156 / 271, 156 / 271 / 300 / 333, 95 / 168 / 200, 168 / 271, 95 / 200 / 333, 189 / 200 / 271 / 289 / 333, 95 / 168 / 189 / 333 / 337, 189, 189 / 333 / 337, 156 / 189 / 271 / 289 / 333, 289,95 / 156 / 200 / 271 / 289、289 / 300 / 333、95 / 189 / 289 / 337、95 / 156 / 168 / 189 / 289、271 / 289、333、168 / 337、156 / 189 / 271 / 289、95 / 156 / 189 / 337、156 / 189 / 271 / 300、156 / 168 / 289 / 333、168 / 189 / 289 / 333、95 / 333、333 / 343、156 / 189 / 289、95 / 289 / 300、95 / 156 / 189 / 200 / 300 / 333 / 337、189 / 200 / 271 / 289 / 337、95 / 156 / 168 / 189 / 289 / 337、95 / 289、156 / 289 / 333 / 337、156 / 289 / 337、337、168 / 289、95 / 156 / 300、95 / 156 / 289、95 / 189 / 271 / 333 / 337、95 / 168 / 300、95 / 156 / 168 / 271 / 333 / 337、95 / 189 / 289 / 333 / 337、156 / 168 / 200 / 289 / 334、95 / 156 / 168 / 189 / 271 / 289、156 / 189 / 200 / 289 / 337、200 / 289、200 / 289 / 333 / 337、95 / 168 / 189 / 200 / 289、168 / 200、156 / 168 / 271 / 289、189 / 206 / 289、156 / 200 / 289 / 333 / 335、156 / 200 / 289 / 333、189 / 200 / 289 / 333、189 / 289 / 335、189 / 254 / 289 / 333、167 / 189 / 206 / 289 / 333 / 335、156 / 200 / 333、156 / 200 / 289 / 335、156 / 189 / 289 / 335、335、156 / 167 / 189 / 289、156 / 189 / 200 / 289 / 335、200 / 289 / 333 / 335、156 / 189 / 200 / 289 / 333、254 / 333 / 335、189 / 254 / 333 / 335、189 / 335、156 / 189 / 254 / 289、156 / 289 / 333、289 / 335、189 / 200 / 254 / 289 / 333、156 / 189 / 289 / 333、25 / 156 / 189 / 289、156 / 189 / 206 / 289 / 333 / 335、289 / 333 / 335、156 / 254 / 289 / 333、156 / 289 / 333 / 335、254 / 289、200 / 289 / 333、156 / 189 / 200, 156 / 254 / 333 / 335, 156, 254 / 289 / 335, 156 / 206 / 289, 156 / 289 / 335, 206 / 289 / 333 / 335, 156 / 189 / 254 / 289 / 333, 156 / 254 / 289, 200 / 289 / 335, 156 / 189 / 206 Substitutions or sets of substitutions at / 289, 156 / 189 / 200 / 206 / 254 / 289 / 333, 25 / 254 / 333, 189 / 289 / 333 / 335, 156 / 189 / 200 / 289 / 333 / 335, 254 / 289 / 333, or 156 / 200 / 333 / 335, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0051] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0052] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions or sets of substitutions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0053] 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 higher sequence identity with a reference sequence, said reference sequence comprising substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein said amino acid positions are relative to residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to the reference sequence corresponding to SEQ ID NO: Reference sequences of 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0054] In some embodiments, the engineered RNA ligase comprises an amino acid sequence containing residues 12 to 343 of SEQ ID NO. with even numbers in SEQ ID NO: 4-220, 224-252, and 270-958, or an amino acid sequence containing SEQ ID NO. with even numbers in SEQ ID NO: 4-220, 224-252, and 270-958.

[0055] In some embodiments, the engineered RNA ligase comprises an amino acid sequence of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or an amino acid sequence of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0056] In some embodiments, the engineered RNA ligase has RNA ligase activity and at least one improved property compared to a reference RNA ligase. In some embodiments, the improved property of the engineered RNA ligase compared to a reference RNA ligase is selected from i) increased activity, ii) increased product yield, iii) increased product yield for polynucleotides having a phosphate thioester nucleoside internucleotide bond, iv) increased product yield for oligonucleotides having a 2'-modified form, and v) increased expression, or any combination of i), ii), iii), iv), and v).

[0057] In some embodiments, the improved properties of the engineered RNA ligase are compared with those of a reference RNA ligase having sequences of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or sequences corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582. In some embodiments, the reference RNA ligase has sequences of residues 12 to 343 corresponding to SEQ ID NO: 2, or sequences corresponding to SEQ ID NO: 2.

[0058] In some embodiments, the engineered RNA ligase comprises a fusion protein. In some embodiments, the engineered RNA ligase is fused with a tag or affinity peptide.

[0059] In some other embodiments, the engineered RNA ligase is purified. In some embodiments, the engineered RNA ligase is provided in solution or immobilized on the surface of a substrate such as a solid substrate or a membrane or particle.

[0060] On the other hand, this disclosure provides recombinant polynucleotides encoding any of the engineered RNA ligases disclosed herein.

[0061] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551 or 581, wherein the recombinant polynucleotide encodes an RNA ligase.

[0062] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence having nucleotide residues 34 to 1029 of SEQ ID NO. corresponding to odd-numbered numbers in SEQ ID NO: 3-219, 223-251, and 269-957, or a reference polynucleotide sequence corresponding to odd-numbered numbers in SEQ ID NO: 3-219, 223-957, and 269-553, having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the recombinant polynucleotide encodes an RNA ligase.

[0063] In some implementations, the multinucleotide sequence encoding the recombinant polynucleotide encoding the engineered RNA ligase is codon-optimized for expression in an organism or its cell type (e.g., bacterial, fungal, or mammalian cells).

[0064] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 34 to 1029 of SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or a polynucleotide sequence containing SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581.

[0065] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 34 to 1029 of SEQ ID NO. with odd numbers in SEQ ID NO: 3-219, 223-251 and 269-957, or a polynucleotide sequence containing SEQ ID NO. with odd numbers in SEQ ID NO: 3-219, 223-251 and 269-957.

[0066] In another aspect, this disclosure provides expression vectors comprising recombinant polynucleotides encoding engineered RNA ligases provided herein. In some embodiments, the recombinant polynucleotides of the expression vector are operatively linked to a control sequence. In some embodiments, the control sequence comprises at least a promoter, particularly a heterologous promoter.

[0067] In another aspect, this disclosure also provides host cells comprising the expression vectors provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell, such as Escherichia coli or Bacillus subtilis.

[0068] In another aspect, this disclosure provides a method for producing engineered RNA ligase peptides, the method comprising culturing the host cells described herein under suitable culture conditions to produce at least one engineered RNA ligase. In some embodiments, the method further includes recovering or isolating the engineered RNA ligase from the culture and / or host cells. In some embodiments, the method further includes purifying the engineered RNA ligase.

[0069] In another aspect, this disclosure provides a composition comprising the engineered RNA ligase disclosed herein. In some embodiments, the composition comprises at least a buffer. In some embodiments, the composition further comprises one or more of a nucleotide substrate (e.g., ATP or dATP) and a polynucleotide ligase substrate.

[0070] In another aspect, this disclosure provides a method for ligating polynucleotide substrates, particularly double-stranded polynucleotide substrates. In some embodiments, the method of ligating at least a first polynucleotide chain and a second polynucleotide chain includes contacting the first and second polynucleotide chains with an engineered RNA ligase described herein in the presence of a nucleotide substrate under conditions suitable for ligating the first polynucleotide chain to the second polynucleotide chain, wherein the first polynucleotide chain includes a ligation-capable 5' end and the second polynucleotide chain includes a 3' end capable of attaching to the 5' end of the first polynucleotide chain. In some embodiments, the first and / or second polynucleotide chains comprise RNA or a mixture of RNA and DNA.

[0071] In some embodiments, the method further includes a third polynucleotide chain, wherein the first and second polynucleotide chains hybridize adjacently to each other on the third polynucleotide chain to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain to form a ligase-compatible nick. In some embodiments, the third polynucleotide chain is continuous with either the first or second polynucleotide chain. In some embodiments, the third polynucleotide chain is continuous with both the first and second polynucleotide chains to form a single continuous polynucleotide ligase substrate. In some embodiments, the 3' end region of the second polynucleotide chain hybridizing with the third polynucleotide chain is at least 4, 6, 8, or more base pairs long. In some embodiments, the 5' end region of the first polynucleotide chain hybridizing with the third polynucleotide chain is at least 2, 3, 4, 6, 8, or more base pairs long.

[0072] In some embodiments of the method, the third polynucleotide chain comprises a splint or bridging polynucleotide, wherein the 5' end sequence of the first polynucleotide chain and the 3' end sequence of the second polynucleotide chain hybridize adjacent to each other on the splint or bridging polynucleotide to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain.

[0073] In some embodiments, the first polynucleotide chain hybridizes with a third polynucleotide chain to form a first double-stranded fragment, and the second polynucleotide chain hybridizes with a fourth polynucleotide chain to form a second double-stranded fragment, wherein the first and second double-stranded fragments have complementary ends that can base-pair to form a substrate for an engineered RNA ligase. In some embodiments, the first and second double-stranded fragments have complementary overhangs or complementary single-stranded ends that can base-pair to form a double-stranded nick, which serves as a substrate for the engineered RNA ligase.

[0074] In some embodiments of the method, at least 2, 3, 4, 5, or 6 or more double-stranded fragments, each having a complementary end that can pair with at least one other double-stranded fragment having a complementary end, serve as a substrate for RNA ligase.

[0075] In some embodiments, the polynucleotide substrate comprises one or more modified nucleotides. In some embodiments, the modification comprises modified sugar residues, modified nucleotides, and / or modified phosphate groups. In some embodiments, the modified sugar residues are modified at the 2-position of the sugar moiety. In some embodiments, the modified 2-position is a 2'-halogenated group or a 2'-O-alkyl group, preferably a lower alkyl group, such as methyl or ethyl. In some embodiments, the modified nucleotide is a modified phosphate ester group, such as a thiophosphate ester group. In some embodiments, the modified phosphate ester group is located at the 5' end of the polynucleotide substrate. In some embodiments, the modified phosphate ester group is located at the nucleoside internucleotide bond of the polynucleotide substrate. In some embodiments, the modified nucleotide has modified nucleotides. In some embodiments, the polynucleotide substrate comprises a reverse nucleotide (e.g., a 5'-5' or 3'-3' reverse bond). In some embodiments, the modified nucleotide is modified with a cell-targeting moiety (such as GalNac or a lipid (e.g., cholesterol)). In some embodiments, the modified nucleotide is modified with a linker moiety. In some embodiments, the modification using the cell-targeting or linker portion is located on a nucleobase or sugar residue. In some embodiments, the cell-targeting or linker portion is attached to the 5' or 3' end of the polynucleotide substrate.

[0076] In some embodiments, engineered RNA ligases are used in methods for synthesizing RNA or DNA / RNA polynucleotides by ligating shorter RNA or DNA / RNA oligonucleotides. In some embodiments, engineered RNA ligases are used to ligate the 3' OH of RNA to the 5' phosphate ester of DNA or RNA. In some embodiments, engineered RNA ligases are used to ligate the 3' OH of RNA to the 5' phosphate ester of DNA in the construction of double-stranded NGS RNA libraries. In some embodiments, engineered RNA ligases are used in methods for preparing RNA loops. In some embodiments, engineered RNA ligases are used to repair nicks in dsRNA or dsRNA / DNA. In some embodiments, engineered RNA ligases are used to synthesize modified RNA oligonucleotides.

[0077] In another aspect, this disclosure also provides a kit comprising at least one engineered RNA ligase disclosed herein. In some embodiments, the kit further comprises a buffer, a nucleotide substrate (e.g., ATP or dATP), and / or one or more polynucleotide ligase substrates. Detailed Implementation

[0078] This disclosure provides engineered RNA ligase peptides and compositions thereof, as well as polynucleotides encoding the engineered RNA ligase peptides. This disclosure also provides methods for using the engineered RNA ligase peptides and compositions thereof for molecular biology, diagnostic, and other purposes. In some embodiments, the engineered RNA ligase peptides exhibit, in particular, increased activity, increased stability, increased thermal stability, and / or increased activity against polynucleotides containing one or more modified nucleotides or nucleotide analogs.

[0079] Abbreviations and Definitions For the purposes of this invention, unless otherwise expressly defined, the technical and scientific terms used in this description will have the meanings commonly understood by one of ordinary skill in the art. Therefore, the following terms are intended to have the following meanings and will be described more fully by reference to the entire application.

[0080] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, references to “a polypeptide” include more than one polypeptide.

[0081] Similarly, “comprise” and “include” are interchangeable and are not intended to be restrictive. Therefore, as used herein, the term “comprise” and its cognates are used in their inclusive sense (i.e., equivalent to the term “include” and its corresponding cognates).

[0082] It should also be understood that, in the context of the use of the term "comprising" when describing various embodiments, those skilled in the art will understand that, in certain specific cases, the language "substantially consisting of" or "consisting of" may be used to alternatively describe the embodiment.

[0083] Furthermore, the numerical range includes the number that defines the range. Therefore, each numerical range disclosed herein is intended to encompass every narrower numerical range falling within such a wider numerical range, as if such a narrower numerical range were explicitly stated herein. Each maximum (or minimum) numerical limit disclosed herein is also intended to include every lower (or higher) numerical limit, as if such lower (or higher) numerical limits were explicitly stated herein.

[0084] As used herein, the term “about” refers to the acceptable error for a particular value. In some cases, “about” means within 0.05%, 0.5%, 1.0%, or 2.0% of the given value range. In other cases, “about” means within 1, 2, 3, or 4 standard deviations of the given value.

[0085] Furthermore, the headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the entire application. Therefore, the terms defined below are defined more fully by reference to the entire application.

[0086] "EC" refers to the enzyme nomenclature system of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system of enzymes based on enzyme-catalyzed chemical reactions.

[0087] "ATCC" refers to the American Type Culture Collection, whose biobank collection includes genes and strains.

[0088] "NCBI" refers to the National Center for Biotechnology Information and the sequence database it provides.

[0089] RNA ligases are enzymes that covalently link the 5'-phosphoryl terminus of RNA or DNA to the 3'-hydroxy terminus. Two families of RNA ligases are known to exist in nature. RNA ligase 1 catalyzes the covalent linking of the 5'-phosphoryl terminus of a single strand of RNA or DNA to the 3'-hydroxy terminus of the same strand. RNA ligase 2 also catalyzes the covalent linking of the 3'-hydroxy terminus of RNA to 5'-phosphorylated RNA or DNA, but shows a preference for double-stranded substrates. RNA ligases include those classified as EC 6.5.1.3.

[0090] Although some DNA ligases can act on DNA or RNA as 3'-hydroxyl chain substrates, RNA ligase 2, which acts at the double-strand cleavage, preferentially acts on the 3'-hydroxyl chain of RNA, but does not know whether the 5-phosphoryl chain is DNA or RNA. Furthermore, in some embodiments, the 3'-hydroxyl chain may include deoxyribonucleotides if a 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 chain, particularly by the two terminal ribonucleotides on the 3'-hydroxyl side of the cleavage (see, for example, Nandakumar et al., Mol. Cell., 2004, 16:211-221). It should be understood that the ligation reaction is not limited to naturally occurring RNA and DNA substrates, but also includes polynucleotide substrates containing modified nucleotides and / or nucleotide analogs.

[0091] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably to refer to polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0092] Amino acids are referred to in this document by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides are referred to by their generally accepted single-letter codes. The abbreviations used for gene-encoding amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile 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 (Val or V). When using a three-letter abbreviation, unless specifically preceded by "L" or "D" or clearly indicated from the context of its use, amino acids can refer to α-carbon (C) α The amino acid sequence is represented by its L- or D-configuration. For example, "Ala" indicates alanine without specifying the configuration with respect to the α-carbon, "D-Ala" and "L-Ala" indicate D-alanine and L-alanine, respectively. When using single-letter abbreviations, uppercase letters indicate amino acids with the L-configuration with respect to the α-carbon, and lowercase letters indicate amino acids with the D-configuration with respect to the α-carbon. For example, "A" indicates L-alanine, and "a" indicates D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is conventionally presented in the direction from amino (N) to carboxyl (C).

[0093] "Fusion protein," "chimeric protein," and "chimera" refer to hybrid proteins produced by linking two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are produced using recombinant technologies (e.g., molecular biology techniques known in the art).

[0094] The terms “polynucleotide,” “nucleic acid,” or “oligonucleotide” are used herein to refer to polymers comprising at least two nucleotides, wherein the nucleotides are deoxyribonucleotides or ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for gene-encoding nucleosides are conventional and follow the following: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, the abbreviation for nucleosides may be ribonucleoside or 2'-deoxyribonucleoside. Nucleosides may be designated as ribonucleoside or 2'-deoxyribonucleoside on a single or collective basis. When a polynucleotide, nucleic acid, or oligonucleotide sequence is presented as a string of single-letter abbreviations, the sequence is presented in the 5' to 3' orientation according to common practice and does not indicate phosphate esters. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. Polynucleotides or nucleic acids may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions.

[0095] In some embodiments, the terms "polynucleotide," "nucleic acid," and "oligonucleotide" encompass polynucleotides or nucleic acids or oligonucleotide analogs, which in particular include nucleosides linked together via phosphodiester bonds that are different from standard phosphodiester bonds, such as non-standard aminophosphate bonds, thiophosphate bonds, amide bonds, etc.; nucleosides / nucleotides having non-standard nucleobases (including modified and / or synthetic nucleobases), such as inosine, xanthine, hypoxanthine, etc.; nucleosides having modified sugar residues, such as 2'-O-alkyl, 2'-halo, 2,3-dideoxy, 2'-halo-2'deoxy, β-D-ribose-LNA, α-L-ribose-LNA (locked nucleic acid), etc.; and / or 5'-phosphate analogs, particularly including thiophosphates, phosphate acetates, aminophosphates, monomethyl phosphates, methylphosphonates, or phosphonoylcarboxylic acids.

[0096] "Double strand" and "ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule composed of two single-stranded polynucleotides that are complementary in sequence (e.g., A to T or U, C to G), arranged in an antiparallel 5' to 3' orientation, and held together by hydrogen bonds between nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]). In some embodiments, the double strand or double-stranded nucleic acid is formed using modified nucleobases or nucleobase analogs.

[0097] The term "complementary" is used in this paper to describe the structural relationship between nucleotide bases that can form base pairs with each other. For example, purine nucleotide bases that are complementary to the pyrimidine nucleotide bases on a polynucleotide can pair with each other by forming hydrogen bonds. Complementary nucleotide bases can pair via Watson / Crick base pairing or in any other way except to form stable double strands or other nucleic acid structures.

[0098] "Watson / Crick base pairing" refers to a pattern of specific nucleobase and analog pairs that are bound together by sequence-specific hydrogen bonds, such as A pairing with T or U, and G pairing with C.

[0099] "Annealing" or "hybridization" refers to the base-pairing interaction between one nucleobase polymer (e.g., polynucleotides and oligonucleotides) and another nucleobase polymer, resulting in the formation of double-stranded, triple-stranded, or quaternary structures. Annealing or hybridization can occur via Watson-Crick base-pairing interactions, but can also be mediated by other hydrogen-bonding interactions such as Hoogsteen base-pairing. In some embodiments, the nucleobase polymer annealed or hybridized with another nucleobase polymer is a single nucleobase polymer, while in other embodiments, these nucleobase polymers are individual nucleobase polymers.

[0100] When used in relation to cells, polynucleotides, or peptides, “engineered,” “recombinant,” “non-natural,” and “variant” refer to a material or a material corresponding to the natural or native form of that material that has been modified in a manner not present in or similar to that in nature, but produced or derived from synthetic materials and / or manipulated using recombinant techniques.

[0101] "Wild-type" and "naturally occurring" refer to forms found in nature. For example, wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified by humans.

[0102] "Coding sequence" refers to the part of a nucleic acid (e.g., a gene) that encodes the amino acid sequence of a protein.

[0103] "Sequence identity percentage (%)" refers to a comparison between polynucleotides and peptides, and is determined by comparing two optimally aligned sequences within a comparison window. For optimal alignment, the portion of the polynucleotide or peptide sequence in the comparison window may contain additions or deletions (i.e., vacancies) compared to the reference sequence. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears, or by aligning the nucleic acid base or amino acid residue with vacancies to obtain a matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will understand that many established algorithms exist for aligning two sequences. The optimal alignment of sequences for comparison can be performed as is known in the art, for example, by Smith and Waterman’s local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by Needleman and Wunsch’s homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by Pearson and Lipman’s similarity search method (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. Examples of algorithms suitable for determining sequence identity percentages and sequence similarity include, but are not limited to, the BLAST and BLAST2.0 algorithms (see, for example, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence. These short words match or satisfy a positive threshold score “T” when compared to words of the same length in a database sequence. T is called the neighborhood word score threshold (see Altschul et al., ibid.). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.The word hit then extends in both directions along each sequence, as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction stops when: the cumulative alignment score decreases from its maximum realized value by an amount "X"; the cumulative score becomes zero or lower due to the accumulation of one or more negative score 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) defaults to a word length of 11 (W), an expected value of 10 (E), M=5, N=-4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (see, for example, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) with the provided default parameters.

[0104] A “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Since each of two polynucleotides or polypeptides can (1) contain sequences similar to the two sequences (i.e., a portion of the complete sequence) and (2) also contain sequences different from the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides on 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, wherein the reference sequence is a sequence that may have one or more variations in the primary sequence. For example, the phrase “reference sequence corresponding to SEQ ID NO: 14 with leucine at the residue corresponding to X14” (or “reference sequence corresponding to SEQ ID NO: 14 with lysine at the residue corresponding to position 14”) refers to a reference sequence in which the corresponding residue (e.g., alanine) at position X14 in SEQ ID NO: 14 has been changed to lysine.

[0105] A “comparison window” refers to a conceptual segment of adjacent nucleotide positions or amino acid residues, where the sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein, for optimal alignment of the two sequences, the sequence portion within the comparison window may contain 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which does not contain additions or deletions). In some embodiments, the comparison window may be longer than 15-20 consecutive residues, and optionally include windows of 30, 40, 50, 100, or longer.

[0106] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," and "relative to" refer to specifying the number of residues in the reference sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbering or position of a given polymer is specified relative to the reference sequence, rather than by the actual numerical position of residues within the given amino acid or polynucleotide sequence. For example, residue matching between two sequences can be optimized by introducing vacancies, such as aligning a given amino acid sequence (e.g., the amino acid sequence of an engineered RNA ligase) to a reference sequence. In these cases, the numbering of residues in the given amino acid or polynucleotide sequence is relative to the reference sequence with which it is aligned, despite the presence of vacancies. In some embodiments, the sequence is tagged (e.g., with histidine tags).

[0107] A "mutation" refers to an alteration in the sequence of a nucleic acid. In some embodiments, a mutation results in a change in the sequence of the encoded polypeptide (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation includes substitution, resulting in a different amino acid. In some alternative embodiments, a mutation includes addition, resulting in an amino acid being added (e.g., inserted) into the original polypeptide sequence. In some further embodiments, a mutation includes deletion, resulting in an amino acid being removed from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0108] "Amino acid difference" and "residue difference" refer to the difference of an amino acid residue at a position in a polypeptide sequence relative to the corresponding amino acid residue at a position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X17 compared to SEQ ID NO:2" (or "residue difference at position 17 compared to SEQ ID NO:2") refers to the difference of an amino acid residue at the polypeptide position corresponding to position 17 of SEQ ID NO:2. Therefore, if the reference polypeptide of SEQ ID NO:2 has a serine residue at position 17, then "residue difference at position X17 compared to SEQ ID NO:2" refers to the substitution of any amino acid residue other than serine at the polypeptide position corresponding to position 17 of SEQ ID NO:2. In some cases herein, a specific amino acid residue difference at a position is represented as "XnY," where "Xn" specifies the corresponding residue and position of the reference polypeptide (as described above), and "Y" is a single-letter identifier of the amino acid found in the engineered polypeptide (i.e., a residue different from that in the reference polypeptide). In some cases (e.g., in the tables of the embodiments), this disclosure also provides specific amino acid differences represented by the conventional symbol “AnB”, where A is a single-letter identifier of a residue in the reference sequence, “n” is the position number of the residue in the reference sequence, and B is a single-letter identifier of a residue substitution in the sequence of the engineered polypeptide. In some cases, amino acid residue differences or substitutions may be deletions and may be represented by “-”. In some embodiments, amino acid differences (e.g., substitutions) are represented by the abbreviation “nB” without an identifier of a residue in the reference sequence. In some embodiments, the phrase “amino acid residue nB” indicates the presence of an amino residue in the engineered polypeptide, the presence of which may or may not be a substitution in the context of the reference polypeptide or amino acid sequence.

[0109] In some cases, the polypeptide of this disclosure may include one or more amino acid residue differences relative to a reference sequence, indicated by a list of designated positions where residue differences exist relative to the reference sequence. In some embodiments, where more than one amino acid may be used at a particular residue position of the polypeptide, the various amino acid residues that may be used are separated by " / " (e.g., T33A / T33K, T33A / K, or 33A / K).

[0110] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, the substitution set contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to a group of amino acid substitutions present in any of the variant RNA ligase polypeptides listed in any of the tables in the examples. In these substitution sets, each substitution is separated by a semicolon (";"; for example, N141T;A184P) or a forward slash (" / "; for example, N141T / A184P or 141T / 184P).

[0111] "Conservative amino acid substitution" refers to the substitution of a residue with a different residue having a similar side chain, and therefore generally involves the substitution of an amino acid in a polypeptide with an amino acid within the same or similar defined amino acid class. 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 a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be substituted with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid may be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0112] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can use amino acids between rather than within groups and affect: (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine); (b) charge or hydrophobicity; and / or (c) the body of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0113] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, representing up to 10% or up to 20% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the improved properties of the engineered RNA ligase. Deletions may target internal and / or terminal portions of the peptide. In various embodiments, deletions may include continuous segments or may be discontinuous. Deletions are indicated by "-" and may be present in a substitution set.

[0114] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. Insertion can occur within the polypeptide itself, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be continuous segments of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0115] The terms “functional fragment” and “bioactive fragment” are used interchangeably herein and refer to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the sequence being compared (e.g., the full-length engineered RNA ligase of the present invention) and substantially retains all the activities of the full-length polypeptide.

[0116] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other naturally occurring contaminants, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis). Recombinant RNA ligase polypeptides may be present intracellularly, in cell culture media, or prepared in various forms, such as lysates or isolated formulations. Therefore, in some embodiments, the recombinant RNA ligase polypeptides provided herein are isolated polypeptides.

[0117] "Substantially pure polypeptide" refers to a composition in which the polypeptide is the dominant substance (i.e., it is more abundant than any other individual macromolecule in the composition, based on molar or weight), and is typically substantially pure when the target substance constitutes at least about 50% (in molar or weight %) of the present macromolecules. Typically, a substantially pure RNA ligase composition will contain 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 macromolecules present in the composition, based on molar or weight %. In some embodiments, the target substance is purified to substantially homogeneity (i.e., contaminants are not detectable in the composition by conventional detection methods), wherein the composition consists substantially of a single macromolecule. Solvents, small molecules (<500 Daltons), and elemental ions are not considered macromolecules. In some embodiments, the isolated recombinant RNA ligase polypeptide is a substantially pure polypeptide composition.

[0118] "Improved enzyme properties" refers to an engineered RNA ligase peptide exhibiting improved enzyme properties compared to a reference RNA ligase peptide, such as a wild-type RNA ligase peptide or another engineered RNA ligase peptide. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermal activity, increased thermal stability, increased stability, increased enzyme activity, 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.

[0119] "Increased enzyme activity" and "enhanced catalytic activity" refer to improved properties of engineered RNA ligase peptides, which can be expressed as an increase in specific activity (e.g., product / time / protein weight produced) and / or the percentage of substrate-to-product conversion (e.g., the percentage of substrate-to-product conversion using a specified amount of RNA ligase over a specified time period) compared to a reference RNA ligase (e.g., wild-type RNA ligase and / or another engineered RNA ligase). Exemplary methods for determining enzyme activity are provided in the examples. Any properties related to enzyme activity may be affected, including K m , V max or k catThe classic characteristics of an enzyme can be altered to lead to increased enzyme activity. Improvements in enzyme activity can range from approximately 1.1 times the activity of the corresponding wild-type enzyme to approximately 1.5 times, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, or more than the activity of another engineered RNA ligase derived from a naturally occurring RNA ligase or RNA ligase polypeptide.

[0120] "Hybridization stringency" refers to the hybridization conditions in nucleic acid hybridization, such as washing conditions. Typically, hybridization reactions are performed under low stringency conditions, followed by washing under different but higher stringency conditions (see, for example, 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 "moderate stringency hybridization" refers to conditions that allow target DNA to bind to a complementary nucleic acid that has approximately 60% identity with the target DNA, preferably approximately 75% or 85% identity, and greater than approximately 90% identity with the target polynucleotide. An exemplary moderate stringency condition is equivalent to hybridization at 42°C in 50% formamide, 5× Denhart solution, 5× SSPE, and 0.2% SDS, followed by washing at 42°C in 0.2× SSPE and 0.2% SDS. "High stringency hybridization" generally refers to conditions close to the pyrolysis temperature. T m Conditions of approximately 10°C or lower, such as those used to determine the defined polynucleotide sequences under solution conditions. In some embodiments, high stringency conditions refer to conditions that allow hybridization only of those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e., if a hybrid is unstable in 0.018 M NaCl at 65°C, it will be unstable under high stringency conditions, as contemplated herein). For example, high stringency conditions can be provided by hybridization at 42°C in a solution equivalent to 50% formamide, 5× Denhart solution, 5× SSPE, and 0.2% SDS, followed by washing at 65°C in 0.1× SSPE and 0.1% SDS. Another high stringency condition includes hybridization equivalent to hybridization at 65°C in 5× SSC containing 0.1% (w:v) SDS and washing at 65°C in 0.1× SSC containing 0.1% SDS. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

[0121] "Codon optimization" refers to changing the codons of polynucleotides encoding proteins to codons that are preferentially used in a particular organism, making the encoded protein more effectively expressed in that organism. Although the genetic code is degenerate, as most amino acids are represented by a few codons (called "synonyms"), codon usage in a particular organism is known to be non-random and biased towards specific codon triples. This codon bias can be even higher for a given gene, genes with a common function or ancestral origin, highly expressed proteins versus low-copy-number proteins, and aggregated protein-coding regions of an organism's genome. In some implementations, polynucleotides encoding RNA ligases are codon-optimized for optimal production from a selected host organism for expression.

[0122] "Control sequence" herein refers to all components that are essential or advantageous for the expression of the polynucleotides and / or polypeptides disclosed herein. Each control sequence may be native or exogenous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. These control sequences contain at least a promoter and transcription and translation termination signals. In some embodiments, the control sequence is provided with an adapter to introduce a specific restriction site that facilitates the attachment of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0123] "Operably linked" refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) relative to the polynucleotide of interest, such that the control sequence directs or regulates the expression of the polynucleotide encoding the polypeptide of interest.

[0124] A "promoter" or "promoter sequence" is a nucleic acid sequence, such as a coding sequence, that is recognized by the host cell for the expression of a polynucleotide of interest. A promoter sequence contains a transcriptional control sequence that mediates the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to those of the host cell.

[0125] "Suitable reaction conditions" or "suitable conditions" refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme load, substrate load, temperature, pH, buffer, co-solvent, etc.) under which the RNA ligase polypeptide of this disclosure can convert a polynucleotide substrate into the desired ligation product polynucleotide. Exemplary "suitable reaction conditions" are provided herein (see Examples).

[0126] In the context of enzymatic conversion processes, "product" refers to the compound or molecule produced by the action of RNA ligase polypeptide on the substrate.

[0127] “Cultivation” refers to the growth of a microbial cell population under suitable conditions using any suitable culture medium (e.g., liquid, gel, or solid).

[0128] A “vector” is a recombinant construct used to introduce a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector operatively linked to a suitable control sequence capable of enabling expression of the polynucleotide or a polypeptide encoded in the polynucleotide in a suitable host. In some embodiments, the “expression vector” has a promoter sequence operatively linked to the polynucleotide (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0129] "Expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers peptide secretion from cells.

[0130] "Production" refers to the production of proteins and / or other compounds by cells. This term is intended to encompass any steps involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of peptides from cells.

[0131] "Heterogeneous" or "recombinant" refers to a relationship between two or more nucleic acid or polypeptide sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that are derived from different sources and are essentially unrelated.

[0132] "Host cell" and "host strain" refer to a suitable host for an expression vector containing a polynucleotide (e.g., a polynucleotide sequence encoding at least one RNA ligase variant) provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA technology, as is known in the art.

[0133] Engineered RNA ligase peptide In one aspect, this disclosure provides RNA ligases, including engineered RNA ligase peptide variants. In some embodiments, the RNA ligase and the engineered RNA ligase peptide variants can be used to ligate polynucleotide substrates, such as for the preparation of polynucleotides from short oligonucleotides, and for diagnostic and other purposes. The engineered RNA ligase variants are available in solution and immobilization embodiments. In some embodiments, the engineered RNA ligase can be prepared and used as a non-fusion peptide or a fusion peptide.

[0134] In some embodiments, the engineered RNA ligase or its functional fragment 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 2-220, 224-252 and 270-958. The reference sequences 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

[0135] 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582. The reference sequences NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

[0136] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or with respect 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 343 of SEQ ID NO: 2 or with respect to the reference sequence corresponding to SEQ ID NO: 2.

[0137] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0138] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 4-220, 224-252, and 270-958, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0139] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, 156, 167, 168, 170, 171, 17 Substitutions at 4, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0140] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least the substituted or amino acid residues 11P, 12S, 14F / R / T / W, 16R, 17P, 23M, 25A / G / K / L / W, 26F / G / H / L / P / R / S / V, 30A / C / H / R, 32R, 33A / K, 38D, 40E, 52L, 57T, 60R, 68W, 69L, 71K, 73T, 75L / Q / T, 79W, 82I, 83P, and 92A / E. / G / M / N / S, 93A, 95E / V, 96A / S, 98D / E / P / R / S, 100R, 101G, 102T, 103I, 111L, 114F / G / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 118G / L / Q, 119A / E / S / T, 120P, 135Q / T, 136A, 141A / C / E / T, 142H / R, 145D, 147L, 156C / Y, 167A, 168A, 170E, 171E, 174Q, 177I / L, 179F, 183V, 184L / M / P / R, 185H / S, 188S, 189L / V, 190F, 191K , 192V, 193N, 196K, 200S / T, 202W, 205R / V, 206R, 207S / T, 209E / L, 221E, 229A, 254G, 255E / S, 259V, 270A, 271 I, 287A, 288A, 289A / H / S / T, 292E / Y, 295R, 296E / L / R / V / W, 300E / K, 307A, 320D / K, 327I / Q / R / W, 332R, 333A / E / S / T, 334S, 335E / H / K / L, 336S, 337G, 339K / R, 342I / T, 343P, 339K / R or 342I / T or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0141] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substituted or amino acid residues G11P, M12S, K14F / R / T / W, Y16R, S17P, Y23M, S25A / G / K / L / W, K26F / G / H / L / P / R / S / V, K30A / C / H / R, Y32R, T33A / K, T38D, V40E, F52L, E57T, N60R, G68W, P69L, L71K, A73T, D75L / Q / T, Y79W, V82I, L83P, T 92A / E / G / M / N / S, V93A, K95E / V, F96A / S, Y98D / E / P / R / S, A100R, R101G, A102T, V103I, F111L, G114F / K / N / P / R / S / V, Q117 A / C / F / G / H / I / K / L / M / R / S / T / V / W, K118G / L / Q, G119A / E / S / T, V120P, N135Q / T, T136A, N141A / C / E / T, T142H / R, T145D, Y14 7L, F156C / Y, G167A, T168A, D170E, S171E, M174Q, N177I / L, L179F, L183V, A184L / M / P / R, A185H / S, A188S, T189L / V, A19 0F, S191K, E192V, D193N, E196K, C200S / T, F202W, N205R / V, V206R, I207S / T, D209E / L, F221E, T229A, V254G, P255E / S, I2 59V, Y270A, V271I, T287A, P288A, K289A / H / S / T, G292E / Y, M295R, G296E / L / R / V / W, Q300E / K, S307A, N320D / K, V327I / Q / R / W, D332R, V333A / E / S / T, L334S, R335E / H / K / L, P336S, A337G, I339K / R, V342I / T or S343P or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0142] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 3, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0143] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 33A / K, 38D, 71K, 73T, 75L / Q / T, 96A / S, 98D / E / P / R / S, 101G, 114F / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 136A, 156C / Y, 179F, 184L / M / P / R, 191K, 196K, 221E, 289A / H / S / T, 292E / Y, 296E / L / R / V / W, 320D / K, 335E / H / K / L, 336S, or 339K / R, or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: The reference sequence of residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0144] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substituted or amino acid residues 33A, 38D, 71K, 73T, 75T, 96S, 98S, 101G, 114N, 117W, 136A, 156C, 179F, 184M, 191K, 196K, 221E, 289H, 292E, 296W, 320D, 335E / K, 336S, or 339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0145] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues T33A, T38D, L71K, A73T, D75T, F96S, Y98S, R101G, G114N, Q117W, T136A, F156C, L179F, A184M, S191K, E196K, F221E, K289H, G292E, G296W, N320D, R335E / K, P336S, or I339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0146] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid position 179, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution or amino acid residue 179F, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution L179F, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0147] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 33, 101, 221, 320, 335, 336, or 339, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least substitutions at 33A, 101G, 221E, 320D, 335E, 336S, or 339R, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least substitutions for T33A, R101G, F221E, N320D, R335E, P336S, or I339R, or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a set of substitutions at amino acid positions 33 / 101 / 221 / 320 / 335 / 336 / 339, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the set of substitutions or amino acid residues 33A / 101G / 221E / 320D / 335E / 336S / 339R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least the set of substitutions or amino acid residues T33A / R101G / F221E / N320D / R335E / P336S / I339R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0148] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 38, 71, 114, 184, 191, or 196, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least substitutions or amino acid residues 38D, 71K, 114N, 184M, 191K, or 196K, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least the substitutions or amino acid residues T38D, L71K, G114N, A184M, S191K, or E196K, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least the set of substitutions at amino acid positions 38 / 71 / 114 / 184 / 191 / 196, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions or amino acid residues 38D / 71K / 114N / 184M / 191K / 196K, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions or amino acid residues T38D / L71K / G114N / A184M / S191K / E196K or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0149] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid position 117, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution or amino acid residue 117W, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution Q117W, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0150] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid position 292, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution 292E, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution G292E, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0151] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid positions 75, 136, or 296, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residues 75T, 136A, or 296W, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residues D75T, T136A, or G296W, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions at amino acid positions 75 / 136 / 296 or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions or amino acid residues 75T / 136A / 296W or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions or amino acid residues D75T / T136A / G296W or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0152] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid position 98, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residue 98S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residue Y98S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0153] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid positions 73, 96, 98, 156, or 335, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residues 73T, 96S, 98Y, 156C, or 335K, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residues A73T, F96S, S98Y, F156C, or E335K, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions at amino acid positions 73 / 96 / 98 / 156 / 335 or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions or amino acid residues 73T / 96S / 98Y / 156C / 335K or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a set of substitutions or amino acid residues A73T / F96S / S98Y / F156C / E335K or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0154] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a substitution at amino acid position 289, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residue 289H, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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 includes at least a substitution or amino acid residue K289H, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0155] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 14, 32, 33, 38, 40, 57, 60, 71, 75, 82, 92, 96, 98, 101, 114, 141, 142, 145, 170, 171, 174, 179, 184, 191, 193, 196, 202, 205, 207, 209, 221, 255, 320, 327, 339, or 342, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0156] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least the substituted or amino acid residues 179F, 184P, 196K, 92A, 40E, 82I, 32R, 207T, 33K, 114K, 171E, 33A, 221E, 207S, 202W, 101G, 184R, 141C, 60R, 184L, 98R, 96S, 205V, 191K, 193 N, 141A, 320K, 174Q, 98D, 71K, 142R, 57T, 320D, 205R, 209L, 145D, 14R, 209E, 342I, 255E, 327Q, 170E, 141E, 98P, 339K, 38D, 142H, 75Q, 184M, 342T, or 339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0157] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least the substituted or amino acid residues L179F, A184P, E196K, T92A, V40E, V82I, Y32R, I207T, T33K, G114K, S171E, T33A, F221E, I207S, F202W, R101G, A184R, N141C, N60R, A184L, Y98R, F96S, N205V, S191K, and D193. N, N141A, N320K, M174Q, Y98D, L71K, T142R, E57T, N320D, N205R, D209L, T145D, K14R, D209E, V342I, P255E, V327Q, D170E, N141E, Y98P, I339K, T38D, T142H, D75Q, A184M, V342T, or I339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0158] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0159] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0160] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions or sets of substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2.

[0161] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0162] 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 higher sequence identity with a reference sequence, said reference sequence corresponding to substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein said amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0163] In some embodiments, the engineered RNA ligase comprises a reference sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 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 higher sequence identity.

[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%, 97%, 98%, 99% or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs in SEQ ID NOs: 4-220, 224-252, and 270-958.

[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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence is identical with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or ... The reference sequences 4, 106, 218, 286, 396, 436, 520, 552, or 582 contain one or more substitutions.

[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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4-220, 224-252 and 270-958. The reference sequences 4, 106, 218, 286, 396, 436, 520, 552, or 582 contain one or more substitutions.

[0167] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, 156, 167, 168, 170, 171, 17 Substitutions at 4, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ Reference sequences of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to reference sequences corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0168] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least the substituted or amino acid residues 11P, 12S, 14F / R / T / W, 16R, 17P, 23M, 25A / G / K / L / W, 26F / G / H / L / P / R / S / V, 30A / C / H / R, 32R, 33A / K, 38D, 40E, 52L, 57T, 60R, 68W, 69L, 71K, 73T, 75L / Q / T, 79W, 82I, 83P, 92A / E / G. / M / N / S, 93A, 95E / V, 96A / S, 98D / E / P / R / S / Y, 100R, 101G, 102T, 103I, 111L, 114F / G / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 118G / L / Q, 119A / E / S / T, 120P, 135Q / T, 136A, 141A / C / E / T, 142H / R, 145D, 147L, 15 6C / F / Y, 167A, 168A, 170E, 171E, 174Q, 177I / L, 179F / L, 183V, 184L / M / P / R, 185H / S, 188S, 189L / V, 190F, 191 K, 192V, 193N, 196K, 200S / T, 202W, 205R / V, 206R, 207S / T, 209E / L, 221E, 229A, 254G, 255E / S, 259V, 270A, 271 I, 287A, 288A, 289A / H / K / S / T, 292E / Y, 295R, 296E / L / R / V / W, 300E / K, 307A, 320D / K, 327I / Q / R / W, 332R, 333A / E / S / T, 334S, 335E / H / K / L, 336S, 337G, 339K / R, 342I / T, 343P, 339K / R or 342I / T or combinations thereof, wherein the amino acid position is relative to the corresponding SEQ Reference sequences of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to reference sequences corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0169] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 33, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0170] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 33A / K, 38D, 71K, 73T, 75L / Q / T, 96A / S, 98D / E / P / R / S / Y, 101G, 114F / G / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 136A, 156C / F / Y, 179F / L, 184L / M / P / R, 191K, 196K, 221E, 289A / H / K / S / T, 292E / Y, 296E / L / R / V / W, 320D / K, 335E / H / K / L, 336S, or 339K / R, or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: Reference sequences of residues 12 to 343 of 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to reference sequences corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0171] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substituted or amino acid residues 33A, 38D, 71K, 73T, 75T, 96S, 98S / Y, 101G, 114N, 117W, 136A, 156C, 179F, 184M, 191K, 196K, 221E, 289H, 292E, 296W, 320D, 335E / K, 336S, or 339R, or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0172] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4.

[0173] 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0174] In some embodiments, the engineered RNA ligase comprises amino acid positions 33 / 101 / 221 / 320 / 335 / 336 / 339, 71 / 92 / 171 / 184 / 202, 40 / 92 / 171 / 202 / 327, 196 / 300 / 335 / 339, 33 / 184 / 221 / 300 / 320, 335 / 336 / 339, 184 / 196 / 221 / 336, 33 / 184 / 320 / 335 / 336, 207 / 209 / 221 / 300 / 320 / 336 / 339, 101 / 196 / 221 / 300 / 335, 207 / 209 / 300 / 320, 40 / 170 / 171 / 202 / 327, 33 / 101 / 184 / 196 / 209 / 336 / 339, 40 / 71 / 92 / 171 / 184 / 327, 207 / 335 / 336 / 339, 33 / 101 / 196 / 300 / 320 / 335 / 336 / 339, 57 / 171 / 179 / 184 / 202 / 327, 57 / 92 / 202 / 327, 33 / 101 / 207 / 221 / 300 / 335 / 336 / 339, 33 / 221 / 336, 196 / 221 / 300 / 335 / 336 / 339, 33 / 184 / 221 / 336 / 339, 33 / 184 / 196 / 221 / 300, 101 / 196 / 20 7 / 209 / 221 / 300 / 336 / 339, 101 / 184 / 335 / 336 / 339, 33 / 184 / 196 / 320 / 336 / 339, 33 / 184 / 196 / 300 / 320 / 335 / 336 / 339, 184 / 320 / 335 / 336 / 339, 184 / 196 / 2 07 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 320 / 335 / 336 / 339, 33 / 196 / 209 / 221 / 339, 184 / 320 / 335 / 336 / 339, 101 / 209 / 335 / 336 / 339, 101 / 196 / 209 / 339, 40 / 71 / 82 / 170 / 171 / 179 / 202 / 327、320 / 335 / 336 / 339、101 / 184 / 300 / 335 / 336 / 339、33 / 101 / 196 / 209 / 335 / 336 / 339、33 / 196 / 300 / 320、221 / 335、33 / 184 / 19 6 / 207 / 221, 221 / 335 / 336 / 339, 196 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 300 / 320, 57 / 171 / 202 / 327, 40 / 71 / 171 / 184, 184 / 196 / 221, 33 / 196 / 207 / 335 / 336,The substitution sets at positions 32 / 57 / 71 / 202 / 327 / 342, 33 / 196 / 221 / 336, 101 / 221 / 300 / 320 / 335 / 336 / 339, 33 / 101 / 207 / 221, 101 / 184 / 196 / 209 / 221, 33 / 207 / 209 / 300 / 335 / 336 / 339, or 33 / 83 / 184 / 196 / 300 / 336, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0175] In some embodiments, the amino acid sequence of the engineered RNA ligase includes a set of substitutions or amino acid residues: 33A / 101G / 221E / 320D / 335E / 336S / 339R, 71K / 92A / 171E / 184P / 202W, 40E / 92A / 171E / 202W / 327Q, 196K / 300K / 335E / 339R, 33A / 184R / 221E / 300K / 320D, 335E / 336S / 339R, 184P / 196K / 221E / 336S, 33K / 184R / 320D / 335E / 336S, 207T / 209L / 221E / 300K / 320D / 336S / 3 39R, 101G / 196K / 221E / 300K / 335E, 207T / 209L / 300K / 320D, 40E / 170E / 171E / 202W / 327Q、33K / 101G / 184R / 196K / 209L / 336S / 339R、40E / 71K / 92A / 171E / 1 84P / 327Q, 207T / 335E / 336S / 339R, 33K / 101G / 196K / 300K / 320D / 335E / 336S / 339R、57T / 171E / 179L / 184P / 202W / 327Q、57T / 92A / 202W / 327Q、33K / 101G / 2 07T / 221E / 300K / 335E / 336S / 339R, 33K / 221E / 336S, 196K / 221E / 300K / 335E / 336S / 339R、33K / 184P / 221E / 336S / 339R、33A / 184P / 196K / 221E / 300K、101G / 196K / 207T / 209L / 221E / 300K / 336S / 339R、101G / 184R / 335E / 336S / 339R、3 3A / 184L / 196K / 320D / 336S / 339R, 33A / 184P / 196K / 300K / 320D / 335H / 336S / 3 39R、184L / 320D / 335E / 336S / 339R、184L / 196K / 207T / 209L / 300K / 335E / 336 S / 339R, 184R / 196K / 320D / 335E / 336S / 339R, 33K / 196K / 209L / 221E / 339R, 18 4R / 320D / 335E / 336S / 339R, 101G / 209L / 335E / 336S / 339R, 101G / 196K / 209L / 339R、40E / 71K / 82I / 170E / 171E / 179L / 202W / 327Q、320D / 335E / 336S / 339R、101G / 184L / 300K / 335E / 336S / 339R, 33K / 101G / 196K / 209L / 335E / 336S / 339 R, 33K / 196K / 300K / 320D, 221E / 335E, 33K / 184R / 196K / 207S / 221E, 221E / 33 5E / 336S / 339R, 196K / 209L / 300K / 335E / 336S / 339R, 184P / 196K / 300K / 320D , 57T / 171E / 202W / 327Q, 40E / 71K / 171E / 184P, 184L / 196K / 221E, 33A / 196K / 2 07S / 335E / 336S, 32R / 57T / 71K / 202W / 327Q / 342I, 33A / 196K / 221E / 336S, 101G / 221E / 300K / 320D / 335E / 336S / 339R, 33K / 184P / 320D / 335H / 336S, 33K / 101G / 207S / 221E, 101G / 184R / 196K / 209L / 221E, 33A / 207S / 209L / 300K / 335E / 336S / 339R, or 33K / 83P / 184P / 196K / 300K / 336S, or combinations thereof, wherein the amino acid position is relative to the corresponding SEQ The reference sequence of residues 12 to 343 of IDNO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0176] In some embodiments, the amino acid sequence of the engineered RNA ligase includes a set of substitutions or amino acid residues: T33A / R101G / F221E / N320D / R335E / P336S / I339R, L71K / T92A / S171E / A184P / F202W, V40E / T92A / S171E / F202W / V327Q, E196K / Q300K / R335E / I339R, T33A / A184R / F221E / Q300K / N320D, R335E / P336S / I339R, A184P / E196K / F221E / P336S, T33K / A184R / N320D / R335E / P336S, I207T / D209L / F221E / Q300K / N320D / P336S / I339R, R101G / E 196K / F221E / Q300K / R335E, I207T / D209L / Q300K / N320D, V40E / D170E / S17 1E / F202W / V327Q, T33K / R101G / A184R / E196K / D209L / P336S / I339R, V40E / L71K / T92A / S171E / A184P / V327Q, I207T / R335E / P336S / I339R, T33K / R101G / E196K / Q300K / N320D / R335E / P336S / I339R, E57T / S171E / F179L / A184P / F 202W / V327Q, E57T / T92A / F202W / V327Q, T33K / R101G / I207T / F221E / Q300K / R335E / P336S / I339R, T33K / F221E / P336S, E196K / F221E / Q300K / R335E / P 336S / I339R, T33K / A184P / F221E / P336S / I339R, T33A / A184P / E196K / F221E / Q300K, R101G / E196K / I207T / D209L / F221E / Q300K / P336S / I339R, R101G / A184R / R335E / P336S / I339R, T33A / A184L / E196K / N320D / P336S / I339R, T3 3A / A184P / E196K / Q300K / N320D / R335H / P336S / I339R, A184L / N320D / R335 E / P336S / I339R, A184L / E196K / I207T / D209L / Q300K / R335E / P336S / I339R,A184R / E196K / N320D / R335E / P336S / I339R, T33K / E196K / D209L / F221E / I339R, A184R / N320D / R335E / P336S / I339R, R101G / D209L / R335E / P336S / I339R, R101G / E196K / D209L / I339R, V40E / L71K / V82I / D170E / S171E / F179L / F202W / V327Q, N320D / R3 35E / P336S / I339R, R101G / A184L / Q300K / R335E / P336S / I339R, T33K / R101G / E196K / D209L / R335E / P336S / I339R, T33K / E19 6K / Q300K / N320D, F221E / R335E, T33K / A184R / E196K / I207S / F221E, F221E / R335E / P336S / I339R, E196K / D209L / Q300K / R33 5E / P336S / I339R, A184P / E196K / Q300K / N320D, E57T / S171E / F202W / V327Q, V40E / L71K / S171E / A184P, A184L / E196K / F221E , T33A / E196K / I207S / R335E / P336S, Y32R / E57T / L71K / F202W / V327Q / V342I, T33A / E196K / F221E / P336S, R101G / F221E / Q30 0K / N320D / R335E / P336S / I339R, T33K / A184P / N320D / R335H / P336S, T33K / R101G / I207S / F221E, R101G / A184R / E196K / D209L / F221E, T33A / I207S / D209L / Q300K / R335E / P336S / I339R or T33K / L83P / A184P / E196K / Q300K / P336S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0177] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO: 106, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO: 106.

[0178] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 218-220 and 224-252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0179] In some embodiments, the amino acid sequence of the engineered RNA ligase includes amino acid positions 38 / 71 / 114 / 184 / 191 / 196, 141 / 170 / 171 / 174, 114, 57 / 60 / 170 / 171 / 174 / 192 / 196 / 320, 60 / 92 / 170 / 171, 141 / 174 / 196, 57 / 170 / 171 / 174, 141 / 184, 57 / 60 The substitutions or sets of substitutions at / 141 / 170 / 171 / 174 / 192, 60 / 170 / 174, 141 / 170 / 171, 69 / 71 / 114 / 184, 38 / 40, 40 / 71 / 98 / 184 / 259, 32 / 60 / 141 / 170 / 171 / 174 / 196 / 320, 32 / 170 / 207 / 320 or 196, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0180] In some embodiments, the amino acid sequence of the engineered RNA ligase contains substitutions or sets of substitutions or amino acid residues such as 38D / 71K / 114N / 184M / 191K / 196K, 141A / 170E / 171E / 174Q, 114R, 57T / 60R / 170E / 171E / 174Q / 192V / 196K / 320K, 60R / 92A / 170E / 171E, 141A / 174Q / 196K, 57T / 170E / 171E / 174Q, 141T / 184P. 57T / 60R / 141A / 170E / 171E / 174Q / 192V, 60R / 170E / 174Q, 141C / 170E / 171E, 69L / 71K / 114N / 184M, 38D / 40E, 40E / 71K / 98R / 184M / 259V, 32R / 60R / 141A / 170E / 171E / 174Q / 196K / 320K, 32R / 170E / 207T / 320K or 196K, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0181] In some embodiments, the amino acid sequence of the engineered RNA ligase contains substitutions or sets of substitutions or amino acid residues: T38D / L71K / G114N / A184M / S191K / E196K, N141A / D170E / S171E / M174Q, G114R, E57T / N60R / D170E / S171E / M174Q / E192V / E196K / D320K, N60R / T92A / D170E / S171E, N141A / M174Q / E196K, E57T / D170E / S171E / M174Q, N141T / A184P, E5 7T / N60R / N141A / D170E / S171E / M174Q / E192V, N60R / D170E / M174Q, N141C / D170E / S171E, P69L / L71K / G114N / A184M, T38D / V40E, V40E / L71K / Y98R / A184M / I259V, Y32R / N60R / N141A / D170E / S171E / M174Q / E196K / D320K, Y32R / D170E / I207T / D320K or E196K, wherein the amino acid position is relative to the corresponding SEQ Reference sequence of residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0182] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO: 218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO: 218.

[0183] 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%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 270-312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 218 or relative to the reference sequence corresponding to SEQ ID NO: 218.

[0184] In some embodiments, the amino acid sequence of the engineered RNA ligase includes a set of substitutions or substitutions at amino acid positions 17, 190, 16, 68, 23, 270, 117 / 229, or 117, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to a reference sequence corresponding to SEQ ID NO: 218.

[0185] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises substitutions or sets of substitutions or amino acid residues 102T, 17P, 190F, 16R, 68W, 23M, 270A, 117K / 229A, 117W, 117T, 117H, 117A, 117M, 117G, 117I, 117K, 117R, 117S, 117V, 117F, 117C, or 117L, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to a reference sequence corresponding to SEQ ID NO: 218.

[0186] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises substitutions or sets of substitutions or amino acid residues A102T, S17P, A190F, Y16R, G68W, Y23M, Y270A, Q117K / T229A, Q117W, Q117T, Q117H, Q117A, Q117M, Q117G, Q117I, Q117K, Q117R, Q117S, Q117V, Q117F, Q117C, or Q117L, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to a reference sequence corresponding to SEQ ID NO: 218.

[0187] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO: 286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO: 286.

[0188] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 286 or the reference sequence corresponding to SEQ ID NO: 286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or relative to the reference sequence corresponding to SEQ ID NO: 286.

[0189] In some embodiments, the amino acid sequence of the engineered RNA ligase includes substitutions or sets of substitutions at amino acid positions 296, 114 / 117, 26, 30, 117 / 118, 117 / 119, 14, 183, 117, 185, 307, 12, 93 / 135, 135, 92, 288, 117 / 120, 79, 100, 287, 292, 25, 75, 327, or 111, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286, or relative to a reference sequence corresponding to SEQ ID NO: 286.

[0190] In some embodiments, the amino acid sequence of the engineered RNA ligase includes substitutions or sets of substitutions or amino acid residues 296E, 114V / 117K, 26S, 30C, 117K / 118G, 114P / 117K, 114S / 117K, 296L, 117K / 119S, 26P, 14F, 114G / 117K, 114F / 117K, 117K / 119A, 183V, 26R, 117K, 296R, 185S, 26F, 117K / 118L, 307A, 30R, 1 2S, 93A / 135T, 117K / 118Q, 185H, 135Q, 92M, 288A, 92N, 14W, 117K / 119T, 92S, 92G, 117K / 120P, 79W, 100R, 287A, 117K / 119E, 296V, 292E, 26H, 296W, 30H, 26G, 30A, 25W, 25L, 26V, 26L, 92E, 14T, 75T, 25G, 327W, or 111L, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286, or relative to the reference sequence corresponding to SEQ ID NO: 286.

[0191] In some embodiments, the amino acid sequence of the engineered RNA ligase includes substitutions or sets of substitutions or amino acid residues such as G296E, N114V / W117K, K26S, K30C, W117K / K118G, N114P / W117K, N114S / W117K, G296L, W117K / G119S, K26P, K14F, N114G / W117K, N114F / W117K, W117K / G119A, L183V, K26R, W117K, G296R, A185S, K26F, W117K / K118L, S307A, K30R, M12S. V93A / N135T, W117K / K118Q, A185H, N135Q, T92M, P288A, T92N, K14W, W117K / G119T, T92S, T92G, W117K / V120P, Y79W, A100R, T287A, W117K / G119E, G296V, G292E, K26H, G296W, K30H, K26G, K30A, S25W, S25L, K26V, K26L, T92E, K14T, D75T, S25G, V327W or F111L, wherein the amino acid position is relative to the corresponding SEQ The reference sequence of residues 12 to 343 of SEQ ID NO:286, or relative to the reference sequence corresponding to SEQ ID NO:286.

[0192] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396.

[0193] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396.

[0194] In some embodiments, the amino acid sequence of the engineered RNA ligase includes amino acid positions 102 / 119, 75 / 102, 75 / 102 / 117, 75 / 119, 75 / 136 / 296, 75 / 327, 75 / 117 / 119, 119, 119 / 296, 14 / 102 / 119, 14 / 102 / 296, 14 / 75, 14 / 75 / 119, 14 / 75 / 119 / 327, 14 / 75 / 117 / 119, The substitutions or sets of substitutions at 14 / 75 / 117 / 119 / 296, 14 / 119, 14 / 296, 14 / 30 / 270, 14 / 25, 14 / 117, 14 / 117 / 119, 14 / 117 / 119 / 296, 14 / 117 / 296 / 327, 26 / 75 / 327, 25 / 75, 25 / 30 / 102, 117, 117 / 119, or 117 / 119 / 296, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

[0195] In some embodiments, the amino acid sequence of the engineered RNA ligase includes substitutions or sets of substitutions or amino acid residues such as 102T / 119E, 75T / 102T, 75T / 102T / 117M, 75T / 119E, 75T / 136A / 296W, 75T / 327W, 75T / 117K / 119E, 119E, 119E / 296W, 14T / 102T / 119E, 14T / 102T / 296W, 14T / 75T, 14T / 75T / 119E, 14T / 75T / 119E / 327W, 14T / 75T / 117M / 119E, 1 4T / 75T / 117M / 119E / 296W, 14T / 119E, 14T / 296W, 14T / 30H / 270A, 14T / 25G, 14T / 117K, 14T / 117K / 119E, 14T / 117M / 119E, 14T / 117M / 119E / 296W, 14T / 117M / 296W / 327W, 26V / 75T / 327W, 25L / 75T, 25W / 30H / 102T, 117M, 117M / 119E, or 117M / 119E / 296W, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

[0196] In some embodiments, the amino acid sequence of the engineered RNA ligase includes substitutions or sets of substitutions or amino acid residues: A102T / G119E, D75T / A102T, D75T / A102T / W117M, D75T / G119E, D75T / T136A / G296W, D75T / V327W, D75T / W117K / G119E, G119E, G119E / G296W, K14T / A102T / G119E, K14T / A102T / G296W, K14T / D75T, K14T / D75T / G119E, K14T / D75T / G119E / V327W, K14T / D75T / W117M / G119E, K14T / D75T / W117M / G119E / G296W, K14T / G119E, K14T / G296W, K14T / K30H / Y270A, K14 T / S25G, K14T / W117K, K14T / W117K / G119E, K14T / W117M / G119E, K14T / W117M / G1 19E / G296W, K14T / W117M / G296W / V327W, K26V / D75T / V327W, S25L / D75T, S25W / K30H / A102T, W117M, W117M / G119E or W117M / G119E / G296W, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

[0197] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436.

[0198] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 436 or the reference sequence corresponding to SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or relative to the reference sequence corresponding to SEQ ID NO: 436.

[0199] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least a set of substitutions or substitutions at amino acid positions 332, 156, 177, 333, 96, 11, 189, 327, 25, 73, 98, 335, 95, 188, or 52, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to a reference sequence corresponding to SEQ ID NO: 436.

[0200] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substituted or amino acid residues 332R, 156C, 177L, 333A, 96A, 11P, 156Y, 189L, 177I, 327I, 333T, 189V, 25K, 73T, 333S, 98S, 335L, 327R, 95E, 188S, 98E, 52L, or 335K or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to the reference sequence corresponding to SEQ ID NO: 436.

[0201] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues D332R, F156C, N177L, V333A, F96A, G11P, F156Y, T189L, N177I, V327I, V333T, T189V, S25K, A73T, V333S, Y98S, E335L, V327R, K95E, A188S, Y98E, F52L, or E335K or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to the reference sequence corresponding to SEQ ID NO: 436.

[0202] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520.

[0203] 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%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520.

[0204] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the set of substitutions at amino acid positions 96 / 98 / 156 / 189 / 335, 73 / 96 / 98 / 189 / 335, 73 / 177 / 189, 73 / 98 / 177 / 189 / 335, 25 / 73 / 95 / 96 / 98 / 189 / 327, 25 / 98 / 189, 73 / 95 / 96 / 156 / 177, 73 / 335, 73 / 96 / 98 / 156 / 335, or 73 / 177 / 189 / 333, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0205] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least the set of substitutions or amino acid residues 96S / 98Y / 156C / 189V / 335K, 73T / 96S / 98E / 189L / 335K, 73T / 177L / 189V, 73T / 98E / 177L / 189V / 335K, 25A / 73T / 95E / 96A / 98E / 189V / 327I, 25K / 98Y / 189V, 73T / 95E / 96S / 156Y / 177L, 73T / 335K, 73T / 96S / 98Y / 156C / 335K, or 73T / 177L / 189V / 333T, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520, or relative to the sequence corresponding to SEQ ID NO: 520. Reference sequence for ID NO: 520.

[0206] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least a set of substitutions or amino acid residues: F96S / S98Y / F156C / T189V / E335K, A73T / F96S / S98E / T189L / E335K, A73T / N177L / T189V, A73T / S98E / N177L / T189V / E335K, S25A / A73T / K. 95E / F96A / S98E / T189V / V327I, S25K / S98Y / T189V, A73T / K95E / F96S / F156Y / N177L, A73T / E335K, A73T / F96S / S98Y / F156C / E335K or A73T / N177L / T189V / V333T, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0207] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552.

[0208] 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%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552.

[0209] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least substitutions at amino acid positions 292, 147, 255, 295, 75, 300, 333, 200, 289, 95, or 168, or combinations thereof, wherein said amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to a reference sequence corresponding to SEQ ID NO: 552.

[0210] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substituted or amino acid residues 292Y, 147L, 255S, 295R, 75L, 300E, 333E, 200S, 289S, 95V, 289A, 289T, 168A, 289H, or 200T, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to the reference sequence corresponding to SEQ ID NO: 552.

[0211] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substituted or amino acid residues E292Y, Y147L, P255S, M295R, T75L, Q300E, V333E, C200S, K289S, K95V, K289A, K289T, T168A, K289H, or C200T, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to the reference sequence corresponding to SEQ ID NO: 552.

[0212] 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 higher sequence identity with the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582.

[0213] 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%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0214] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 95 / 156 / 337, 189 / 200 / 271, 200 / 271 / 289 / 337, 200 / 271 / 289, 189 / 200, 156 / 200 / 289, 156 / 168 / 189 / 271 / 300, 156 / 189 / 200 / 289, 156 / 168 / 289, 95 / 156 / 271 / 289, 156 / 333, 95 / 200 / 289 / 337, 168 / 200 / 289, 189 / 200 / 289, 333 / 337, 103 / 156 / 271, 95 / 156 / 200 / 30 0 / 333, 156 / 271 / 289 / 333, 95 / 156 / 271, 95 / 189 / 289, 95 / 156 / 168 / 189 / 271 / 289 / 300, 189 / 271 / 289, 95 / 168 / 271 / 300, 156 / 189 / 289 / 337, 200 / 271, 15 6 / 189 / 333, 189 / 289, 156 / 189 / 200 / 300 / 333 / 337, 156 / 189 / 200 / 271 / 289 / 337, 156 / 200 / 271 / 289 / 333 / 337, 168 / 271 / 300, 289 / 337, 156 / 168 / 337, 15 6 / 168 / 189 / 289, 156 / 271 / 289, 189 / 300, 156 / 289 / 300 / 337, 168 / 189 / 289, 95 / 156 / 168 / 300, 156 / 200 / 300 / 337, 289 / 333 / 337, 95 / 189 / 200 / 300 / 337 95 / 156 / 189 / 200 / 271 / 333, 200 / 271 / 289 / 300, 289 / 333, 189 / 271 / 300, 95 / 300, 95 / 189 / 333 / 337, 95 / 189 / 271 / 300, 95 / 189 / 200 / 289, 168, 156 / 200 / 2 71 / 289 / 337, 95 / 168 / 289, 156 / 289, 95 / 156 / 189 / 271 / 289, 156 / 168 / 189 / 200 / 289 / 333, 156 / 300 / 333 / 337, 95 / 156 / 189 / 200 / 271 / 337, 271, 156 / 271, 156 / 271 / 300 / 333, 95 / 168 / 200, 168 / 271, 95 / 200 / 333, 189 / 200 / 271 / 289 / 333, 95 / 168 / 189 / 333 / 337, 189, 189 / 333 / 337, 156 / 189 / 271 / 289 / 333, 289,95 / 156 / 200 / 271 / 289、289 / 300 / 333、95 / 189 / 289 / 337、95 / 156 / 168 / 189 / 289、271 / 289、333、168 / 337、156 / 189 / 271 / 289、95 / 156 / 189 / 337、156 / 189 / 271 / 300、156 / 168 / 289 / 333、168 / 189 / 289 / 333、95 / 333、333 / 343、156 / 189 / 289、95 / 289 / 300、95 / 156 / 189 / 200 / 300 / 333 / 337、189 / 200 / 271 / 289 / 337、95 / 156 / 168 / 189 / 289 / 337、95 / 289、156 / 289 / 333 / 337、156 / 289 / 337、337、168 / 289、95 / 156 / 300、95 / 156 / 289、95 / 189 / 271 / 333 / 337、95 / 168 / 300、95 / 156 / 168 / 271 / 333 / 337、95 / 189 / 289 / 333 / 337、156 / 168 / 200 / 289 / 334、95 / 156 / 168 / 189 / 271 / 289、156 / 189 / 200 / 289 / 337、200 / 289、200 / 289 / 333 / 337、95 / 168 / 189 / 200 / 289、168 / 200、156 / 168 / 271 / 289、189 / 206 / 289、156 / 200 / 289 / 333 / 335、156 / 200 / 289 / 333、189 / 200 / 289 / 333、189 / 289 / 335、189 / 254 / 289 / 333、167 / 189 / 206 / 289 / 333 / 335、156 / 200 / 333、156 / 200 / 289 / 335、156 / 189 / 289 / 335、335、156 / 167 / 189 / 289、156 / 189 / 200 / 289 / 335、200 / 289 / 333 / 335、156 / 189 / 200 / 289 / 333、254 / 333 / 335、189 / 254 / 333 / 335、189 / 335、156 / 189 / 254 / 289、156 / 289 / 333、289 / 335、189 / 200 / 254 / 289 / 333、156 / 189 / 289 / 333、25 / 156 / 189 / 289、156 / 189 / 206 / 289 / 333 / 335、289 / 333 / 335、156 / 254 / 289 / 333、156 / 289 / 333 / 335、254 / 289、200 / 289 / 333、156 / 189 / 200, 156 / 254 / 333 / 335, 156, 254 / 289 / 335, 156 / 206 / 289, 156 / 289 / 335, 206 / 289 / 333 / 335, 156 / 189 / 254 / 289 / 333, 156 / 254 / 289, 200 / 289 / 335, 156 / 189 / 2 The substitution set at 06 / 289, 156 / 189 / 200 / 206 / 254 / 289 / 333, 25 / 254 / 333, 189 / 289 / 333 / 335, 156 / 189 / 200 / 289 / 333 / 335, 254 / 289 / 333, or 156 / 200 / 333 / 335, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0215] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least substitutions or sets of substitutions or amino acid residues: 95V / 156Y / 337G, 189V / 200T / 271I, 200S / 271I / 289A / 337G, 200T / 271I / 289A, 189V / 200S, 156Y / 200S / 289T, 156F / 168A / 189V / 271I / 300E, 156Y / 189V / 200T / 289A, 156F / 168A / 289K, 95V / 156Y / 271I / 289K, 156F / 333E, 95V / 200T / 289K / 337G, 168A / 200S / 289A, 189V / 200T / 289K, 333E / 337G, 103I / 156F / 271I, 95V / 156F / 200S / 3 00E / 333E, 156F / 271I / 289T / 333E, 95V / 156F / 271I, 95V / 189V / 289K, 95V / 1 56Y / 168A / 189V / 271I / 289T / 300E, 189V / 271I / 289A, 95V / 168A / 271I / 300 E, 156Y / 189V / 289A / 337G, 200S / 271I, 156F / 189V / 333E, 189V / 289T, 156Y / 189V / 200T / 300E / 333E / 337G, 156Y / 200T / 289A, 156F / 189V / 200S / 271I / 2 89K / 337G, 156Y / 200S / 271I / 289A / 333E / 337G, 168A / 271I / 300E, 289T / 337 G, 156Y / 168A / 337G, 156F / 168A / 189V / 289T, 156Y / 271I / 289T, 189V / 300E ,156Y / 289T / 300E / 337G, 168A / 189V / 289T, 95V / 156F / 168A / 300E, 156Y / 20 0S / 300E / 337G, 289A / 333E / 337G, 95V / 189V / 200T / 300E / 337G, 95V / 156F / 189V / 200T / 271I / 333E, 200S / 271I / 289A / 300E, 289A / 333E, 189V / 271I / 30 0E, 95V / 300E, 95V / 189V / 333E / 337G, 95V / 189V / 271I / 300E, 95V / 189V / 20 0S / 289A, 168A, 156Y / 200S / 271I / 289K / 337G, 95V / 168A / 289T, 156F / 289A,95V / 156Y / 189V / 271I / 289K、156F / 168A / 189V / 200S / 289A / 333E、156F / 300 E / 333E / 337G、95V / 156F / 189V / 200S / 271I / 337G、156F / 189V / 200T / 289K、1 56Y / 289T、271I、156Y / 271I、156F / 271I / 300E / 333E、95V / 168A / 200S、189V / 200S / 289A、168A / 271I、95V / 200T / 333E、189V / 200S / 271I / 289A / 333E、95 V / 168A / 189V / 333E / 337G、189V、189V / 333E / 337G、156F / 189V / 271I / 289A / 333E、289A、95V / 156F / 200T / 271I / 289K、289A / 300E / 333E、95V / 189V / 289A / 337G、95V / 156F / 168A / 189V / 289A、271I / 289T、333E、168A / 337G、156Y / 28 9A、156Y / 189V / 271I / 289K、95V / 156F / 189V / 337G、189V / 289K、189V / 289A、1 56Y / 189V / 271I / 300E、156F / 168A / 289A / 333E、168A / 189V / 289K / 333E、95V / 333E、333E / 343P、156F / 189V / 289A、95V / 289A / 300E、95V / 156Y / 189V / 200 S / 300E / 333E / 337G、189V / 200T / 271I / 289K / 337G、95V / 156Y / 168A / 189V / 2 89K / 337G、156F / 289K、95V / 289A、156Y / 168A / 289A、156F / 289K / 333E / 337G 、156F / 289K / 337G、289A / 337G、337G、168A / 289A、95V / 156F / 300E、95V / 156 Y / 289K、95V / 189V / 271I / 333E / 337G、95V / 168A / 300E、95V / 156Y / 168A / 271 I / 333E / 337G、95V / 189V / 289A / 333E / 337G、156F / 168A / 200S / 289A / 334S、9 5V / 156F / 168A / 189V / 271I / 289T、156Y / 189V / 200T / 289T / 337G、156F / 271I、289K / 300E / 333E, 95V / 156Y / 271I, 200S / 289A, 200S / 289K / 333E / 337G, 95V / 168A / 189V / 200T / 289A, 156Y / 271I / 289T / 333E, 168A / 200S, 156F / 168A / 271I / 289A, 189L / 206R / 289S, 156F / 200S / 289S / 333E / 335E, 156F / 200S / 289S / 333E, 189V / 200S / 289K / 333E, 189L / 289K / 335E, 189V / 254G / 289S / 333 E、189L / 289S、167A / 189L / 206R / 289S / 333E / 335E、156F / 200S / 333E、189L / 200S / 289S、156Y / 200S / 289K / 335E、156F / 189L / 200S / 289K、156Y / 189L / 2 89K / 335E, 156Y / 200S / 289K / 333E, 335E, 156F / 200S / 289K / 333E, 156F / 167A / 189V / 289T, 156Y / 189L / 200S / 289S / 335E, 200S / 289S / 333E / 335E, 156Y / 189V / 200S / 289T / 333E, 254G / 333E / 335E, 189L / 254G / 333E / 335E, 289S, 189V / 289S, 189V / 335E, 156F / 289T, 156F / 189V / 254G / 289S, 156Y / 289K / 333 E、289S / 335E、189L / 200S / 254G / 289S / 333E、156F / 189L / 289S / 333E、156Y / 189V / 289S / 335E、25A / 156Y / 189L / 289S、156Y / 189V / 206R / 289K / 333E / 335 E, 289S / 333E / 335E, 189V / 200S / 289S, 156F / 254G / 289K / 333E, 156Y / 289T / 333E / 335E, 254G / 289T, 200S / 289T / 333E, 156Y / 189V / 200S, 156Y / 254G / 3 33E / 335E, 156F, 254G / 289K / 335E, 200S / 289T, 200S / 289S, 156Y / 206R / 289S, 156F / 289S / 335E, 206R / 289T / 333E / 335E, 156F / 189V / 254G / 289K / 333E156F / 254G / 289T, 200S / 289T / 335E, 156Y / 189V / 206R / 289S, 156Y / 189V / 200S / 206R / 254 G / 289T / 333E, 156Y / 333E, 156Y / 189L / 254G / 289T / 333E, 25A / 254G / 333E, 189L / 289S / 33 3E / 335E, 200S / 289K / 335E, 156Y / 189V / 289S, 156F / 189L / 200S / 289K / 333E / 335E, 289T, 156F / 200S / 289T / 335E, 254G / 289S / 333E, or 156F / 200S / 333E / 335E, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0216] In some embodiments, the amino acid sequence of the engineered RNA ligase contains at least substitutions or sets of substitutions or amino acid residues: K95V / C156Y / A337G, T189V / C200T / V271I, C200S / V271I / H289A / A337G, C200T / V271I / H289A, T189V / C200S, C156Y / C200S / H289T, C156F / T168A / T189V / V271I / Q300E, C156Y / T189V / C200T / H289A, C156F / T168A / H289K, K95V / C156Y / V271I / H289K, C156F / V3 33E, K95V / C200T / H289K / A337G, T168A / C200S / H289A, T189V / C200T / H289K , V333E / A337G, V103I / C156F / V271I, K95V / C156F / C200S / Q300E / V333E, C15 6F / V271I / H289T / V333E, K95V / C156F / V271I, K95V / T189V / H289K, K95V / C1 56Y / T168A / T189V / V271I / H289T / Q300E, T189V / V271I / H289A, K95V / T168A / V271I / Q300E, C156Y / T189V / H289A / A337G, C200S / V271I, C156F / T189V / V3 33E, T189V / H289T, C156Y / T189V / C200T / Q300E / V333E / A337G, C156Y / C200T / H289A, C156F / T189V / C200S / V271I / H289K / A337G, C156Y / C200S / V271I / H 289A / V333E / A337G, T168A / V271I / Q300E, H289T / A337G, C156Y / T168A / A337 G. C156F / T168A / T189V / H289T, C156Y / V271I / H289T, T189V / Q300E, C156Y / H289T / Q300E / A337G, T168A / T189V / H289T, K95V / C156F / T168A / Q300E, C156 Y / C200S / Q300E / A337G, H289A / V333E / A337G, K95V / T189V / C200T / Q300E / A3 37G, K95V / C156F / T189V / C200T / V271I / V333E, C200S / V271I / H289A / Q300E,H289A / V333E、T189V / V271I / Q300E、K95V / Q300E、K95V / T189V / V333E / A337G、K95V / T189V / V271I / Q300E、K95V / T189V / C200S / H289A、T168A、C156Y / C200S / V271I / H289K / A337G、K95V / T168A / H289T、C156F / H289A、K95V / C156Y / T189V / V271I / H289K、C156F / T168A / T189V / C200S / H289A / V333E、C156F / Q300E / V333E / A337G、K95V / C156F / T189V / C200S / V271I / A337G、C156F / T189V / C200T / H289K、C156Y / H289T、V271I、C156Y / V271I、C156F / V271I / Q300E / V333E、K95V / T168A / C200S、T189V / C200S / H289A、T168A / V271I、K95V / C200T / V333E、T189V / C200S / V271I / H289A / V333E、K95V / T168A / T189V / V333E / A337G、T189V、T189V / V333E / A337G、C156F / T189V / V271I / H289A / V333E、H289A、K95V / C156F / C200T / V271I / H289K、H289A / Q300E / V333E、K95V / T189V / H289A / A337G、K95V / C156F / T168A / T189V / H289A、V271I / H289T、V333E、T168A / A337G、C156Y / H289A、C156Y / T189V / V271I / H289K、K95V / C156F / T189V / A337G、T189V / H289K、T189V / H289A、C156Y / T189V / V271I / Q300E、C156F / T168A / H289A / V333E、T168A / T189V / H289K / V333E、K95V / V333E、V333E / S343P、C156F / T189V / H289A、K95V / H289A / Q300E、K95V / C156Y / T189V / C200S / Q300E / V333E / A337G、T189V / C200T / V271I / H289K / A337G、K95V / C156Y / T168A / T189V / H289K / A337G, C156F / H289K, K95V / H289A, C156Y / T168A / H289A, C156F / H289K / V333E / A337G, C156F / H289K / A337G, H289A / A337G、A337G、T168A / H289A、K95V / C156F / Q300E、K95V / C156Y / H289K、K9 5V / T189V / V271I / V333E / A337G、K95V / T168A / Q300E、K95V / C156Y / T168A / V 271I / V333E / A337G, K95V / T189V / H289A / V333E / A337G, C156F / T168A / C200S / H289A / L334S, K95V / C156F / T168A / T189V / V271I / H289T, C156Y / T189V / C200T / H289T / A337G、C156F / V271I、H289K / Q300E / V333E、K95V / C156Y / V2 71I、C200S / H289A、C200S / H289K / V333E / A337G、K95V / T168A / T189V / C200T / H289A、C156Y / V271I / H289T / V333E、T168A / C200S、C156F / T168A / V271I / H289A、T189L / V206R / H289S、C156F / C200S / H289S / V333E / K335E、C156F / C2 00S / H289S / V333E, T189V / C200S / H289K / V333E, T189L / H289K / K335E, T189V / V254G / H289S / V333E, T189L / H289S, G167A / T189L / V206R / H289S / V333E / K335E、C156F / C200S / V333E、T189L / C200S / H289S、C156Y / C200S / H289K / K335E、C156F / T189L / C200S / H289K、C156Y / T189L / H289K / K335E、C156Y / C2 00S / H289K / V333E, K335E, C156F / C200S / H289K / V333E, C156F / G167A / T189V / H289T, C156Y / T189L / C200S / H289S / K335E, C200S / H289S / V333E / K335EC156Y / T189V / C200S / H289T / V333E, V254G / V333E / K335E, T189L / V254G / V333E / K335E, H289S, T189V / H289S, T189V / K335E, C156F / H289T, C156 F / T189V / V254G / H289S, C156Y / H289K / V333E, H289S / K335E, T189L / C20 0S / V254G / H289S / V333E, C156F / T189L / H289S / V333E, C156Y / T189V / H2 89S / K335E, S25A / C156Y / T189L / H289S, C156Y / T189V / V206R / H289K / V3 33E / K335E, H289S / V333E / K335E, T189V / C200S / H289S, C156F / V254G / H 289K / V333E, C156Y / H289T / V333E / K335E, V254G / H289T, C200S / H289T / V333E, C156Y / T189V / C200S, C156Y / V254G / V333E / K335E, C156F, V254G / H289K / K335E, C200S / H289T, C200S / H289S, C156Y / V206R / H289S, C156F / H289S / K335E, V206R / H289T / V333E / K335E, C156F / T189V / V254G / H289 K / V333E, C156F / V254G / H289T, C200S / H289T / K335E, C156Y / T189V / V20 6R / H289S, C156Y / T189V / C200S / V206R / V254G / H289T / V333E, C156Y / V33 3E, C156Y / T189L / V254G / H289T / V333E, S25A / V254G / V333E, T189L / H289S / V333E / K335E, C200S / H289K / K335E, C156Y / T189V / H289S, C156F / T189L / C200S / H289K / V333E / K335E, H289T, C156F / C200S / H289T / K335E, V254G / H289S / V333E or C156F / C200S / V333E / K335E, wherein the amino acid position is relative to the corresponding SEQ The reference sequence of residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0217] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0218] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0219] In some embodiments, the amino acid sequence of the engineered RNA ligase includes at least the substitutions or sets of substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0220] In some embodiments, the amino acid sequence of the engineered RNA ligase comprises at least substitutions or sets of substitutions for the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0221] 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 higher sequence identity with a reference sequence, said reference sequence comprising substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein said amino acid positions are relative to residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to the reference sequence corresponding to SEQ ID NO: Reference sequences of 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0222] 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 higher sequence identity with residues 12 to 343 of the following sequence: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 1 34, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 224, 226, 228, 230, 232, 234, 236, 238, 240 242, 244, 246, 248, 250, 252, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 3 62, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 59 0, 592, 594, 596, 598, 600, 602, 604, 506, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 6 52, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 83 6, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 8 98, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, or 958.

[0223] In some embodiments, the engineered RNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a sequence corresponding to the following sequence: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 336, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 13 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246 248, 250, 252, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598 600, 602, 604, 506, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, or 958.

[0224] In some embodiments, the engineered RNA ligase comprises an amino acid sequence containing residues 12 to 343 of SEQ ID NOs with even numbers in SEQ ID NOs: 4-220, 224-252, and 270-958, or an amino acid sequence containing SEQ ID NOs with even numbers in SEQ ID NOs: 4-220, 224-252, and 270-958. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions.

[0225] In some embodiments, the engineered RNA ligase comprises an amino acid sequence containing residues 12 to 343 of the following sequence: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 336, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 14 6, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 270, 272, 274, 276, 278, 280, 282 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 506, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, or 958. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions.Nine or at most ten substitutions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions.

[0226] In some embodiments, the engineered RNA ligase comprises an amino acid sequence containing the following sequence: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 336, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 1 48, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 270, 272, 274, 276, 278, 280, 282, 284, 2 86, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 506, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 6 34, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 74 2, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956 or 958. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions.Nine or at most ten substitutions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions.

[0227] In some embodiments, the engineered RNA ligase comprises an amino acid sequence of residues 12 to 343 containing SEQ ID NO: 2, 106, 218, 286, 396, 436, 520, 552, or 582, or an amino acid sequence containing SEQ ID NO: 2, 106, 218, 286, 396, 436, 520, 552, or 582. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some implementations, the amino acid sequence of the engineered RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions.

[0228] In some embodiments, the engineered RNA ligases of this disclosure possess RNA ligase activity against single-stranded and / or double-stranded polynucleotide substrates. In some embodiments, the engineered RNA ligases possess RNA ligase activity and one or more improved or enhanced properties compared to a reference RNA ligase. Exemplary improved properties are provided in the embodiments.

[0229] In some embodiments, the engineered RNA ligase exhibits increased activity compared to a reference RNA ligase. In some embodiments, the engineered RNA ligase exhibits 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10 times or more of activity compared to a reference RNA ligase. In some embodiments, the assay conditions used to measure ligase activity are described in the examples. In some embodiments, the increase in activity occurs at a double-stranded nick formed by a double-stranded fragment having complementary ends and base pairs to form a double-stranded nick, particularly where at least one or both strands of the double-stranded fragment contain modified nucleotides.

[0230] In some embodiments, engineered RNA ligases exhibit increased product yields compared to reference RNA ligases. In some embodiments, engineered RNA ligases exhibit increased product yields for polynucleotide substrates having phosphate-thioester nucleoside internucleotide bonds compared to reference RNA ligases. In some embodiments, engineered RNA ligases exhibit increased product yields for polynucleotide substrates with 2'-modifications (e.g., 2'-O-methyl and / or 2'-fluorine) compared to reference RNA ligases.

[0231] Specifically, engineered RNA ligases exhibit increased activity for polynucleotide substrates with a 2'-modified sugar moiety on the 3' and / or 5' end nucleotides, wherein the 3' end nucleotide on the polynucleotide acceptor chain is ligated to the 5' end nucleotide of the polynucleotide donor chain substrate.

[0232] In some embodiments, engineered RNA ligases exhibit ligation efficiencies of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher on double-stranded ligase substrates to polynucleotide receptor substrates containing a modified nucleoside at the 3' terminal nucleoside of the polynucleotide receptor chain. In some embodiments, engineered RNA ligases exhibit ligation efficiencies of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher on polynucleotide receptor chains having a modified 3' terminal nucleoside of 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-fluorocytidine, 2'-fluorouridine, 2'-fluorouridine, 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, or 2'-O-methyluridine.

[0233] In some embodiments, the engineered RNA ligase exhibits an efficiency of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher on a polynucleotide donor substrate, wherein the polynucleotide donor substrate contains a modified nucleoside at the 5' end of the polynucleotide acceptor chain. In some implementations, engineered RNA ligases exhibit ligation efficiencies of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher for polynucleotide donor chains containing modified 5'-terminal nucleosides of 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-fluorocytidine, 2'-fluorouridine, 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine or 2'-O-methyluridine.

[0234] In some embodiments, engineered RNA ligases exhibit ligation efficiencies of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher on polynucleotide substrates containing a modified nucleoside at the 3' end of the polynucleotide acceptor chain. In some embodiments, engineered RNA ligases exhibit ligation efficiencies of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher for ligation of substrates containing the following: A polynucleotide acceptor chain with a modified 3'-terminal nucleoside of 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-uridine, 2'-O-methyl-adenosine, 2'-O-methyl-guanosine, 2'-O-methyl-cytidine or 2'-O-methyl-uridine; A polynucleotide donor chain having a modified 5'-terminal nucleoside of 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-fluorocytidine, 2'-fluorouridine, 2'-fluorouridine, 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, or 2'-O-methyluridine; and A polynucleotide complementary to a polynucleotide acceptor chain and a polynucleotide donor chain, wherein the polynucleotide complementary to the polynucleotide acceptor chain and the polynucleotide donor chain contains a complementary 2'-fluorine or 2'-O-methyl nucleotide at a position complementary to the 3' terminal nucleoside of the polynucleotide acceptor chain, and / or contains a complementary 2-fluorine or 2-O-methyl nucleotide at a position complementary to the 5' terminal nucleoside of the polynucleotide donor chain.

[0235] In some embodiments, the reference RNA ligase used for comparing RNA ligase properties has a sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or a sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582. In some embodiments, the reference RNA ligase has a sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or a sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0236] In some embodiments, the engineered RNA ligase, compared to a reference RNA ligase, has one or more improved properties selected from: i) increased activity, ii) increased product yield, iii) increased product yield for polynucleotides having a phosphate thioester nucleoside internucleotide bond, iv) increased product yield for oligonucleotides having a 2'-modification, and v) increased expression, or any combination of i), ii), iii), iv), and v). In some embodiments, the reference RNA ligase has a sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or a sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582. In some embodiments, the reference RNA ligase has a sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0237] In some embodiments, the engineered RNA ligase is expressed as a fusion protein. In some embodiments, the engineered RNA ligase described herein can be fused to a variety of polypeptide sequences, such as, but not limited to, polypeptide tags that can be used for detection and / or purification. In some embodiments, the fusion protein of the engineered RNA ligase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion protein of the engineered RNA ligase comprises polylysine, for example, 2-12 consecutive lysine residues. In some embodiments, the fusion protein of the engineered RNA ligase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered RNA ligase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, it is fused to the amino (N-) terminus of the engineered RNA ligase polypeptide. In some embodiments, it is fused to the carboxyl (C-) terminus of the engineered RNA ligase polypeptide.

[0238] In some embodiments, this disclosure also provides functional or bioactive fragments of the engineered RNA ligase polypeptide described herein. Therefore, for each and every embodiment of the engineered RNA ligase herein, a functional or bioactive fragment of the engineered RNA ligase is provided herein. In some embodiments, the functional or bioactive fragment of the engineered RNA ligase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the RNA ligase polypeptide from which it is derived (i.e., the RNA-parent ligase). In some embodiments, the functional or bioactive fragment comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parental sequence of the RNA ligase. In some embodiments, the functional fragment is truncated to less than 5, 10, 15, 25, 30, or 35 amino acids.

[0239] In some embodiments, the functional or bioactive fragment of the engineered RNA ligase polypeptide described herein includes at least a mutation or set of mutations in the amino acid sequence of the engineered RNA ligase described herein. Therefore, in some embodiments, the functional or bioactive fragment of the engineered RNA ligase exhibits enhanced or improved properties associated with the mutation or set of mutations in the parental RNA ligase.

[0240] In some embodiments, the engineered RNA ligase is purified, as described herein. In some embodiments, the purified preparation has an engineered RNA ligase comprising at least 60%, 70%, 80%, 85%, 90%, or 95% or higher of the protein content of the preparation.

[0241] In some embodiments, the engineered RNA ligase described herein is provided as immobilized on a substrate or supporting medium such as a solid substrate, a porous substrate, a membrane, or particles. The polypeptide may be encapsulated in a matrix or membrane. In some embodiments, the matrix comprises polymeric materials such as calcium alginate, agar, k-carrageenan, polyacrylamide, and collagen. In some embodiments, solid matrices particularly include activated carbon, porous ceramics, and diatomaceous earth. In some embodiments, the matrix is ​​a particle, membrane, or fiber. Types of membranes particularly include nylon, cellulose, polysulfone, or polyacrylate.

[0242] In some embodiments, engineered RNA ligases are immobilized on the surface of a support material. In some embodiments, peptides are adsorbed onto the support material. In some embodiments, peptides are immobilized on the support material via covalent attachment. Support materials include, in particular, inorganic materials such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion exchange resins such as Amberlite, Sephadex, and Dowex.

[0243] Polynucleotides encoding engineered peptides, expression vectors, and host cells In another aspect, this disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered RNA ligase described herein. In some embodiments, the recombinant polynucleotide is operatively linked to one or more control sequences to produce a recombinant polynucleotide construct capable of expressing the engineered RNA ligase. In some embodiments, an expression construct encoding an engineered RNA ligase polypeptide is introduced into a suitable host cell to express the encoded RNA ligase polypeptide.

[0244] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide a description of all polynucleotides capable of encoding the subject RNA ligase polypeptide. The degeneracy of the genetic code (where the same amino acid is encoded by substitutional or synonymous codons) allows for the preparation of extremely large quantities of nucleic acids, all of which encode the engineered RNA ligase of this disclosure. Therefore, this disclosure provides methods and compositions for producing each and every possible variant of the polynucleotide encoding the engineered RNA ligase polypeptide described herein, which can be prepared by selecting combinations based on possible codon selection, and all such variants are considered to be specifically disclosed for any polypeptide described herein, including the examples (e.g., Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12) and the amino acid sequences presented in the sequence listing.

[0245] In some embodiments, the codons are preferably optimized for use by the selected host cell in protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered RNA ligase polypeptide described herein contains preferred codons at approximately 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0246] Therefore, by way of example and not limitation, in some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising a reference sequence of residues 12 to 343 of SEQ ID NO. corresponding to even-numbered numbers in SEQ ID NO: 2-220, 224-252, and 270-958, or a reference sequence corresponding to even-numbered numbers in SEQ ID NO: 2-220, 224-252, and 270-958, having an amino acid sequence identity of 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 higher, wherein the amino acid sequence is relative to the sequence corresponding to SEQ ID NO: The reference sequence of residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 or the reference sequence containing one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 97%, 98%, or 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582. The reference sequence of residues 12 to 343 of 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 or the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contains one or more substitutions.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or with respect 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 343 of SEQ ID NO: 2 or with respect to the reference sequence corresponding to SEQ ID NO: 2.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or with respect 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 343 of SEQ ID NO: 2 or with respect to the reference sequence corresponding to SEQ ID NO: 2.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 4-220, 224-252, and 270-958, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, and 156. Substitutions at residues 167, 168, 170, 171, 174, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least substitutions at amino acid positions 33, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least one substitution at amino acid positions 14, 32, 33, 38, 40, 57, 60, 71, 75, 82, 92, 96, 98, 101, 114, 141, 142, 145, 170, 171, 174, 179, 184, 191, 193, 196, 202, 205, 207, 209, 221, 255, 320, 327, 339, or 342, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the substitutions or sets of substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0258] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least a substitution or set of substitutions for the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 97%, 98%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs in SEQ ID NOs: 4-220, 224-252, and 270-958.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 97%, 98%, or 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582. The reference sequence of residues 12 to 343 of 4, 106, 218, 286, 396, 436, 520, 552 or 582 or the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582 contains one or more substitutions.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising a reference 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4-220, 224-252 and 270-958. Reference sequences NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

[0263] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, and 156. Substitution at SEQ ID NO: 167, 168, 170, 171, 174, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: 167, 168, 170, 171, 174, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 34 Reference sequences of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to reference sequences corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence, the amino acid sequence comprising at least the amino acid positions 33 / 101 / 221 / 320 / 335 / 336 / 339, 71 / 92 / 171 / 184 / 202, 40 / 92 / 171 / 202 / 327, 196 / 300 / 335 / 339, 33 / 184 / 221 / 300 / 320, 335 / 336 / 339, 184 / 196 / 221 / 336, 33 / 184 / 320 / 335 / 336, 207 / 209 / 221 / 300 / 320 / 336 / 339, 1 01 / 196 / 221 / 300 / 335, 207 / 209 / 300 / 320, 40 / 170 / 171 / 202 / 327, 33 / 101 / 184 / 196 / 209 / 336 / 339, 40 / 71 / 92 / 171 / 184 / 327, 207 / 335 / 336 / 339, 33 / 10 1 / 196 / 300 / 320 / 335 / 336 / 339, 57 / 171 / 179 / 184 / 202 / 327, 57 / 92 / 202 / 327, 33 / 101 / 207 / 221 / 300 / 335 / 336 / 339, 33 / 221 / 336, 196 / 221 / 300 / 335 / 336 / 339, 33 / 184 / 221 / 336 / 339, 33 / 184 / 196 / 221 / 300, 101 / 196 / 207 / 209 / 221 / 300 / 336 / 339, 101 / 184 / 335 / 336 / 339, 33 / 184 / 196 / 320 / 336 / 339, 33 / 18 4 / 196 / 300 / 320 / 335 / 336 / 339, 184 / 320 / 335 / 336 / 339, 184 / 196 / 207 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 320 / 335 / 336 / 339, 33 / 196 / 209 / 221 / 339, 184 / 320 / 335 / 336 / 339, 101 / 209 / 335 / 336 / 339, 101 / 196 / 209 / 339, 40 / 71 / 82 / 170 / 171 / 179 / 202 / 327, 320 / 335 / 336 / 339, 101 / 184 / 300 / 335 / 336 / 339, 3 3 / 101 / 196 / 209 / 335 / 336 / 339, 33 / 196 / 300 / 320, 221 / 335, 33 / 184 / 196 / 207 / 221, 221 / 335 / 336 / 339, 196 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 300 / 320,The substitution set at 57 / 171 / 202 / 327, 40 / 71 / 171 / 184, 184 / 196 / 221, 33 / 196 / 207 / 335 / 336, 32 / 57 / 71 / 202 / 327 / 342, 33 / 196 / 221 / 336, 101 / 221 / 300 / 320 / 335 / 336 / 339, 33 / 101 / 207 / 221, 101 / 184 / 196 / 209 / 221, 33 / 207 / 209 / 300 / 335 / 336 / 339 or 33 / 83 / 184 / 196 / 300 / 336, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, or relative to the sequence corresponding to SEQ ID NO: Reference sequence for 4.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO: 106, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO: 106.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered SEQ ID NOs in SEQ ID NOs: 218-220 and 224-252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the amino acid positions 38 / 71 / 114 / 184 / 191 / 196, 141 / 170 / 171 / 174, 114, 57 / 60 / 170 / 171 / 174 / 192 / 196 / 320, 60 / 92 / 170 / 171, 141 / 174 / 196, 57 / 170 / The substitutions or sets of substitutions at positions 171 / 174, 141 / 184, 57 / 60 / 141 / 170 / 171 / 174 / 192, 60 / 170 / 174, 141 / 170 / 171, 69 / 71 / 114 / 184, 38 / 40, 40 / 71 / 98 / 184 / 259, 32 / 60 / 141 / 170 / 171 / 174 / 196 / 320, 32 / 170 / 207 / 320 or 196, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO: 106.

[0270] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO: 218, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO: 218.

[0271] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 270-312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 218 or relative to the reference sequence corresponding to SEQ ID NO: 218.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least one substitution or set of substitutions at amino acid positions 102, 17, 190, 16, 68, 23, 270, 117 / 229, or 117, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to a reference sequence corresponding to SEQ ID NO: 218.

[0273] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO: 286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO: 286.

[0274] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or relative to the reference sequence corresponding to SEQ ID NO: 286.

[0275] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least one of the following amino acid positions: 296, 114 / 117, 26, 30, 117 / 118, 117 / 119, 14, 183, 117, 185, 307, 12, 93 / 135, 135, 92, 288, 117 / 120, 79, 100, 287, 292, 25, 75, 327, or 111, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286, or relative to a reference sequence corresponding to SEQ ID NO: 286.

[0276] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO: 396.

[0277] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 428-488, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 396 or relative to the reference sequence corresponding to SEQ ID NO: 396.

[0278] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the amino acid positions 102 / 119, 75 / 102, 75 / 102 / 117, 75 / 119, 75 / 136 / 296, 75 / 327, 75 / 117 / 119, 119, 119 / 296, 14 / 102 / 119, 14 / 102 / 296, 14 / 75, 14 / 75 / 119, and 14 / 75 / 119. The substitutions or sets of substitutions at 9 / 327, 14 / 75 / 117 / 119, 14 / 75 / 117 / 119 / 296, 14 / 119, 14 / 296, 14 / 30 / 270, 14 / 25, 14 / 117, 14 / 117 / 119, 14 / 117 / 119 / 296, 14 / 117 / 296 / 327, 26 / 75 / 327, 25 / 75, 25 / 30 / 102, 117, 117 / 119, or 117 / 119 / 296, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO: 396.

[0279] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436.

[0280] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or relative to the reference sequence corresponding to SEQ ID NO: 436.

[0281] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least substitutions at amino acid positions 332, 156, 177, 333, 96, 11, 189, 327, 25, 73, 98, 335, 95, 188, or 52, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to a reference sequence corresponding to SEQ ID NO: 436.

[0282] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO: 520.

[0283] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 536-554, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0284] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the set of substitutions at amino acid positions 96 / 98 / 156 / 189 / 335, 73 / 96 / 98 / 189 / 335, 73 / 177 / 189, 73 / 98 / 177 / 189 / 335, 25 / 73 / 95 / 96 / 98 / 189 / 327, 25 / 98 / 189, 73 / 95 / 96 / 156 / 177, 73 / 335, 73 / 96 / 98 / 156 / 335, or 73 / 177 / 189 / 333, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520, or relative to a reference sequence corresponding to SEQ ID NO: 520.

[0285] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552.

[0286] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or relative to the reference sequence corresponding to SEQ ID NO: 552.

[0287] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least substitutions at amino acid positions 292, 147, 255, 295, 75, 300, 333, 200, 289, 95, or 168, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to a reference sequence corresponding to SEQ ID NO: 552.

[0288] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582.

[0289] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582.

[0290] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence at least contained in the amino acid positions 95 / 156 / 337, 189 / 200 / 271, 200 / 271 / 289 / 337, 200 / 271 / 289, 189 / 200, 156 / 200 / 289, 156 / 168 / 189 / 271 / 300, 156 / 189 / 200 / 289, 156 / 168 / 289, 95 / 156 / 271 / 289, 156 / 333, 95 / 200 / 289 / 337, 168 / 200 / 289, 189 / 200 / 289, 333 / 337, 103 / 156 / 271, 95 / 156 / 200 / 300 / 333, 156 / 271 / 289 / 333, 95 / 156 / 271, 95 / 189 / 289, 95 / 156 / 168 / 189 / 271 / 289 / 300, 189 / 271 / 289, 95 / 168 / 271 / 300, 156 / 189 / 289 / 337, 200 / 271, 156 / 189 / 333, 189 / 289, 156 / 189 / 200 / 300 / 333 / 337, 156 / 189 / 200 / 271 / 289 / 337, 156 / 200 / 271 / 289 / 333 / 3 37, 168 / 271 / 300, 289 / 337, 156 / 168 / 337, 156 / 168 / 189 / 289, 156 / 271 / 289, 189 / 300, 156 / 289 / 300 / 337, 168 / 189 / 289, 95 / 156 / 168 / 300, 156 / 200 / 30 0 / 337, 289 / 333 / 337, 95 / 189 / 200 / 300 / 337, 95 / 156 / 189 / 200 / 271 / 333, 200 / 271 / 289 / 300, 289 / 333, 189 / 271 / 300, 95 / 300, 95 / 189 / 333 / 337, 95 / 189 / 271 / 300, 95 / 189 / 200 / 289, 168, 156 / 200 / 271 / 289 / 337, 95 / 168 / 289, 156 / 289, 95 / 156 / 189 / 271 / 289, 156 / 168 / 189 / 200 / 289 / 333, 156 / 300 / 333 / 337 95 / 156 / 189 / 200 / 271 / 337, 271, 156 / 271, 156 / 271 / 300 / 333, 95 / 168 / 200, 168 / 271, 95 / 200 / 333, 189 / 200 / 271 / 289 / 333, 95 / 168 / 189 / 333 / 337, 189,189 / 333 / 337、156 / 189 / 271 / 289 / 333、289、95 / 156 / 200 / 271 / 289、289 / 300 / 333、95 / 189 / 289 / 337、95 / 156 / 168 / 189 / 289、271 / 289、333、168 / 337、156 / 189 / 271 / 289、95 / 156 / 189 / 337、156 / 189 / 271 / 300、156 / 168 / 289 / 333、168 / 189 / 289 / 333、95 / 333、333 / 343、156 / 189 / 289、95 / 289 / 300、95 / 156 / 189 / 200 / 300 / 333 / 337、189 / 200 / 271 / 289 / 337、95 / 156 / 168 / 189 / 289 / 337、95 / 289、156 / 289 / 333 / 337、156 / 289 / 337、337、168 / 289、95 / 156 / 300、95 / 156 / 289、95 / 189 / 271 / 333 / 337、95 / 168 / 300、95 / 156 / 168 / 271 / 333 / 337、95 / 189 / 289 / 333 / 337、156 / 168 / 200 / 289 / 334、95 / 156 / 168 / 189 / 271 / 289、156 / 189 / 200 / 289 / 337、200 / 289、200 / 289 / 333 / 337、95 / 168 / 189 / 200 / 289、168 / 200、156 / 168 / 271 / 289、189 / 206 / 289、156 / 200 / 289 / 333 / 335、156 / 200 / 289 / 333、189 / 200 / 289 / 333、189 / 289 / 335、189 / 254 / 289 / 333、167 / 189 / 206 / 289 / 333 / 335、156 / 200 / 333、156 / 200 / 289 / 335、156 / 189 / 289 / 335、335、156 / 167 / 189 / 289、156 / 189 / 200 / 289 / 335、200 / 289 / 333 / 335、156 / 189 / 200 / 289 / 333、254 / 333 / 335、189 / 254 / 333 / 335、189 / 335、156 / 189 / 254 / 289、156 / 289 / 333、289 / 335、189 / 200 / 254 / 289 / 333、156 / 189 / 289 / 333、25 / 156 / 189 / 289、156 / 189 / 206 / 289 / 333 / 335、289 / 333 / 335、156 / 254 / 289 / 333、156 / 289 / 333 / 335, 254 / 289, 200 / 289 / 333, 156 / 189 / 200, 156 / 254 / 333 / 335, 156, 254 / 289 / 335, 156 / 206 / 289, 156 / 289 / 335, 206 / 289 / 333 / 335, 156 / 189 / 254 / 289 / 333, 156 / 254 / 289, 200 / 2 Substitutions or sets of substitutions at positions 89 / 335, 156 / 189 / 206 / 289, 156 / 189 / 200 / 206 / 254 / 289 / 333, 25 / 254 / 333, 189 / 289 / 333 / 335, 156 / 189 / 200 / 289 / 333 / 335, 254 / 289 / 333, or 156 / 200 / 333 / 335, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO: 582.

[0291] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the substitutions at the amino acid positions shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0292] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0293] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least the substitutions or sets of substitutions at the amino acid positions shown in 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0294] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence comprising at least a substitution or set of substitutions for the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0295] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase 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 higher sequence identity with a reference sequence, wherein the reference sequence comprises substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to the reference sequence corresponding to SEQ ID NO: Reference sequences of 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582.

[0296] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising residues 12-343 of SEQ ID NOs (even-numbered in SEQ ID NOs: 4-220, 224-252, and 270-958), or an amino acid sequence containing SEQ ID NOs (even-numbered in SEQ ID NOs: 4-220, 224-252, and 270-958).

[0297] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence of residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or an amino acid sequence of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0298] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence having nucleotide residues 34 to 1029 of SEQ ID NO. corresponding to an odd number in SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or 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 higher sequence identity with the reference polynucleotide corresponding to SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, wherein the recombinant polynucleotide encodes an RNA ligase.

[0299] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence having nucleotide residues 34 to 1029 of SEQ ID NO. corresponding to odd-numbered numbers in SEQ ID NO: 3-219, 223-251, and 269-957, or a reference polynucleotide sequence corresponding to odd-numbered numbers in SEQ ID NO: 3-219, 223-251, and 269-957 having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, wherein the recombinant polynucleotide encodes an RNA ligase.

[0300] In some embodiments, the recombinant polynucleotide comprises a polynucleotide 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 higher sequence identity with a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1029 of the following sequence: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 1 33, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 23 7, 239, 241, 243, 245, 247, 249, 251, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461,463、465、467、469、471、473、475、477、479、481、483、485、487、489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、925、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955 or 957, wherein the recombinant polynucleotide encodes an RNA ligase.

[0301] In some embodiments, the recombinant polynucleotide comprises a polynucleotide 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 higher sequence identity with a reference sequence corresponding to the following sequence: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 1 35, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 24 1, 243, 245, 247, 249, 251, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 7 19, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 7 81, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 84 3, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, or 957, wherein the recombinant polynucleotide encodes an RNA ligase.

[0302] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 34 to 1029 of SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or a polynucleotide sequence containing SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581.

[0303] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 34 to 1029 of SEQ ID NO. with odd numbers in SEQ ID NO: 3-219, 223-251 and 269-957, or a polynucleotide sequence containing SEQ ID NO. with odd numbers in SEQ ID NO: 3-219, 223-251 and 269-957.

[0304] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 34 to 1029 of the following sequence: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 269, 271, 273, 275, 277, 279 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 6 27, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 73 7, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, or 957.

[0305] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing the following sequence: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523,525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 6 33, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 74 1, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, or 957.

[0306] In some embodiments, this disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions with a reference polynucleotide encoding an engineered RNA ligase polypeptide described herein, such as the recombinant polynucleotides or their reverse complementary sequences provided in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions with a reference polynucleotide corresponding to nucleotide residues 34 to 1029 of SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or with a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or their reverse complementary sequence. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions with reference polynucleotides of nucleotide residues 34 to 1029 corresponding to odd-numbered SEQ ID NOs in SEQ ID NOs: 3-219, 223-251, and 269-957, or with polynucleotide sequences containing odd-numbered SEQ ID NOs in SEQ ID NOs: 3-219, 223-251, and 269-957, or their reverse complementary sequences.

[0307] In some embodiments, this disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions with the reverse complementary sequence of a reference polynucleotide encoding an engineered RNA ligase polypeptide described herein, wherein the recombinant polynucleotide hybridizing under stringent conditions encodes an RNA ligase polypeptide comprising an amino acid sequence having one or more amino acid differences at residue positions selected from any of the positions shown in Tables 3, 4, 106, 218, 286, 396, 436, 520, 552, or 582 compared to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582. In some embodiments, a recombinant polynucleotide hybridizing under highly stringent conditions with the reverse complementary sequence of a reference polynucleotide encoding an engineered RNA ligase polypeptide described herein encodes an RNA ligase polypeptide having one or more amino acid differences present in an amino acid sequence having residues 12 to 343 of SEQ ID NO. corresponding to even numbers in SEQ ID NO: 4-220, 224-252, and 270-958, or in an engineered RNA ligase containing an amino acid sequence of SEQ ID NO. corresponding to even numbers in SEQ ID NO: 4-220, 224-252, and 270-958, wherein the amino acid difference is relative to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

[0308] In some embodiments, the recombinant polynucleotide hybridized under highly stringent conditions comprises a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1029 of SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or its reverse complementary 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 higher sequence identity. In some embodiments, the recombinant polynucleotide hybridized under highly stringent conditions comprises a reference polynucleotide sequence of nucleotide residues 34-1029 of SEQ ID NO. corresponding to odd-numbered SEQ ID NO. in SEQ ID NO: 3-219, 223-251 and 269-957, or a polynucleotide sequence of SEQ ID NO. corresponding to odd-numbered SEQ ID NO. in SEQ ID NO: 3-219, 223-251 and 269-957, or its reverse complementary 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 higher sequence identity.

[0309] In some other embodiments, the polynucleotide hybridized under highly stringent conditions comprises an inverse complementary sequence to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1029 of SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, or a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551, or 581, encoding an engineered RNA ligase polypeptide. In some other embodiments, the polynucleotide hybridized under highly stringent conditions comprises an inverse complementary sequence to a reference polynucleotide sequence of nucleotide residues 34-1029 of SEQ ID NO. corresponding to odd-numbered SEQ ID NO. in SEQ ID NO: 3-219, 223-251, and 269-957, or a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO. in SEQ ID NO: 3-219, 223-251, and 269-957, encoding an engineered RNA ligase polypeptide.

[0310] In some embodiments, recombinant polynucleotides encoding any of the RNA ligases provided herein are manipulated in a variety of ways to provide polypeptide expression. In some embodiments, the recombinant polynucleotide encoding the polypeptide is provided as an expression vector, wherein one or more control sequences are operatively linked to the recombinant polynucleotide to regulate the expression of the polynucleotide and / or the encoded polypeptide. In some embodiments, the control sequences particularly include promoter sequences, Kozak sequences, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, regulatory elements, and transcription terminators. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are known in the art.

[0311] In some implementations, a suitable promoter can be selected based on the host cell used. For bacterial host cells, suitable promoters for directing the transcription of the nucleic acid constructs of this disclosure include, but are not limited to, promoters derived from the *E. coli* *lac* operon, the *Streptomyces coelicolor* agarase gene (dagA), the *Bacillus subtilis* fructan sucrase gene (sacB), the *Bacillus licheniformis* α-amylase gene (amyL), the *Bacillus stearothermophilus* maltose amylase gene (amyM), the *Bacillus samyloliquefaciens* α-amylase gene (amyQ), the *Bacillus licheniformis* penicillinase gene (penP), the *Bacillus subtilis* xylA and xylB genes, and prokaryotic β-lactamase genes (see, for example, Villa-Kamaroff et al., *Proc. Natl Acad. Sci. USA, 1978,*). 75:3727-3731), and the tac promoter (see, for example, DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80: 21-25). Exemplary promoters for fungal host cells include, but are not limited to, promoters derived from the genes of Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamase, and Fusarium oxysporum trypsin-like protease (see, for example, WO 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters of the genes of Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase), as well as their mutant, truncated, and hybrid promoters. Exemplary yeast cell promoters can be derived from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphate glycerate kinase.Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for insect cells include, but are not limited to, polyhedroprotein, p10, ELT, OpIE2, and hr5 / ie1 promoters. Exemplary promoters for mammalian cells include, but are not limited to, promoters from cytomegalovirus (CMV), chicken β-actin fused with a CMV enhancer, simian vacuolar virus 40 (SV40), promoters from Homo sapiens phosphoglycerate kinase, β-actin, elongation factor-1a, or glyceraldehyde-3-phosphate dehydrogenase, or promoters from chicken β-actin.

[0312] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operatively linked to the 3' end of a nucleic acid sequence encoding a leucine decarboxylase polypeptide. Any suitable terminator that is functional in the selected host cell can be used in this invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that depends on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require the transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., ibid.). Exemplary terminators for mammalian cells include, but are not limited to, terminators derived from cytomegalovirus (CMV), simian virus 40 (SV40), human growth hormone hGH, bovine growth hormone BGH, and human or rabbit β-globulin.

[0313] In some embodiments, the control sequence is also a suitable leader sequence (i.e., the untranslated region of mRNA important for translation by the host cell). In some embodiments, the leader sequence is operatively linked to the 5' end of a nucleic acid sequence encoding an RNA ligase polypeptide. Any suitable leader sequence that is functional in the selected host cell can be used in this invention. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for yeast host cells are obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leader sequences for mammalian host cells include, but are not limited to, the 5'-UTR element present in orthopoxvirus mRNA.

[0314] In some embodiments, the control sequence includes a 3' untranslated nucleic acid region and a polyadenylated tail nucleic acid sequence, which are operatively linked to the 3' end of a protein-coding nucleic acid sequence to mediate binding to proteins involved in mRNA transport and translation, as well as mRNA half-life. Any polyadenylated sequence and 3' UTR that function in a selected host cell can be used in this invention. Exemplary polyadenylated sequences for filamentous fungal host cells include, but are not limited to, those from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylated sequences for yeast host cells are also known in the art (see, for example, Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3' UTR sequences in mammalian host cells include, but are not limited to, the 3'-UTR of α- and β-globin mRNAs, which contain several sequence elements that increase mRNA stability and translation.

[0315] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence linked to the amino terminus of a polypeptide and directing the encoded polypeptide into the cellular secretion pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in the translation reading frame to a segment encoding the coding region of the secretory polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is exogenous to the coding sequence. Any suitable signal peptide coding region directing the expressed polypeptide into the secretion pathway of a selected host cell can be used to express the engineered polypeptide. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from genes producing maltose amylase from Bacillus NCIB 11837, α-amylase from Bacillus stearothermophilus, subtilisin from Bacillus licheniformis, β-lactamase from Bacillus licheniformis, neutral proteases (nprT, nprS, nprM) from Bacillus stearothermophilus, and prsA from Bacillus subtilis. Other signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, the effective signal peptide coding regions of filamentous fungal host cells include, but are not limited to, signal peptide coding regions derived from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Rhizopus oryzae aspartic protease, Pythium spp. cellulase, and Pythium spp. lipase. Useful signal peptides of yeast host cells include, but are not limited to, those derived from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Useful signal peptides of mammalian host cells include, but are not limited to, those derived from the genes of immunoglobulin γ (IgG) and signal peptides in human secreted proteins (such as human β-galactosidase polypeptide).

[0316] In some implementations, the control sequence is a regulatory sequence that promotes the regulation of expression of recombinant polynucleotides and / or encoded polypeptides. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operon systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter. Exemplary inducible promoters regulated by exogenous factors include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible promoter, the mouse mammary tumor virus (MMTV) promoter, the ecdysone insect promoter, the tetracycline inducible promoter system, the RU486 inducible promoter system, and the rapamycin inducible promoter system.

[0317] In another aspect, this disclosure provides an expression vector comprising a recombinant polynucleotide encoding an RNA ligase polypeptide, wherein the recombinant polynucleotide is operatively or operatively linked to control sequences, such as promoters and terminators, origins of replication, etc., depending on the type of host to which they will be introduced. The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can readily undergo a recombinant DNA procedure and induce expression of the RNA ligase polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell to which it is to be introduced. The vector can be a linear or closed circular plasmid.

[0318] In some implementations, the expression vector is a self-replicating vector (i.e., a vector existing as an extrachromosomal entity whose replication does not depend on chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome). The vector may contain any means to ensure self-replication. In some alternative implementations, the vector may be one that, upon introduction into a host cell, is integrated into the genome and replicates along with the chromosome into which the vector is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used together containing the total DNA of the genome to be introduced into the host cell.

[0319] In one implementation, the recombinant polynucleotide can be provided on a non-replicating expression vector or plasmid. In some implementations, the non-replicating expression vector or plasmid may be based on a replication-defective viral vector (see, for example, Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0320] In some implementations, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A “selective marker” is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophic to auxotrophic traits, etc. Examples of bacterial selective markers include, but are not limited to, the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for host cells of filamentous fungi include, but are not limited to, amdS (acetamase; e.g., from Aspergillus nidulans or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinicotinic acetyltransferase; e.g., from Streptomyces hygromycin), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase; e.g., from Aspergillus nidulans or Aspergillus oryzae), sC (adenosyl sulfate transferase), and trpC (o-aminobenzoic acid synthase) and their equivalents.

[0321] In another aspect, this disclosure provides a host cell comprising at least one recombinant polynucleotide encoding an RNA ligase polypeptide of the present disclosure, said recombinant polynucleotide being operatively linked to one or more control sequences for expressing the RNA ligase polypeptide. In some embodiments, the host cell suitable for expressing the polypeptide encoded by the expression vector is a prokaryotic or eukaryotic cell known in the art, and includes, but is not limited to, bacterial cells such as *Escherichia coli*, *Vibrio fluvialis*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichiapastoris* (ATCC Registry No. 201178)); insect cells such as *Drosophila* S2 and *Spodoptera* Sf9 cells; animal (e.g., mammalian) cells such as CHO, COS, BHK, 293, and Bos melanoma cells; and plant cells. Exemplary host cells also include various strains of Escherichia coli (e.g., W3110 (ΔfhuA) and BL21).

[0322] In another aspect, this disclosure provides a method for generating an RNA ligase polypeptide, wherein the method comprises culturing a host cell containing an expression vector capable of expressing or generating the RNA ligase polypeptide under suitable culture conditions, thereby expressing or generating the RNA ligase polypeptide. In some embodiments, the method includes isolating the RNA ligase from the culture medium and / or host cells, as described herein. In some other embodiments, the method further includes purifying the expressed RNA ligase polypeptide.

[0323] In some embodiments, RNA ligase peptides expressed in host cells are recovered from cells and / or culture media using any one or more known techniques for protein purification, including lysozyme or detergent treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, as described herein.

[0324] Chromatographic techniques used for the isolation / purification of RNA ligase peptides include, in particular, reversed-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying RNA ligases depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity chromatography can be used to isolate RNA ligases. For affinity chromatography purification, antibodies that specifically bind to RNA ligase peptides can be used. In some embodiments, an affinity tag (e.g., a His tag) can be introduced into the RNA ligase peptide for isolation / purification purposes.

[0325] Appropriate culture media and growth conditions for the host cells described above are well known in the art. Polynucleotides for expressing RNA ligases can be introduced into cells using a variety of methods known in the art. Techniques include, in particular, electroporation, bioballistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0326] In some embodiments, the polynucleotide encoding the RNA ligase polypeptide can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, the polynucleotide fragments can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired continuous sequence (see, for example, Hughes et al., Cold Spring Harb Perspect Biol. 2017 Jan; 9(1):a023812). For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classic phosphoramide method (see, for example, Beaucage et al., Tetra. Lett., 1981, 22:1859-69; and Mattes et al., EMBO J., 1984, 3:801-05), as it is generally practiced in automated synthetic methods.

[0327] In some embodiments, a method for preparing an RNA ligase may include: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequences of RNA ligases, such as those described in the tables of the examples; and (b) expressing an engineered RNA ligase encoded by the polynucleotide. In some embodiments of this method, the amino acid sequence encoded by the polynucleotide may optionally have one or more (e.g., up to 3, 4, 5, or up to 10) amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence may optionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the substitution can be conserved or non-conserved. Desired properties of the expressed peptide, such as RNA ligase activity against one or more oligonucleotide substrates, as described in the examples, can be evaluated.

[0328] RNA ligase composition In another aspect, this disclosure provides compositions of engineered RNA ligases disclosed herein. In some embodiments, the composition comprises at least one engineered RNA ligase polypeptide described herein, such as the engineered RNA ligases provided in or in the sequence listings of Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12. In some embodiments, the composition comprises an RNA ligase comprising an amino acid sequence containing residues 12 to 343 of SEQ ID NO: 2, an amino acid sequence containing SEQ ID NO: 2, or an amino acid sequence containing residues 12 to 343 of SEQ ID NO: 4-220, 224-252, and 270-958, or an amino acid sequence containing even-numbered SEQ ID NO: 4-220, 224-252, and 270-958. In some embodiments, the composition comprises an engineered RNA ligase containing residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582, or an amino acid sequence containing SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582. In some embodiments, the engineered RNA ligase polypeptide in the composition is isolated or purified. In some embodiments, the RNA ligase is combined with other components and compounds to provide compositions and formulations containing engineered RNA ligase polypeptides suitable for various applications and uses.

[0329] In some embodiments, the composition further comprises one or more of a buffer, a nucleotide substrate (e.g., ATP or dATP), and / or at least one or more polynucleotide ligase substrates. In some embodiments, the polynucleotide substrate is single-stranded or double-stranded, or comprises both single-stranded and double-stranded polynucleotide substrates. In some embodiments, the polynucleotide substrate comprises at least two oligonucleotide fragments, wherein the oligonucleotide fragments include double-stranded oligonucleotide fragments, and the at least two double-stranded oligonucleotide fragments are base-paired to serve as substrates for RNA ligases. In some embodiments, the composition further comprises at least 2, 3, 4, 5, 6, or more double-stranded oligonucleotide fragments, each of which can serve as substrates for RNA ligases. In some embodiments, the polynucleotide substrate is a modified polynucleotide. In some embodiments, the modified polynucleotide comprises modifications on sugar residues, such as at the 2'-position; modified phosphate ester groups, such as thiophosphates; or modified nucleobases, or any combination of modifications as further described herein.

[0330] In some embodiments, the RNA ligase substrate concentration in the composition is about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3-0.6 mM. In some embodiments, the RNA ligase substrate is about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, or 1 mM. In some embodiments, the RNA ligase substrate concentration is about 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, the engineered RNA ligase exhibits increased product yield at concentrations of about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

[0331] In some embodiments, the composition further comprises a nucleotide cofactor or substrate used by an RNA ligase to catalyze the ligation reaction. In some embodiments, the nucleotide cofactor or substrate is ATP and / or dATP. Preferably, the nucleotide cofactor or substrate is ATP. In some embodiments, the nucleotide cofactor or substrate (e.g., ATP) is present at concentrations of about 0.05-25 mM, 1-20 mM, 2-18 mM, or 5-15 mM. In some embodiments, the nucleotide cofactor or substrate is present at concentrations of about 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 20 mM, or 25 mM.

[0332] In some embodiments, the composition further comprises a buffer solution, which in some embodiments particularly includes borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris). In some embodiments, the buffer solution concentration is 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM.

[0333] In some embodiments, the composition further comprises additives or linker enhancers, particularly including one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., dextran 6000). In some embodiments, the composition comprises 1% to 40% v / v DMSO. In some embodiments, the composition comprises 0.1 M to 3 M betaine. In some embodiments, the composition comprises 0.5% to 20% w / v PEG (e.g., PEG6000 or PEG8000).

[0334] In some embodiments, the composition further comprises an RNase inhibitor, particularly porcine RNase inhibitors, human placental RNase inhibitors, human liver RNase inhibitors, mouse RNase inhibitors, rat RNase inhibitors, and recombinantly expressed RNase inhibitors thereof.

[0335] In some embodiments, the engineered RNA ligase described herein is provided in solution or immobilized on a substrate. In some embodiments, the substrate is a solid substrate, membrane, or particle. The enzyme may be embedded in a matrix or membrane. In some embodiments, the matrix includes polymeric materials such as calcium alginate, agar, k-carrageenan, polyacrylamide, and collagen, or solid matrices such as activated carbon, porous ceramics, and diatomaceous earth. In some embodiments, the matrix is ​​a particle, membrane, or fiber. Types of membranes particularly include nylon, cellulose, polysulfone, or polyacrylate.

[0336] In some embodiments, the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed onto the support material. In some embodiments, the enzyme is immobilized on the support material by covalent attachment. Support materials include, in particular, inorganic materials such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion exchange resins such as Amberlite, Sephadex, and Dowex.

[0337] Uses and methods On the other hand, this disclosure provides the use of engineered RNA ligases for polynucleotide synthesis, RNA repair, or other molecular biology applications.

[0338] In some embodiments, engineered RNA ligases are used to ligate polynucleotides and oligonucleotides. In some embodiments, engineered RNA ligases are used to synthesize polynucleotides from shorter oligonucleotides. In some embodiments, a method of ligating at least a first polynucleotide chain and a second polynucleotide chain includes contacting the first and second polynucleotide chains with the engineered RNA ligase described herein in the presence of a nucleotide substrate under conditions suitable for ligating the first polynucleotide chain to the second polynucleotide chain, wherein the first polynucleotide chain includes a ligation-capable 5' end and the second polynucleotide chain includes a 3' end capable of ligating to the 5' end of the first polynucleotide chain.

[0339] In some embodiments, the second polynucleotide chain, which includes a 3'-terminus, has at least 2, 3, 4, 5, or 6 ribonucleotides at its 3' end. In some embodiments of the method, the 3' end of the second polynucleotide chain is a 3'-OH. In some embodiments of the method, the 5' end of the first polynucleotide chain is a 5'-phosphate ester. In some embodiments of the method, the internucleotide bonds contained in the first and / or second polynucleotide chains include phosphate ester bonds.

[0340] In some embodiments, the first polynucleotide chain has at least a 5' terminal nucleotide having a 2'-modified sugar moiety. In some embodiments, at least two, three, four, or more nucleotides at the 5' terminal region of the first polynucleotide chain have a 2'-modified sugar moiety. In some embodiments, the first polynucleotide chain has at least a 5' terminal nucleotide having a non-natural nucleoside internucleotide bond with an adjacent 3'-nucleotide. In some embodiments, at least two, three, four, or more nucleotides at the 5' terminal region of the first polynucleotide chain have a non-natural nucleoside internucleotide bond. In some embodiments, the non-natural bond is a phosphate thioester nucleoside internucleotide bond.

[0341] In some embodiments, the second polynucleotide chain has at least a 3' terminal nucleotide having a 2'-modified sugar moiety. In some embodiments, at least two, three, four, or more nucleotides in the 3' terminal region of the second polynucleotide chain have a 2'-modified sugar moiety. In some embodiments, the second polynucleotide chain has at least a 3' terminal nucleotide having a non-natural nucleoside internucleotide bond with an adjacent 5'-nucleotide. In some embodiments, at least two, three, four, or more nucleotides in the 3' terminal region of the second polynucleotide chain have a non-natural nucleoside internucleotide bond. In some embodiments, the non-natural bond is a phosphate thioester nucleoside internucleotide bond.

[0342] In some embodiments, the first polynucleotide chain has at least a 5' terminal nucleotide having a 2'-modified sugar moiety, and the second polynucleotide chain has at least a 3' terminal nucleotide having a 2'-modified sugar moiety. In some embodiments, at least two, three, four, or more nucleotides in the 5' terminal region of the first polynucleotide chain have a 2'-modified sugar moiety, and at least two, three, four, or more nucleotides in the 3' terminal region of the second polynucleotide chain have a 2'-modified sugar moiety. As further discussed below, in some embodiments, the 2'-modified sugar moiety is 2'-O-methyl, 2'-O-ethyl, or 2'-fluoro. In some embodiments, the first polynucleotide chain has at least a 5' terminal nucleotide having a non-natural nucleoside internucleotide bond with an adjacent 3'-nucleotide, and the second polynucleotide chain has at least a 3' terminal nucleotide having a non-natural nucleoside internucleotide bond with an adjacent 5'-nucleotide. In some embodiments, at least 2, 3, 4, or more nucleotides at the 5' end region of the first polynucleotide chain and at least 2, 3, 4, or more nucleotides at the 3' end region of the second polynucleotide chain have non-natural nucleoside bonds. In some embodiments, the non-natural nucleoside bonds are phosphate thioester nucleoside bonds.

[0343] In some embodiments, the method further includes a third polynucleotide chain, wherein the first and second polynucleotide chains hybridize adjacently to each other on the third polynucleotide chain to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain. In some embodiments of the method, the third polynucleotide chain is continuous with the first or second polynucleotide chain. In some embodiments of the method, the third polynucleotide chain is continuous with the first and second polynucleotide chains to form a single continuous polynucleotide ligase substrate. In some embodiments, the third polynucleotide chain is RNA, DNA, or a mixture of RNA and DNA.

[0344] In some embodiments of the method, the third polynucleotide comprises a splint or bridging polynucleotide, wherein the 5' end sequence of the first polynucleotide chain and the 3' end sequence of the second polynucleotide chain hybridize adjacently to each other on the splint or bridging polynucleotide to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain. In some embodiments, the splint or bridging polynucleotide is RNA, DNA, or a mixture of RNA and DNA.

[0345] In some embodiments, the second polynucleotide chain, which includes a 3'-terminus, has at least 4, 5, or 6 ribonucleotides at its 3' end. In some embodiments of the method, the 3' end of the second polynucleotide chain is a 3'-OH. In some embodiments of the method, the 5' end of the first polynucleotide chain is a 5'-phosphate ester. In some embodiments of the method, the internucleotide bonds contained in the first and / or second polynucleotide chains include phosphate ester bonds.

[0346] In some embodiments of the method, the third polynucleotide chain comprises a splint or bridging polynucleotide, wherein the 5' end sequence of the first polynucleotide chain and the 3' end sequence of the second polynucleotide chain hybridize adjacent to each other on the splint or bridging polynucleotide to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain.

[0347] In some implementations, one or more additional polynucleotide chains may hybridize with a third polynucleotide chain, wherein the additional polynucleotide chain hybridizes adjacent to the 3' end of the first polynucleotide chain or adjacent to the 5' end of the second polynucleotide chain, or two additional polynucleotide chains hybridize adjacent to the 3' end of the first polynucleotide chain and adjacent to the 5' end of the second polynucleotide chain, to form a nick that can be ligated to an engineered RNA ligase. The additional polynucleotide chains may hybridize and ligate in a similar manner to prepare longer polynucleotide chains.

[0348] In some embodiments, when hybridizing with a third polynucleotide chain and adding a fourth polynucleotide chain that hybridizes with a single-stranded region of a first or second polynucleotide chain, the first and / or second polynucleotide chains may have single-stranded regions (e.g., protruding ends or unpaired regions), wherein the fourth polynucleotide chain hybridizes adjacent to the 5' or 3' end of the third polynucleotide chain to form a nick that can then be ligated with an engineered RNA ligase. In some embodiments, when hybridizing with a first or second polynucleotide chain on a third polynucleotide chain and adding a fifth polynucleotide chain that hybridizes with a single-stranded region of the fourth polynucleotide chain, the fourth polynucleotide chain may have single-stranded regions (e.g., protruding ends or unpaired regions), wherein the fifth polynucleotide chain hybridizes adjacent to the 3' end of the first polynucleotide chain or the 5' end of the second polynucleotide chain to form a nick that can be ligated with an engineered RNA ligase. Such sequential addition of polynucleotide chains can be used to generate longer polynucleotide products (see, for example, Paul et al., ACS Chem. Biol. 2023, 18, 2183-2187, which is incorporated herein by reference).

[0349] In some embodiments, the first polynucleotide chain hybridizes with a third polynucleotide chain to form a first double-stranded fragment, and the second polynucleotide chain hybridizes with a fourth polynucleotide chain to form a second double-stranded fragment, wherein the first and second double-stranded fragments are base-pairable to form a substrate for an engineered RNA ligase. In some embodiments, the first and second double-stranded fragments have complementary overhangs or complementary single-stranded ends, also known as sticky or viscous ends, which are base-pairable and form a double-stranded nick between the first and second double-stranded fragments, which serves as a substrate for the engineered RNA ligase.

[0350] In some embodiments, the complementary end (sticky or cohesive end) is of sufficient length to allow the double-stranded fragments to pair bases and form a suitable substrate for an engineered RNA ligase. In some embodiments, the complementary single-stranded end comprises at least 1-50, 2-40, 3-35, 4-30, 5-25, 6-20, or 8-15 or more nucleotides. In some embodiments, the complementary single-stranded end is 1-10, 2-8, or 4-6 nucleotides long. In some embodiments, the complementary single-stranded end is 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more nucleotides long.

[0351] In some embodiments, the length of the double-stranded region or complementary portion of each of the double-stranded fragments is 4-50, 6-45, 8-40, 10-35, 12-30, or 14-25 nucleotides. In some embodiments, the length of the double-stranded region is at least 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50 nucleotides.

[0352] In some embodiments of this method, the ligase substrate comprises at least 2, 3, 4, 5, or 6 or more double-stranded fragments, each having a complementary end that can pair with at least one other double-stranded fragment having a complementary end to form a substrate for the RNA ligase. In some embodiments, the double-stranded fragments have complementary or sticky ends, for example, at the 5' and 3' ends, such that the double-stranded fragments can be joined to form a multiplex. In some embodiments, when the number of double-stranded fragments is 3, at least one of these double-stranded fragments has complementary ends at the 5' and 3' ends of the fragment, wherein the double-stranded fragment pairs with two other different double-stranded fragments to form a substrate that can be linked to a product containing three double-stranded fragments. It should be understood that when linking 3 or more double-stranded fragments, the complementary ends of the double-stranded fragments can be designed to link all the different double-stranded fragments.

[0353] In some embodiments, the polynucleotide substrate comprises one or more modified nucleotides. In some embodiments, the modification comprises a modified sugar residue, a modified nucleotide base, and / or a modified phosphate group. In some embodiments, the modified sugar residue is modified at the 2-position of the sugar moiety. In some embodiments, the modified 2-position is a 2'-halogenated group or a 2'-O-alkyl group, preferably a lower C1-C4 alkyl group, such as methyl or ethyl. In some embodiments, the modified nucleotide is a modified phosphate ester group, such as a thiophosphate ester group. In some embodiments, the modified phosphate ester group is located at the 5' end of the polynucleotide substrate. In some embodiments, the modified phosphate ester group is located at the nucleoside internucleotide bond of the polynucleotide substrate. In some embodiments, the modified nucleotide has a modified nucleotide base. In some embodiments, the polynucleotide substrate comprises a reverse nucleotide or a debased nucleotide (e.g., a 5'-5' bond or a 3'-3' bond), including 3'-3' reverse dA, dT, dG, dC, dU or a debased deoxyribonucleotide; and / or 5'-5' reverse dA, dT, dG, dC, dU or a debased deoxyribonucleotide.

[0354] In some embodiments of the method, the first polynucleotide chain and / or the second polynucleotide chain comprises one or more modified nucleotides or nucleotide analogs, wherein the modified nucleotides or nucleotide analogs comprise nucleobase analogs, modified nucleoside sugar residues, modified nucleoside internucleotides, modified 5' phosphate groups, and / or modified 3' hydroxyl groups.

[0355] In some embodiments, the polynucleotide ligase substrate comprises a modified 5'-phosphate group, wherein the modified phosphate group is a phosphate analog. In some embodiments of the method, the first polynucleotide comprises a 5'-phosphate analog. In some embodiments, the 5'-phosphate analog is a thiophosphate, aminophosphate, monomethylphosphate, methylphosphonate, vinylphosphonate, or phosphonocarboxylic acid ester.

[0356] In some embodiments of this method, the polynucleotide ligase substrate comprises one or more modified nucleotide sugar residues. In some embodiments of this method, the first polynucleotide and / or the second polynucleotide comprises one or more modified nucleotide sugar residues. In some embodiments, the modified nucleotide sugar residues are 2'-O-alkyl, 2'-halogenated, β-D-ribose-LNA, or α-L-ribose-LNA (locked nucleic acid). In some embodiments, the modified nucleotide sugar residues are particularly 2'-O-methyl, 2'-O-ethyl, or 2'-O-propyl. In some embodiments, the modified nucleotide sugar residues are 2'-fluorine, 2'-bromine, or 2'-chloro, preferably 2'-fluorine. In some embodiments, the sugar residues are modified with ligands (such as cell-targeting ligands) or cell-penetrating moieties (e.g., GalNAc and lipid moieties). In some embodiments, the sugar residues are modified with linker moieties.

[0357] In some embodiments of this method, the polynucleotide ligase substrate comprises one or more modified nucleotide residues having modified nucleosides or nucleobase analogs. In some embodiments of this method, the first polynucleotide and / or the second polynucleotide comprises one or more modified nucleotide residues having modified nucleosides or nucleobase analogs. In some embodiments, the nucleobase analogs are, in particular, xanthine, hypoxanthine, inosine, 6-methyladenine, 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-deazoguanine. In some embodiments, the nucleosides are modified with ligands (such as chemically labeled, cell-targeting ligands) or cell-penetrating portions. In some embodiments, the nucleosides are modified with linker portions, for example, for conjugating ligands.

[0358] In some embodiments of this method, the polynucleotide ligase substrate contains one or more modified or non-standard nucleoside internucleotides, i.e., nucleoside internucleotides other than phosphate esters. In some embodiments, the first polynucleotide and / or the second polynucleotide contains one or more modified or non-standard nucleoside internucleotides. In some embodiments, the nucleoside internucleotide is a thiophosphate, phosphate acetate, aminophosphate, methylphosphonate, or phosphonoacylcarboxylate. In some embodiments, at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nucleoside internucleotides are non-standard nucleoside internucleotides. In some embodiments, the non-standard nucleoside internucleotide is present at 1, 2, 3, 4, or 5 nucleotides at the 5' and / or 3' ends of the polynucleotide substrate. In some embodiments, the non-standard nucleoside internucleotide (such as thiophosphate) is diastereomeric, e.g., S or P configuration.

[0359] In some embodiments of this method, the polynucleotide ligase substrate comprises a modified 3'-hydroxyl group. In some embodiments, the 3' end of the polynucleotide ligase substrate is attached to a matrix or surface, such as a solid matrix. In this case, the polynucleotide ligase substrate attached to the matrix has a ligable 5' end. In some embodiments, the 3' end of the ligase substrate is modified with an amine, a halide, a phosphate ester, a phosphate ester analog, an -O-alkyl group, a lipid moiety, or a detectable label. In some embodiments, the 5' end of the polynucleotide substrate is modified with a cell-targeting moiety, a cell-penetrating moiety, or a linker moiety. In some embodiments, the cell-targeting moiety is a GalNAc moiety.

[0360] In some implementations, the polynucleotide ligase substrate contains C-4' modifications, particularly 4'-thio-C2' modifications, 4' / 5' aminoalkyl / C2' modifications, C4'-guanidino-C2' modifications, and C4'-O-Me / C2' modifications (see, for example, Gangopadhyay et al., RNA Biology, 2022, 19:1, 452-467).

[0361] In some embodiments, engineered RNA ligases are used in methods for synthesizing RNA or DNA / RNA polynucleotides by ligating shorter RNA or DNA / RNA oligonucleotides. In some embodiments, engineered RNA ligases are used to ligate the 3' OH of RNA to the 5' phosphate ester of DNA or RNA. In some embodiments, engineered RNA ligases are used to ligate the 3' OH of RNA to the 5' phosphate ester of DNA in the construction of double-stranded NGS RNA libraries. In some embodiments, engineered RNA ligases are used in methods for preparing RNA loops. In some embodiments, engineered RNA ligases are used to repair nicks in dsRNA or dsRNA / DNA. In some embodiments, engineered RNA ligases are used to synthesize modified oligonucleotides, such as shRNA and antisense oligonucleotides.

[0362] In some embodiments, the amount of engineered RNA ligase used is sufficient to ligate the oligonucleotide substrate or to ligate a nick formed from the oligonucleotide substrate to the desired level. The amount of enzyme used can also be adjusted based on the activity of the ligase and the type of substrate being ligated. In some embodiments, the amount of enzyme used is about 0.1 mg / mL to about 5 mg / mL, about 0.5 mg / mL to about 4 mg / mL, 1 mg / mL to about 3 mg / mL, or about 1.5 to about 2.5 mg / mL. In some embodiments, the engineered RNA ligase is provided at concentrations of about 0.01 g / L to about 50 g / L; about 0.01 to about 0.1 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L.

[0363] In some embodiments, the method includes a nucleotide substrate used by an RNA ligase to catalyze the ligation reaction. In some embodiments, the nucleotide substrate is ATP and / or dATP. Preferably, the nucleotide substrate is ATP. In some embodiments, the reaction conditions for ligation include additional components such as Mg. -2 Buffer solutions and / or salts.

[0364] In some embodiments, the nucleotide cofactor or substrate (e.g., ATP) is present at concentrations of about 0.05-25 mM, 1-20 mM, 2-18 mM, or 5-15 mM. In some embodiments, the nucleotide cofactor or substrate is present at concentrations of about 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 20 mM, or 25 mM.

[0365] In some embodiments, the buffer solution particularly includes borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris). In some embodiments, the buffer solution concentration is 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM.

[0366] In some embodiments, the reaction conditions also include linkage-enhancing agents, particularly polyethylene glycol (e.g., PEG6000 and PEG8000) or other molecular crowding agents such as bovine serum albumin (BSA), dextran, and Ficoll.

[0367] In some embodiments, the ligation reaction is carried out at a suitable temperature and reaction time. In some embodiments, the ligation reaction temperature is from about 2°C to about 60°C. In some embodiments, the ligation reaction temperature is 4°C to 55°C, 4°C to 50°C, 4°C to 45°C, or 10°C to 40°C. In some embodiments, the ligation reaction temperature is 2°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, different temperatures can be used for the ligation reaction, such as the temperature at which a stable hybrid is formed between polynucleotide substrates, followed by a higher temperature to facilitate the completion of the ligation reaction.

[0368] In some embodiments, the ligation reaction time can be sufficient for ligating the polynucleotide substrate. In some embodiments, the ligation reaction time is 0.5-72 hours or longer. In some embodiments, the ligation reaction time is 1-72 hours, 2-48 hours, or 2-24 hours. In some embodiments, the ligation reaction time is 0.5, 1, 2, 4, 5, 12, 24, 48, or 72 hours or longer.

[0369] In some embodiments, the ligase reaction is carried out at a suitable pH. In some embodiments, the pH of the ligase reaction is about pH 5-9, about pH 5.5-8.5, about pH 6-8, or about pH 6.5-7.5. In some embodiments, the pH of the ligase reaction is about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8, about pH 8.5, or about pH 9.

[0370] In some embodiments, the RNA ligase substrate concentration is provided at concentrations of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, the substrate concentration is about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3-0.6 mM. In some embodiments, the substrate concentration is about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any suitable concentration for effectively binding the substrate.

[0371] In some implementations, the polynucleotide substrate is bound to a support, and a ligation reaction is performed using an engineered RNA ligase to ligate the polynucleotide substrate to the support-bound polynucleotide, or to ligate one or more polynucleotide substrates that hybridize with the support-bound polynucleotide substrate.

[0372] In some embodiments, the ligase reaction further includes a nucleotide substrate regeneration system to regenerate the cofactor substrate ATP (or dATP). In some embodiments, the ATP recycling or regeneration system includes a nucleoside monophosphate kinase for converting AMP to ADP in the presence of a phosphate donor. Various nucleoside monophosphate kinases can be used to convert AMP to ADP, including homologues of nucleoside monophosphate kinases. In some embodiments, more than one nucleoside monophosphate kinase can be used in the regeneration system. In some embodiments, the nucleoside monophosphate kinase is adenosine monophosphate kinase (e.g., adenosine kinase), cytidine monophosphate (CMP) kinase, uridine monophosphate (UMP) kinase, or guanylate monophosphate (GMP) kinase.

[0373] In some embodiments, the nucleoside monophosphate kinase suitable for the enzymatic reaction is cytidine monophosphate kinase. Various suitable cytidine monophosphate kinases are known in the art. These include homologues of cytidine monophosphate kinase. In some embodiments, cytidine monophosphate kinases suitable for enzymatic reactions include, in particular, the following: *Thermus thermophilus* (Q5SL35), *Pyrococcus furiosus* (Q8U2L4), *Pseudomonas putida* (AFO48857.1), *Escherichia coli* K-12MG1655 (P0A6I0), *Clostridium acetobutylicum* (Q97I08), *Halobacterium salinarum* (Q9HPA5), *Bacillus acidicola* (WP_066270173), *Acetobacter aceti* (WP_010667744), and *Acidithiobacillus thiooxidans*. (WP_024892761.1), *Acidithiobacillus ferrooxidans* (WP_064220349.1), *Metallosphaerasedula* (WP_011921264.1), *Amphibacillus xylanus* (WP_015009966.1), *Thioalkalivibrio denitrificans* (WP_077278466.1), *Vibrio psychroerythus* (Q482G4), *Pseudoalteromonashaloplanktis* (Q3ILA1), *Psychrobacter arcticus* (Q4FRL5), *Psychromonas ingrahamii* (A1SZ01), Pseudomonas syringae (xQ4ZQ97) and Halobacterium salinarum (Q9HPA5).

[0374] In some embodiments, the nucleoside monophosphate kinase suitable for the enzymatic reaction is uridine monophosphate kinase. Various suitable uridine monophosphate kinases are known in the art. These include homologues of uridine monophosphate kinase. In some embodiments, uridine monophosphate kinases suitable for enzymatic reactions include, in particular, the following: *Pomacea canaliculata* (Q8U122), *Thermophilus thermophilus* (P43891), *Pseudomonas putida* (I7BW46), *Escherichia coli* K-12 MG1655 (P0A7E9), *Aspergillus niger* (A2R195), *Saccharomyces cerevisiae* (P15700), *Clostridium acetobutyricum* (Q97I64) ATCC 824 PyrH, *Halobacterium salina* (Q9HNN8), *Picrophilus torridus* (WP_048059653), *Metrolactone* (WP_012021705), *Acidophilus thermophilus* (WP_010900913), and *Sulfolobus solfataricus*. (WP_009992427), Acetobacter (WP_042788648), Vibrio thioalkalivia species HK1 (WP_081759172.1), Bispores xylan (WP_015010200.1), Vibrio cynomolgus (Q485G8), Pseudomonas halophilus (Q3IIX6), Psychrophilus arcticus (Q4FRH5), Psychrophilus ingrahami (ABM04676.1), Pseudomonas syringae (Q4ZWS6), and Haemophilus salsa (Q9HNN8).

[0375] In some embodiments, the nucleoside monophosphate kinase suitable for the enzymatic reaction is guanosine monophosphate kinase (guanylate kinase). Various suitable guanylate kinases are known in the art. These include homologues of guanylate kinase. In some embodiments, guanylate kinases suitable for enzymatic reactions particularly include the following guanylate kinases: *Thermotoga maritima* (Q9X215), *Thermophilus thermophilus* (Q5SI18), *Pseudomonas putida* (I7C087), *Escherichia coli* K-12 (P60546), *Aspergillus niger* (A2QPV2), *Saccharomyces cerevisiae* (P15454), *Clostridium acetobutyricum* (Q97ID0), *Thiobacillus ferrooxidans* (WP_064219869.1), *Thiobacillus thiooxidans* (WP_010637919.1), *Bacillus acidophilus* (WP_066264774.1), *Acetobacter* (WP_018308252.1), *Bacillus xylanus* (WP_015010280.1), and *Thioalkalivibrio sulfidiphilus*. (WP_018953989.1), Vibrio cynomolgus (Q47UB3), Pseudomonas halophilus (Q3IJH8), Psychrophilus aeruginosa (Q4FQY7), Psychrophilus ingrahami (A1T0P1) and Pseudomonas syringae (Q4ZZY8).

[0376] In some embodiments, the nucleoside monophosphate kinase suitable for the enzymatic reaction is adenosine monophosphate kinase (adenosine kinase). Various suitable adenosine kinases are known in the art. These include homologues of adenosine kinase. In some embodiments, the adenosine kinase is a bacterial, fungal, plant, or animal adenosine kinase. In some embodiments, adenosine kinases suitable for enzymatic reactions include, in particular, the following adenosine kinases: *Thermophilus thermophilus* (Q72125), *Cyclottomella fuciformis* (Q8U207), *Pseudomonas putida* (17CAA9), *Escherichia coli* K-12 W3110 (P69441), *Aspergillus niger* CBS 513.88 (A2QPN9), *Saccharomyces cerevisiae* (P07170), *Clostridium acetobutyricum* (Q97E39), *Haloxylon ammodendron* (Q9HPAT), *Thiobacillus thiooxidans* (WP_024894015.1), *Thiobacillus ferrooxidans* (WP_064218420.1), *Bacillus acidophilus* (WP_066267988.1), *Leucobacterium sulfideum* (WP_009991241.1), *Saccharomyces cerevisiae* (P07170), and *Thermotoga neapolitana*. (Q8GGL2), Escherichia coli (P69441), and Bacillus stearothermophilus (WP_049624206.1). In some embodiments, the adenosine kinase is an engineered adenosine kinase described in International Patent Application No. PCT / US2024 / 051084, filed October 11, 2024, which is incorporated herein by reference.

[0377] In some embodiments, the ATP regeneration system includes at least an enzyme for converting ADP to ATP in the presence of a phosphate donor. In some embodiments, the ATP regeneration system particularly includes acetate kinase, pyruvate kinase, creatine kinase, or polyphosphokinase (see, for example, Endo et al., Adv. Synth. Catal., 2002, 343:521–526; Andexer et al., Chem Bio Chem., 2015, 16:380–386). In the ATP regeneration system, the phosphate donor for converting ADP to ATP is selected based on the ATP regeneration enzyme used. For example (but not limited to), if acetate kinase is used to convert ADP to ATP, the phosphate donor is acetyl phosphate. If pyruvate kinase is used to convert ADP to ATP, the phosphate donor is phosphoenolpyruvate. If creatine kinase is used to convert ADP to ATP, the phosphate donor is creatine phosphate. If polyphosphokinase is used to convert ADP to ATP, the phosphate donor is inorganic polyphosphate.

[0378] Therefore, in some embodiments, the ATP regeneration system includes pyruvate kinase and phosphoenolpyruvate. In some embodiments, the ATP regeneration system includes creatine kinase and creatine phosphate. In some embodiments, the ATP regeneration system includes polyphosphokinase and inorganic polyphosphates. In some embodiments, the ATP regeneration system includes acetate kinase and acetyl phosphate. In some embodiments, the acetate kinase is the following acetate kinase: Escherichia coli K-12 strain MG1655 substrain (NP_416799.1), Corynebacterium jejuni K411 (WP_011272972.1), Lactococcus lactis subsp. lactis KW2 (WP_011835968.1), Lactococcus lactis (WP_004254593.1), Thermosiphomelaniensis species 38H-ov (WP_165147355.1), Thermosipho species KOL6 (WP_101510533.1), Thermosiphomelaniensis (WP_012057479.1), Thermosipho species RQ7 (WP_041844042.1), and Thermosipho africanus (WP_004102380.1). In some embodiments, the acetylkinase is an engineered acetylkinase described in International Patent Application No. PCT / US2024 / 051118, filed October 11, 2024, which is incorporated herein by reference.

[0379] In some embodiments, engineered RNA ligases are used to ligate nicks or related nick structures (see, for example, Cheng et al., Royal Soc Chem Adv., 2019, 9:8620–8627). In some embodiments, engineered RNA ligases are used, for example, to synthesize RNA from shorter oligonucleotides using splice nucleic acids (see, for example, Stark et al., RNA, 2006, 12:2014–2019). In some embodiments, engineered RNA ligases are used to ligate modified oligonucleotides, such as those provided in the examples. Other examples of modified oligonucleotide products synthesized using short oligonucleotide substrates include, in particular, patent disclosures WO22104366, WO22029209, WO22031847, WO20226960, US2022072024, US2021238606, US11286488, US2017305956, WO22212153, WO22192519, WO22125490, WO22072447, WO21257568, and WO211 The shRNA or siRNA described in WO2373, WO21072395, WO21022108, US2022079971, US11034957, US2021332365, US11015201, US10995336, US11091759, US10889813, US10130651, US10513703, WO19232255, WO21108640, WO22147304 and WO21138537.

[0380] In another aspect, this disclosure provides a kit comprising the RNA ligase described herein. In some embodiments, the kit further comprises a buffer, a nucleotide substrate (e.g., ATP or dATP), and / or one or more polynucleotide ligase substrates. In some embodiments, the kit further comprises additives or ligation enhancers, including one or more of polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.) or other molecular crowding agents, bovine serum albumin, Ficoll, and dextran (e.g., dextran 6000).

[0381] The following embodiments, including experiments and results, are provided for illustrative purposes only and should not be construed as limiting the invention.

[0382] Example In the following experimental disclosures, the following abbreviations apply where applicable: ppm (parts per million); M (moles); mM (millimoles), uM and µM (micromoles); nM (nanomoles); mol (moles); gm and g (grams); mg (milligrams); ug and µg (micrograms); L and l (liters); ml and mL (milliliters); ul, ml, uL, mL (microliters); cm (centimeters); mm (millimeters); um and µm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); OD (optical density); MW (molecular weight); rpm (revolutions per minute); rcf (relative centrifugal force); psi and PSI (pounds per square inch); ℃ (degrees Celsius); RT and rt (room temperature); NGS (next-generation sequencing); ds (double-stranded); ss (single-stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (RNA); Escherichia coli W3110 (a commonly used laboratory E. coli strain, available from Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (High Throughput); HPLC (High Performance Liquid Chromatography); FPLC (Rapid Protein Liquid Chromatography); PBS (Phosphate Buffered Saline); BSA (Bovine Serum Albumin); DTT (Dithiothreitol); CAM (Chloramphenicol); CAT (Chloramphenicol Acetyltransferase); IPTG (Isopropyl β-D-1-thiogalactopyranoside); FIOPC (Fold Improvement Relative to Positive Control); FIOP (Fold Improvement Relative to Parent); LB (Luria-Bertani); TB (Super Broth).

[0383] Example 1 E. coli expression host containing recombinant RNA ligase 2 gene To produce the enzyme, the RNA ligase 2 gene was cloned in the pJV110900 vector system (see US10184117B2), which is operatively linked to the lac promoter under the control of the lacI repressor. The expression vector also contains the P15a origin of replication and the chloramphenicol (CAM) resistance gene. The resulting plasmid was transformed into *E. coli* W3110 using standard methods known in the art. Transformants were isolated by subjecting cells to CAM selection, as is known in the art (see, for example, US Patent Nos. 8,383,346 and WO2010 / 144103).

[0384] Example 2 Preparation of wet cell pellet containing HTP RNA ligase 2 *E. coli* cells containing the recombinant RNA ligase 2 encoding gene from a monoclonal colony were seeded into the wells of a 96-well shallow-well microtiter plate in 180 µL of LB growth medium containing 1% glucose and 30 µg / mL CAM. The plate was sealed with a breathable seal and the cultures were incubated overnight at 30°C, 200 rpm, and 85% humidity. Then, 10 µL of each cell culture was transferred to the wells of a 96-well deep-well plate containing 390 mL of TB growth medium and 30 µg / mL CAM. The deep-well plate was sealed with a breathable seal and incubated at 30°C, 250 rpm, and 85% humidity until an OD of 0.6–0.8 was achieved. 600 The cell culture was then induced with IPTG to a final concentration of 1 mM and incubated overnight under the same conditions before induction. The cells were then pelleted by centrifugation at 4,000 rpm for 10 min. The supernatant was discarded and the pellet was frozen at -80°C before lysis.

[0385] Example 3 Recombinant RNA ligase 2 expression and purification in E. coli via shake flask To characterize the improved enzyme variants after each round of evolution, shake-flask production and purification were performed. HTP cultures grown as described above were thawed, and 30 µL of each HTP culture was inoculated into 1 L shake flasks containing: 160 mL of Teknova super broth (TB) medium, 30 μg / mL chloramphenicol, 0.03% (v / v) lactose, and 0.075% (v / v) glucose. The shake flasks were incubated at 32 °C and 250 rpm for 18 h. After this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the precipitate was resuspended in 30 mL of 50 mM Tris-HCl (pH 7.5). The cell suspension was cooled in an ice bath and lysed using a microfluidic cell disruptor (Microfluidics M-110L). The crude lysate was precipitated by centrifugation (10,000 rpm for 90 min, 4°C), and the supernatant was then filtered through a 0.2 μm PES membrane to further clarify the lysate. The clarified lysate was then replenished with 20 mM imidazole and 500 mM NaCl. The lysate was then purified using an AKTA Pure purification system and a 5 mL HisTrap FF column (Cytiva) with the operating parameters provided below (Table 1). The wash buffer consisted of 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, and 0.02% Triton X-100, and the elution buffer consisted of 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 250 mM imidazole, and 0.02% Triton X-100, as follows:

[0386] The most concentrated fractions were identified and combined by UV absorption (280 nm); 3 ml of eluent was dialyzed overnight in 1× ligase storage buffer (40 mM Tris-HCl pH 7.5, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol) in a 3K Slide-A-Lyzer™ dialysis kit (Thermo Fisher) for buffer exchange. The RNA ligase 2 concentration from the formulation was measured by reading the absorbance at 280 nm.

[0387] The purified RNA ligase 2 protein was then screened by observing antisense (AS) conversion, sense (SS) conversion, and total conversion using the analytical methods described in Example 15, and the results are shown in Tables 2.2 to 2.3.

[0388] Example 4 Measurement of the activity of wild-type RNA ligase 2 samples To determine the first-round backbone, a group of nine enzymes were selected based on their homology with SEQ ID NO: 254. To test their activity, the protein concentrations for each test were determined at 0.27 g / L, 0.13 g / L, 0.07 g / L, 0.03 g / L, and 0.0033 g / L in a reaction mixture containing 20 g / L of short-polymer mixture A (where each oligonucleotide was at 1.2 mM (Table 2.1)) and 26.6% (v / v) reaction buffer. The reaction mixture was incubated in a Multitron incubator at 33 °C for 20 h with shaking at 700 rpm. Then, EDTA was added to each reaction to a final concentration of 10 mM, and the samples were analyzed by HPLC to quantify the residual fragment concentration and product yield.

[0389] In the above description of oligonucleotides, "-" indicates a phosphate ester bond; "*-" indicates a thiopho...

Claims

1. An engineered RNA ligase 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or ... The reference sequences 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

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%, 97%, 98%, 99% or higher sequence identity with respect to the sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence is relative to the sequence corresponding to SEQ ID NO: The reference sequence of residues 12 to 343 of 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 or the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 contains one or more substitutions.

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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or with respect 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 343 of SEQ ID NO: 2 or with respect 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%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 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%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 4-220, 224-252, and 270-958, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

6. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase includes at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, 156, 167, 168. Substitutions at residues 170, 171, 174, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

7. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 11P, 12S, 14F / R / T / W, 16R, 17P, 23M, 25A / G / K / L / W, 26F / G / H / L / P / R / S / V, 30A / C / H / R, 32R, 33A / K, 38D, 40E, 52L, 57T, 60R, 68W, 69L, 71K, 73T, 75L / Q / T, 79W, 82I, 83P, 92A / E / G / M / N / S, 93A, 95E / V, 96A / S, 98D / E / P / R / S, 100R, 101G, 102T, 103I, 111L, 114F / G / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 118G / L / Q, 119A / E / S / T, 120P, 135Q / T, 136A, 141A / C / E / T, 142H / R , 145D, 147L, 156C / Y, 167A, 168A, 170E, 171E, 174Q, 177I / L, 179F, 183V, 184L / M / P / R, 185H / S, 188S, 189L / V, 1 90F, 191K, 192V, 193N, 196K, 200S / T, 202W, 205R / V, 206R, 207S / T, 209E / L, 221E, 229A, 254G, 255E / S, 259V, 270 A, 271I, 287A, 288A, 289A / H / S / T, 292E / Y, 295R, 296E / L / R / V / W, 300E / K, 307A, 320D / K, 327I / Q / R / W, 332R, 333A / E / S / T, 334S, 335E / H / K / L, 336S, 337G, 339K / R, 342I / T, 343P, 339K / R or 342I / T or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

8. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions at amino acid positions 33, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

9. The engineered RNA ligase according to any one of claims 1 to 5 and 8, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 33A / K, 38D, 71K, 73T, 75L / Q / T, 96A / S, 98D / E / P / R / S, 101G, 114F / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 136A, 156C / Y, 179F, 184L / M / P / R, 191K, 196K, 221E, 289A / H / S / T, 292E / Y, 296E / L / R / V / W, 320D / K, 335E / H / K / L, 336S, or 339K / R, or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ ID. The reference sequence of residues 12 to 343 of NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

10. The engineered RNA ligase according to any one of claims 1 to 5 and 8, wherein the amino acid sequence of the engineered RNA ligase comprises at least substituted or amino acid residues 33A, 38D, 71K, 73T, 75T, 96S, 98S, 101G, 114N, 117W, 136A, 156C, 179F, 184M, 191K, 196K, 221E, 289H, 292E, 296W, 320D, 335E / K, 336S, or 339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

11. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase comprises at least a set of substitutions or substitutions at amino acid positions 179, 33 / 101 / 221 / 320 / 335 / 336 / 339, 38 / 71 / 114 / 184 / 191 / 196, 117, 292, 75 / 136 / 296, 98, 73 / 96 / 98 / 156 / 335 or 289, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

12. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase comprises at least a set of substitutions or amino acid residues 179F, 33A / 101G / 221E / 320D / 335E / 336S / 339R, 38D / 71K / 114N / 184M / 191K / 196K, 117W, 292E, 75T / 136A / 296W, 98S, 73T / 96S / 98Y / 156C / 335K or 289H, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

13. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions at amino acid positions 14, 32, 33, 38, 40, 57, 60, 71, 75, 82, 92, 96, 98, 101, 114, 141, 142, 145, 170, 171, 174, 179, 184, 191, 193, 196, 202, 205, 207, 209, 221, 255, 320, 327, 339, or 342, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

14. The engineered RNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 179F, 184P, 196K, 92A, 40E, 82I, 32R, 207T, 33K, 114K, 171E, 33A, 221E, 207S, 202W, 101G, 184R, 141C, 60R, 184L, 98R, 96S, 20 5V, 191K, 193N, 141A, 320K, 174Q, 98D, 71K, 142R, 57T, 320D, 205R, 209L, 145D, 14R, 209E, 342I, 255E, 327Q, 170E, 141E, 98P, 339K, 38D, 142H, 75Q, 184M, 342T, or 339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 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, wherein the amino acid sequence of the engineered RNA ligase comprises at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

16. The engineered RNA ligase of claim 1, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or set of substitutions for the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582.

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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs in SEQ ID NOs: 4-220, 224-252, and 270-958.

19. 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582. The reference sequences NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

20. 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 higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or with respect to the reference sequence corresponding to SEQ ID NO: 4-220, 224-252 and 270-958. The reference sequences 4, 106, 218, 286, 396, 436, 520, 552 or 582 contain one or more substitutions.

21. The engineered RNA ligase of claim 19 or 20, wherein the amino acid sequence of the engineered RNA ligase comprises at least the amino acid positions 11, 12, 14, 16, 17, 23, 25, 26, 30, 32, 33, 38, 40, 52, 57, 60, 68, 69, 71, 73, 75, 79, 82, 83, 92, 93, 95, 96, 98, 100, 101, 102, 103, 111, 114, 117, 118, 119, 120, 135, 136, 141, 142, 145, 147, 156, 167, 168, Substitution at 170, 171, 174, 177, 179, 183, 184, 185, 188, 189, 190, 191, 192, 193, 196, 200, 202, 205, 206, 207, 209, 221, 229, 254, 255, 259, 270, 271, 287, 288, 289, 292, 295, 296, 300, 307, 320, 327, 332, 333, 334, 335, 336, 337, 339, 342 or 343 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ. The reference sequence of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

22. The engineered RNA ligase according to any one of claims 19 to 21, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 11P, 12S, 14F / R / T / W, 16R, 17P, 23M, 25A / G / K / L / W, 26F / G / H / L / P / R / S / V, 30A / C / H / R, 32R, 33A / K, 38D, 40E, 52L, 57T, 60R, 68W, 69L, 71K, 73T, 75L / Q / T, 7 9W, 82I, 83P, 92A / E / G / M / N / S, 93A, 95E / V, 96A / S, 98D / E / P / R / S / Y, 100R, 101G, 102T, 103I, 111L, 114F / G / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 118G / L / Q, 119A / E / S / T, 120P, 135Q / T, 136A, 141A / C / E / T, 142H / R, 145D, 147L, 156C / F / Y, 167A, 168A, 170E, 171E, 174Q, 177I / L, 179F / L, 183V, 184L / M / P / R, 185H / S, 188S, 189L / V , 190F, 191K, 192V, 193N, 196K, 200S / T, 202W, 205R / V, 206R, 207S / T, 209E / L, 221E, 229A, 254G, 255E / S, 259V, 27 0A, 271I, 287A, 288A, 289A / H / K / S / T, 292E / Y, 295R, 296E / L / R / V / W, 300E / K, 307A, 320D / K, 327I / Q / R / W, 332R, 333A / E / S / T, 334S, 335E / H / K / L, 336S, 337G, 339K / R, 342I / T, 343P, 339K / R or 342I / T or combinations thereof, wherein the amino acid position is relative to the corresponding SEQ The reference sequence of residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

23. The engineered RNA ligase according to any one of claims 19 to 22, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution at amino acid positions 33, 38, 71, 73, 75, 96, 98, 101, 114, 117, 136, 156, 179, 184, 191, 196, 221, 289, 292, 296, 320, 335, 336, or 339, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582.

24. The engineered RNA ligase according to any one of claims 19 to 23, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 33A / K, 38D, 71K, 73T, 75L / Q / T, 96A / S, 98D / E / P / R / S / Y, 101G, 114F / G / K / N / P / R / S / V, 117A / C / F / G / H / I / K / L / M / R / S / T / V / W, 136A, 156C / F / Y, 179F / L, 184L / M / P / R, 191K, 196K, 221E, 289A / H / K / S / T, 292E / Y, 296E / L / R / V / W, 320D / K, 335E / H / K / L, 336S or 339K / R or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or relative to the reference sequence corresponding to SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

25. The engineered RNA ligase of any one of claims 19 to 23, wherein the amino acid sequence of the engineered RNA ligase comprises at least substituted or amino acid residues 33A, 38D, 71K, 73T, 75T, 96S, 98S / Y, 101G, 114N, 117W, 136A, 156C, 179F, 184M, 191K, 196K, 221E, 289H, 292E, 296W, 320D, 335E / K, 336S, or 339R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: The reference sequence is 4, 106, 218, 286, 396, 436, 520, 552 or 582.

26. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

27. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

28. The engineered RNA ligase of claim 26 or 27, wherein the amino acid sequence of the engineered RNA ligase comprises at least the amino acid positions 33 / 101 / 221 / 320 / 335 / 336 / 339, 71 / 92 / 171 / 184 / 202, 40 / 92 / 171 / 202 / 327, 196 / 300 / 335 / 339, 33 / 184 / 221 / 300 / 320, 335 / 336 / 339, 184 / 196 / 221 / 336, 33 / 184 / 320 / 335 / 336, 207 / 209 / 221 / 300 / 320 / 336 / 339, 101 / 196 / 221 / 300 / 335 207 / 209 / 300 / 320, 40 / 170 / 171 / 202 / 327, 33 / 101 / 184 / 196 / 209 / 336 / 339, 40 / 71 / 92 / 171 / 184 / 327, 207 / 335 / 336 / 339, 33 / 101 / 196 / 300 / 320 / 335 / 336 / 339, 57 / 171 / 179 / 184 / 202 / 327, 57 / 92 / 202 / 327, 33 / 101 / 207 / 221 / 300 / 335 / 336 / 339, 33 / 221 / 336, 196 / 221 / 300 / 335 / 336 / 339, 33 / 184 / 221 / 3 36 / 339, 33 / 184 / 196 / 221 / 300, 101 / 196 / 207 / 209 / 221 / 300 / 336 / 339, 101 / 184 / 335 / 336 / 339, 33 / 184 / 196 / 320 / 336 / 339, 33 / 184 / 196 / 300 / 320 / 335 / 336 / 339, 184 / 320 / 335 / 336 / 339, 184 / 196 / 207 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 320 / 335 / 336 / 339, 33 / 196 / 209 / 221 / 339, 184 / 320 / 335 / 336 / 339 101 / 209 / 335 / 336 / 339, 101 / 196 / 209 / 339, 40 / 71 / 82 / 170 / 171 / 179 / 202 / 327, 320 / 335 / 336 / 339, 101 / 184 / 300 / 335 / 336 / 339, 33 / 101 / 196 / 209 / 33 5 / 336 / 339, 33 / 196 / 300 / 320, 221 / 335, 33 / 184 / 196 / 207 / 221, 221 / 335 / 336 / 339, 196 / 209 / 300 / 335 / 336 / 339, 184 / 196 / 300 / 320, 57 / 171 / 202 / 327,Substitutions or sets of substitutions at positions 40 / 71 / 171 / 184, 184 / 196 / 221, 33 / 196 / 207 / 335 / 336, 32 / 57 / 71 / 202 / 327 / 342, 33 / 196 / 221 / 336, 101 / 221 / 300 / 320 / 335 / 336 / 339, 33 / 101 / 207 / 221, 101 / 184 / 196 / 209 / 221, 33 / 207 / 209 / 300 / 335 / 336 / 339, or 33 / 83 / 184 / 196 / 300 / 336, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO:

4.

29. The engineered RNA ligase according to any one of claims 26 to 28, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions or sets of substitutions or amino acid residues 33A / 101G / 221E / 320D / 335E / 336S / 339R, 71K / 92A / 171E / 184P / 202W, 40E / 92A / 171E / 202W / 327Q, 196K / 300K / 335E / 339R, 33A / 184R / 221E / 300K / 320D, 335E / 336S / 339R, 184P / 196K / 221E / 336S, 33K / 184R / 320D / 335E / 336S, 207T / 209L / 221E / 300K / 320D / 336S / 339R, 101G / 196K / 221E / 300K / 335E, 207T / 209L / 300K / 320D, 40E / 170E / 171E / 202W / 327Q, 33K / 101G / 184 R / 196K / 209L / 336S / 339R, 40E / 71K / 92A / 171E / 184P / 327Q, 207T / 335E / 33 6S / 339R, 33K / 101G / 196K / 300K / 320D / 335E / 336S / 339R, 57T / 171E / 179L / 184P / 202W / 327Q, 57T / 92A / 202W / 327Q, 33K / 101G / 207T / 221E / 300K / 335 E / 336S / 339R, 33K / 221E / 336S, 196K / 221E / 300K / 335E / 336S / 339R, 33K / 1 84P / 221E / 336S / 339R, 33A / 184P / 196K / 221E / 300K, 101G / 196K / 207T / 209 L / 221E / 300K / 336S / 339R, 101G / 184R / 335E / 336S / 339R, 33A / 184L / 196K / 320D / 336S / 339R, 33A / 184P / 196K / 300K / 320D / 335H / 336S / 339R, 184L / 3 20D / 335E / 336S / 339R, 184L / 196K / 207T / 209L / 300K / 335E / 336S / 339R, 18 4R / 196K / 320D / 335E / 336S / 339R, 33K / 196K / 209L / 221E / 339R, 184R / 320D / 335E / 336S / 339R、101G / 209L / 335E / 336S / 339R、101G / 196K / 209L / 339R、40E / 71K / 82I / 170E / 171E / 179L / 202W / 327Q, 320D / 335E / 336S / 339R, 101G / 184L / 3 00K / 335E / 336S / 339R, 33K / 101G / 196K / 209L / 335E / 336S / 339R, 33K / 196K / 300K / 32 0D、221E / 335E、33K / 184R / 196K / 207S / 221E、221E / 335E / 336S / 339R、196K / 209L / 30 0K / 335E / 336S / 339R, 184P / 196K / 300K / 320D, 57T / 171E / 202W / 327Q, 40E / 71K / 171E / 184P、184L / 196K / 221E、33A / 196K / 207S / 335E / 336S、32R / 57T / 71K / 202W / 327Q / 3 42I, 33A / 196K / 221E / 336S, 101G / 221E / 300K / 320D / 335E / 336S / 339R, 33K / 184P / 32 0D / 335H / 336S, 33K / 101G / 207S / 221E, 101G / 184R / 196K / 209L / 221E, 33A / 207S / 209L / 300K / 335E / 336S / 339R or 33K / 83P / 184P / 196K / 300K / 336S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO:

4.

30. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or with respect to the reference sequence corresponding to SEQ ID NO:

106.

31. The engineered RNA ligase of claim 20, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to even-numbered residues of SEQ ID NO: 218-220 and 224-252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106 or relative to the reference sequence corresponding to SEQ ID NO:

106.

32. The engineered RNA ligase of claim 30 or 31, wherein the amino acid sequence of the engineered RNA ligase comprises at least the amino acid positions 38 / 71 / 114 / 184 / 191 / 196, 141 / 170 / 171 / 174, 114, 57 / 60 / 170 / 171 / 174 / 192 / 196 / 320, 60 / 92 / 170 / 171, 141 / 174 / 196, 57 / 170 / 171 / 174, 1 Substitutions or sets of substitutions at positions 41 / 184, 57 / 60 / 141 / 170 / 171 / 174 / 192, 60 / 170 / 174, 141 / 170 / 171, 69 / 71 / 114 / 184, 38 / 40, 40 / 71 / 98 / 184 / 259, 32 / 60 / 141 / 170 / 171 / 174 / 196 / 320, 32 / 170 / 207 / 320, or 196, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO:

106.

33. The engineered RNA ligase according to any one of claims 30 to 32, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions or sets of substitutions or amino acid residues 38D / 71K / 114N / 184M / 191K / 196K, 141A / 170E / 171E / 174Q, 114R, 57T / 60R / 170E / 171E / 174Q / 192V / 196K / 320K, 60R / 92A / 170E / 171E, 141A / 174Q / 196K, 57T / 170E / 171E / 17 4Q, 141T / 184P, 57T / 60R / 141A / 170E / 171E / 174Q / 192V, 60R / 170E / 174Q, 141C / 170E / 171E, 69L / 71K / 114N / 184M, 38D / 40E, 40E / 71K / 98R / 184M / 259V, 32R / 60R / 141A / 170E / 171E / 174Q / 196K / 320K, 32R / 170E / 207T / 320K or 196K, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 106, or relative to the reference sequence corresponding to SEQ ID NO:

106.

34. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO:

218.

35. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218 or with respect to the reference sequence corresponding to SEQ ID NO:

218.

36. The engineered RNA ligase of claim 34 or 35, wherein the amino acid sequence of the engineered RNA ligase comprises at least a set of substitutions or substitutions at amino acid positions 102, 17, 190, 16, 68, 23, 270, 117 / 229 or 117, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to the reference sequence corresponding to SEQ ID NO:

218.

37. The engineered RNA ligase of any one of claims 34 to 36, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substitutions or substitution set 102T, 17P, 190F, 16R, 68W, 23M, 270A, 117K / 229A, 117W, 117T, 117H, 117A, 117M, 117G, 117I, 117K, 117R, 117S, 117V, 117F, 117C, or 117L, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 218, or relative to the reference sequence corresponding to SEQ ID NO:

218.

38. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO:

286.

39. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286 or with respect to the reference sequence corresponding to SEQ ID NO:

286.

40. The engineered RNA ligase of claim 38 or 39, wherein the amino acid sequence of the engineered RNA ligase comprises at least a set of substitutions at amino acid positions 296, 114 / 117, 26, 30, 117 / 118, 117 / 119, 14, 183, 117, 185, 307, 12, 93 / 135, 135, 92, 288, 117 / 120, 79, 100, 287, 292, 25, 75, 327, or 111, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286, or relative to the reference sequence corresponding to SEQ ID NO:

286.

41. The engineered RNA ligase according to any one of claims 38 to 40, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions or sets of substitutions or amino acid residues 296E, 114V / 117K, 26S, 30C, 117K / 118G, 114P / 117K, 114S / 117K, 296L, 117K / 119S, 26P, 14F, 114G / 117K, 114F / 117K, 117K / 119A, 183V, 26R, 117K, 296R, 185S, 26F, 117K / 118G, 114P / 117K, 296R, 185S, 26F, 117K / 118G, 114P / 118K, 114S / 118K, 296L, 117K / 118K, 296L, 118K / 119 ... 8L, 307A, 30R, 12S, 93A / 135T, 117K / 118Q, 185H, 135Q, 92M, 288A, 92N, 14W, 117K / 119T, 92S, 92G, 117K / 120P, 79W, 100R, 287A, 117K / 119E, 296V, 292E, 26H, 296W, 30H, 26G, 30A, 25W, 25L, 26V, 26L, 92E, 14T, 75T, 25G, 327W, or 111L, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 286, or relative to the reference sequence corresponding to SEQ ID NO:

286.

42. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO:

396.

43. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396 or with respect to the reference sequence corresponding to SEQ ID NO:

396.

44. The engineered RNA ligase of claim 42 or 43, wherein the amino acid sequence of the engineered RNA ligase comprises at least the amino acid positions 102 / 119, 75 / 102, 75 / 102 / 117, 75 / 119, 75 / 136 / 296, 75 / 327, 75 / 117 / 119, 119, 119 / 296, 14 / 102 / 119, 14 / 102 / 296, 14 / 75, 14 / 75 / 119, 14 / 75 / 119 / 327, 14 / The substitutions or sets of substitutions at 75 / 117 / 119, 14 / 75 / 117 / 119 / 296, 14 / 119, 14 / 296, 14 / 30 / 270, 14 / 25, 14 / 117, 14 / 117 / 119, 14 / 117 / 119 / 296, 14 / 117 / 296 / 327, 26 / 75 / 327, 25 / 75, 25 / 30 / 102, 117, 117 / 119, or 117 / 119 / 296, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO:

396.

45. The engineered RNA ligase according to any one of claims 42 to 44, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions or sets of substitutions or amino acid residues 102T / 119E, 75T / 102T, 75T / 102T / 117M, 75T / 119E, 75T / 136A / 296W, 75T / 327W, 75T / 117K / 119E, 119E, 119E / 296W, 14T / 102T / 119E, 14T / 102T / 296W, 14T / 75T, 14T / 75T / 119E, 14T / 75T / 119E / 327W, 14T / 75 T / 117M / 119E, 14T / 75T / 117M / 119E / 296W, 14T / 119E, 14T / 296W, 14T / 30H / 270A, 14T / 25G, 14T / 117K, 14T / 117K / 119E, 14T / 117M / 119E, 14T / 117M / 119E / 296W, 14T / 117M / 296W / 327W, 26V / 75T / 327W, 25L / 75T, 25W / 30H / 102T, 117M, 117M / 119E, or 117M / 119E / 296W, wherein the amino acid position is relative to the corresponding SEQ The reference sequence of residues 12 to 343 of SEQ ID NO: 396, or relative to the reference sequence corresponding to SEQ ID NO:

396.

46. ​​The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO: 436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or relative to the reference sequence corresponding to SEQ ID NO:

436.

47. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436 or with respect to the reference sequence corresponding to SEQ ID NO:

436.

48. The engineered RNA ligase of claim 46 or 47, wherein the amino acid sequence of the engineered RNA ligase contains at least substitutions at amino acid positions 332, 156, 177, 333, 96, 11, 189, 327, 25, 73, 98, 335, 95, 188, or 52, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to the reference sequence corresponding to SEQ ID NO:

436.

49. The engineered RNA ligase of any one of claims 46 to 48, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 332R, 156C, 177L, 333A, 96A, 11P, 156Y, 189L, 177I, 327I, 333T, 189V, 25K, 73T, 333S, 98S, 335L, 327R, 95E, 188S, 98E, 52L, or 335K or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 436, or relative to the reference sequence corresponding to SEQ ID NO:

436.

50. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO:

520.

51. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520 or with respect to the reference sequence corresponding to SEQ ID NO:

520.

52. The engineered RNA ligase of claim 50 or 51, wherein the amino acid sequence of the engineered RNA ligase comprises at least a set of substitutions at amino acid positions 96 / 98 / 156 / 189 / 335, 73 / 96 / 98 / 189 / 335, 73 / 177 / 189, 73 / 98 / 177 / 189 / 335, 25 / 73 / 95 / 96 / 98 / 189 / 327, 25 / 98 / 189, 73 / 95 / 96 / 156 / 177, 73 / 335, 73 / 96 / 98 / 156 / 335, or 73 / 177 / 189 / 333, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO:

520.

53. The engineered RNA ligase according to any one of claims 50 to 52, wherein the amino acid sequence of the engineered RNA ligase comprises at least the set of substitutions or amino acid residues 96S / 98Y / 156C / 189V / 335K, 73T / 96S / 98E / 189L / 335K, 73T / 177L / 189V, 73T / 98E / 177L / 189V / 335K, 25A / 73T / 95E / 96A / 98E / 189V / 327I, 25K / 98Y / 189V, 73T / 95E / 96S / 156Y / 177L, 73T / 335K, 73T / 96S / 98Y / 156C / 335K, or 73T / 177L / 189V / 333T, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: The reference sequence of residues 12 to 343 of 520, or relative to the reference sequence corresponding to SEQ ID NO:

520.

54. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO: 552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or relative to the reference sequence corresponding to SEQ ID NO:

552.

55. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552 or with respect to the reference sequence corresponding to SEQ ID NO:

552.

56. The engineered RNA ligase of claim 54 or 55, wherein the amino acid sequence of the engineered RNA ligase contains at least substitutions at amino acid positions 292, 147, 255, 295, 75, 300, 333, 200, 289, 95, or 168, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to the reference sequence corresponding to SEQ ID NO:

552.

57. The engineered RNA ligase according to any one of claims 54 to 56, wherein the amino acid sequence of the engineered RNA ligase comprises at least the substituted or amino acid residues 292Y, 147L, 255S, 295R, 75L, 300E, 333E, 200S, 289S, 95V, 289A, 289T, 168A, 289H, or 200T, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 552, or relative to the reference sequence corresponding to SEQ ID NO:

552.

58. The engineered RNA ligase of claim 19 or 20, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO: 582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or relative to the reference sequence corresponding to SEQ ID NO:

582.

59. The engineered RNA ligase of claim 20, 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%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582 or with respect to the reference sequence corresponding to SEQ ID NO:

582.

60. The engineered RNA ligase of claim 58 or 59, wherein the amino acid sequence of the engineered RNA ligase comprises at least the amino acid positions 95 / 156 / 337, 189 / 200 / 271, 200 / 271 / 289 / 337, 200 / 271 / 289, 189 / 200, 156 / 200 / 289, 156 / 168 / 189 / 271 / 300, 156 / 189 / 200 / 289, 156 / 168 / 289, 95 / 156 / 271 / 289, 156 / 333, 95 / 200 / 289 / 337, 168 / 200 / 289, 189 / 200 / 289, 333 / 337, 103 / 156 / 271, 95 / 156 / 200 / 300 / 333, 156 / 271 / 289 / 333, 95 / 156 / 271, 95 / 189 / 289, 95 / 156 / 168 / 189 / 271 / 289 / 300, 189 / 271 / 289, 95 / 168 / 271 / 300, 156 / 189 / 289 / 337, 200 / 271, 156 / 189 / 333, 189 / 289, 156 / 189 / 200 / 300 / 333 / 337, 156 / 189 / 200 / 271 / 289 / 337, 156 / 200 / 271 / 289 / 333 / 337, 168 / 271 / 300, 289 / 337, 156 / 168 / 337, 156 / 168 / 189 / 289, 156 / 271 / 289, 189 / 300, 156 / 289 / 300 / 337, 168 / 189 / 289, 95 / 156 / 168 / 300, 156 / 200 / 300 / 337, 289 / 333 / 337, 95 / 189 / 200 / 300 / 337, 95 / 156 / 189 / 200 / 271 / 333, 200 / 271 / 289 / 300, 289 / 333, 189 / 271 / 300, 95 / 300, 95 / 189 / 333 / 337, 95 / 189 / 271 / 300, 95 / 18 9 / 200 / 289, 168, 156 / 200 / 271 / 289 / 337, 95 / 168 / 289, 156 / 289, 95 / 156 / 189 / 271 / 289, 156 / 168 / 189 / 200 / 289 / 333, 156 / 300 / 333 / 337, 95 / 156 / 189 / 2 00 / 271 / 337, 271, 156 / 271, 156 / 271 / 300 / 333, 95 / 168 / 200, 168 / 271, 95 / 200 / 333, 189 / 200 / 271 / 289 / 333, 95 / 168 / 189 / 333 / 337, 189, 189 / 333 / 337,156 / 189 / 271 / 289 / 333、289、95 / 156 / 200 / 271 / 289、289 / 300 / 333、95 / 189 / 289 / 337、95 / 156 / 168 / 189 / 289、271 / 289、333、168 / 337、156 / 189 / 271 / 289、95 / 156 / 189 / 337、156 / 189 / 271 / 300、156 / 168 / 289 / 333、168 / 189 / 289 / 333、95 / 333、333 / 343、156 / 189 / 289、95 / 289 / 300、95 / 156 / 189 / 200 / 300 / 333 / 337、189 / 200 / 271 / 289 / 337、95 / 156 / 168 / 189 / 289 / 337、95 / 289、156 / 289 / 333 / 337、156 / 289 / 337、337、168 / 289、95 / 156 / 300、95 / 156 / 289、95 / 189 / 271 / 333 / 337、95 / 168 / 300、95 / 156 / 168 / 271 / 333 / 337、95 / 189 / 289 / 333 / 337、156 / 168 / 200 / 289 / 334、95 / 156 / 168 / 189 / 271 / 289、156 / 189 / 200 / 289 / 337、200 / 289、200 / 289 / 333 / 337、95 / 168 / 189 / 200 / 289、168 / 200、156 / 168 / 271 / 289、189 / 206 / 289、156 / 200 / 289 / 333 / 335、156 / 200 / 289 / 333、189 / 200 / 289 / 333、189 / 289 / 335、189 / 254 / 289 / 333、167 / 189 / 206 / 289 / 333 / 335、156 / 200 / 333、156 / 200 / 289 / 335、156 / 189 / 289 / 335、335、156 / 167 / 189 / 289、156 / 189 / 200 / 289 / 335、200 / 289 / 333 / 335、156 / 189 / 200 / 289 / 333、254 / 333 / 335、189 / 254 / 333 / 335、189 / 335、156 / 189 / 254 / 289、156 / 289 / 333、289 / 335、189 / 200 / 254 / 289 / 333、156 / 189 / 289 / 333、25 / 156 / 189 / 289、156 / 189 / 206 / 289 / 333 / 335、289 / 333 / 335、156 / 254 / 289 / 333、156 / 289 / 333 / 335、254 / 289, 200 / 289 / 333, 156 / 189 / 200, 156 / 254 / 333 / 335, 156, 254 / 289 / 335, 156 / 206 / 289, 156 / 289 / 335, 206 / 289 / 333 / 335, 156 / 189 / 254 / 289 / 333, 156 / 254 / 289, 200 / 289 / 335, 1 Substitutions or sets of substitutions at positions 56 / 189 / 206 / 289, 156 / 189 / 200 / 206 / 254 / 289 / 333, 25 / 254 / 333, 189 / 289 / 333 / 335, 156 / 189 / 200 / 289 / 333 / 335, 254 / 289 / 333, or 156 / 200 / 333 / 335, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO:

582.

61. The engineered RNA ligase according to any one of claims 58 to 60, wherein the amino acid sequence of the engineered RNA ligase comprises at least substitutions or sets of substitutions or amino acid residues 95V / 156Y / 337G, 189V / 200T / 271I, 200S / 271I / 289A / 337G, 200T / 271I / 289A, 189V / 200S, 156Y / 200S / 289T, 156F / 168A / 189V / 271I / 300E, 156Y / 189V / 200T / 289A, 156F / 168A / 289K, 95V / 156Y / 271I / 289K, 156F / 333E, 95V / 200T / 289K / 337G, 168A / 200S / 289A, 189V / 200T / 289K, 333E / 337G, 103I / 1 56F / 271I, 95V / 156F / 200S / 300E / 333E, 156F / 271I / 289T / 333E, 95V / 156F / 271I, 95V / 189V / 289K, 95V / 156Y / 168A / 189V / 271I / 289T / 300E, 189V / 271 I / 289A, 95V / 168A / 271I / 300E, 156Y / 189V / 289A / 337G, 200S / 271I, 156F / 1 89V / 333E, 189V / 289T, 156Y / 189V / 200T / 300E / 333E / 337G, 156Y / 200T / 28 9A, 156F / 189V / 200S / 271I / 289K / 337G, 156Y / 200S / 271I / 289A / 333E / 337G , 168A / 271I / 300E, 289T / 337G, 156Y / 168A / 337G, 156F / 168A / 189V / 289T, 156Y / 271I / 289T, 189V / 300E, 156Y / 289T / 300E / 337G, 168A / 189V / 289T, 95 V / 156F / 168A / 300E, 156Y / 200S / 300E / 337G, 289A / 333E / 337G, 95V / 189V / 200T / 300E / 337G, 95V / 156F / 189V / 200T / 271I / 333E, 200S / 271I / 289A / 300 E, 289A / 333E, 189V / 271I / 300E, 95V / 300E, 95V / 189V / 333E / 337G, 95V / 18 9V / 271I / 300E, 95V / 189V / 200S / 289A, 168A, 156Y / 200S / 271I / 289K / 337G,95V / 168A / 289T、156F / 289A、95V / 156Y / 189V / 271I / 289K、156F / 168A / 189 V / 200S / 289A / 333E、156F / 300E / 333E / 337G、95V / 156F / 189V / 200S / 271I / 337G、156F / 189V / 200T / 289K、156Y / 289T、271I、156Y / 271I、156F / 271I / 3 00E / 333E、95V / 168A / 200S、189V / 200S / 289A、168A / 271I、95V / 200T / 333E、 189V / 200S / 271I / 289A / 333E, 95V / 168A / 189V / 333E / 337G, 189V, 189V / 333E / 337G, 156F / 189V / 271I / 289A / 333E, 289A, 95V / 156F / 200T / 271I / 289K 289A / 300E / 333E, 95V / 189V / 289A / 337G, 95V / 156F / 168A / 189V / 289A, 271I / 289T, 333E, 168A / 337G, 156Y / 289A, 156Y / 189V / 271I / 289K, 95V / 156F / 1 89V / 337G, 189V / 289K, 189V / 289A, 156Y / 189V / 271I / 300E, 156F / 168A / 289A / 333E, 168A / 189V / 289K / 333E, 95V / 333E, 333E / 343P, 156F / 189V / 289A 95V / 289A / 300E、95V / 156Y / 189V / 200S / 300E / 333E / 337G、189V / 200T / 271 I / 289K / 337G、95V / 156Y / 168A / 189V / 289K / 337G、156F / 289K、95V / 289A、15 6Y / 168A / 289A, 156F / 289K / 333E / 337G, 156F / 289K / 337G, 289A / 337G, 337G, 168A / 289A, 95V / 156F / 300E, 95V / 156Y / 289K, 95V / 189V / 271I / 333E / 337 G、95V / 168A / 300E、95V / 156Y / 168A / 271I / 333E / 337G、95V / 189V / 289A / 33 3E / 337G、156F / 168A / 200S / 289A / 334S、95V / 156F / 168A / 189V / 271I / 289T、156Y / 189V / 200T / 289T / 337G、156F / 271I、289K / 300E / 333E、95V / 156Y / 271 I、200S / 289A、200S / 289K / 333E / 337G、95V / 168A / 189V / 200T / 289A、156Y / 2 71I / 289T / 333E, 168A / 200S, 156F / 168A / 271I / 289A, 189L / 206R / 289S, 156F / 200S / 289S / 333E / 335E, 156F / 200S / 289S / 333E, 189V / 200S / 289K / 333E, 1 89L / 289K / 335E, 189V / 254G / 289S / 333E, 189L / 289S, 167A / 189L / 206R / 289S / 333E / 335E, 156F / 200S / 333E, 189L / 200S / 289S, 156Y / 200S / 289K / 335E 156F / 189L / 200S / 289K、156Y / 189L / 289K / 335E、156Y / 200S / 289K / 333E、33 5E、156F / 200S / 289K / 333E、156F / 167A / 189V / 289T、156Y / 189L / 200S / 289S / 335E, 200S / 289S / 333E / 335E, 156Y / 189V / 200S / 289T / 333E, 254G / 333E / 335E, 189L / 254G / 333E / 335E, 289S, 189V / 289S, 189V / 335E, 156F / 289T, 156F / 189V / 254G / 289S、156Y / 289K / 333E、289S / 335E、189L / 200S / 254G / 289S / 333E、156F / 189L / 289S / 333E、156Y / 189V / 289S / 335E、25A / 156Y / 189L / 289S、 156Y / 189V / 206R / 289K / 333E / 335E, 289S / 333E / 335E, 189V / 200S / 289S, 156F / 254G / 289K / 333E, 156Y / 289T / 333E / 335E, 254G / 289T, 200S / 289T / 333E 156Y / 189V / 200S, 156Y / 254G / 333E / 335E, 156F, 254G / 289K / 335E, 200S / 289T, 200S / 289S, 156Y / 206R / 289S, 156F / 289S / 335E, 206R / 289T / 333E / 335E156F / 189V / 254G / 289K / 333E, 156F / 254G / 289T, 200S / 289T / 335E, 156Y / 189V / 206R / 289S, 156Y / 189V / 200S / 206R / 254G / 289T / 333E, 156Y / 333E, 156Y / 189L / 254G / 289T / 333E, 25A / 254G / 333E, 189L / 289S / 333E / 335E, 200S / 289K / 335E, 156Y / 189V / 289S, 156F / 189L / 200S / 289K / 333E / 335E, 289T, 156F / 200S / 289T / 335E, 254G / 289S / 333E, or 156F / 200S / 333E / 335E, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 582, or relative to the reference sequence corresponding to SEQ ID NO:

582.

62. The engineered RNA ligase of claim 1, wherein the amino acid sequence of the engineered RNA ligase comprises at least one substitution shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

63. The engineered RNA ligase of claim 1, wherein the amino acid sequence of the engineered RNA ligase comprises at least a substitution or set of substitutions for the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4, and 12, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582, or relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552, or 582.

64. 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 higher sequence identity with a reference sequence, said reference sequence comprising substitutions or sets of substitutions of the RNA ligase variants shown in Tables 3, 4, 5, 6, 7.1, 7.2, 8, 9, 10, 11.3, 11.4 and 12, wherein said amino acid position is relative to residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582 of said reference sequence, or relative to SEQ ID NO: The reference sequence is 4, 106, 218, 286, 396, 436, 520, 552 or 582.

65. The engineered RNA ligase of claim 1, wherein the engineered RNA ligase comprises an amino acid sequence containing residues 12 to 343 of SEQ ID NO. with even numbers in SEQ ID NO: 4-220, 224-252, and 270-958, or an amino acid sequence containing SEQ ID NO. with even numbers in SEQ ID NO: 4-220, 224-252, and 270-958.

66. The engineered RNA ligase of claim 1, wherein the engineered RNA ligase comprises an amino acid sequence containing residues 12 to 343 of SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582, or an amino acid sequence containing SEQ ID NO: 4, 106, 218, 286, 396, 436, 520, 552 or 582.

67. The engineered RNA ligase according to any one of claims 1 to 66, wherein the engineered RNA ligase has ligase activity for single-stranded and / or double-stranded polynucleotide substrates.

68. The engineered RNA ligase of claim 67, further comprising one or more improved properties compared to a reference RNA ligase.

69. The engineered RNA ligase of claim 68, wherein the improved properties compared to a reference RNA ligase are selected from i) increased activity, ii) increased product yield, iii) increased product yield for polynucleotides having thiophosphate nucleotide internucleotide bonds, iv) increased product yield for oligonucleotides having 2'-modification, and v) increased expression, or any combination of i), ii), iii), iv) and v).

70. The engineered RNA ligase of claim 68 or 69, wherein the reference RNA ligase has a sequence of residues 12 to 343 corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582, or a sequence corresponding to SEQ ID NO: 2, 4, 106, 218, 286, 396, 436, 520, 552 or 582.

71. The engineered RNA ligase according to any one of claims 68 to 70, wherein the reference RNA ligase has a sequence corresponding to residues 12 to 343 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO:

2.

72. The engineered RNA ligase according to any one of claims 1 to 71, wherein the engineered RNA ligase comprises a fusion polypeptide.

73. The engineered RNA ligase according to any one of claims 1 to 72, wherein the engineered RNA ligase is purified.

74. A recombinant polynucleotide encoding an engineered RNA ligase as described in any one of claims 1 to 72.

75. The recombinant polynucleotide of claim 74, comprising a reference polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551 or 581, wherein the recombinant polynucleotide encodes an RNA ligase.

76. The recombinant polynucleotide of claim 74, comprising a reference polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. in SEQ ID NO: 3-219, 223-957 and 269-553, wherein the recombinant polynucleotide encodes an RNA ligase.

77. The recombinant polynucleotide of any one of claims 74 to 76, wherein the polynucleotide sequence is codon-optimized for expression of the encoded engineered RNA ligase.

78. The recombinant polynucleotide of claim 74, comprising a polynucleotide sequence containing nucleotide residues 34 to 1029 of SEQ ID NO. 3, 105, 217, 285, 395, 435, 519, 551 or 581, or a polynucleotide sequence containing SEQ ID NO: 3, 105, 217, 285, 395, 435, 519, 551 or 581.

79. The recombinant polynucleotide of claim 74, comprising a polynucleotide sequence containing nucleotide residues 34 to 1029 of SEQ ID NO. with odd numbers from SEQ ID NO: 3-219, 223-251, and 269-957, or a polynucleotide sequence containing SEQ ID NO. with odd numbers from SEQ ID NO: 3-219, 223-251, and 269-957.

80. An expression vector comprising any one of claims 74 to 79.

81. The expression vector of claim 80, wherein the polynucleotide is operatively linked to a control sequence.

82. The expression vector of claim 81, wherein the control sequence comprises at least a promoter.

83. A host cell comprising the expression vector as described in any one of claims 80 to 82.

84. The host cell of claim 83, comprising a prokaryotic cell or a eukaryotic cell.

85. The host cell of claim 84, comprising bacterial cells.

86. A method for producing engineered RNA ligase polypeptides in host cells, comprising culturing host cells as described in any one of claims 83 to 85 under suitable culture conditions to produce at least one engineered RNA ligase.

87. The method of claim 86, further comprising recovering at least one engineered RNA ligase from the culture and / or the host cell.

88. The method of claim 86 or 87, further comprising purifying the engineered RNA ligase.

89. A composition comprising the RNA ligase as described in any one of claims 1 to 73.

90. The composition of claim 89, further comprising a buffer solution, ATP or dATP, and one or more polynucleotide ligase substrates.

91. A method of linking at least a first polynucleotide chain and a second polynucleotide chain, comprising contacting the first polynucleotide chain and the second polynucleotide chain with an engineered RNA ligase as described in any one of claims 1 to 73 in the presence of a nucleotide substrate under conditions suitable for linking the first polynucleotide chain to the second polynucleotide chain, wherein the first polynucleotide chain includes a 5' end capable of being linked and the second polynucleotide chain includes a 3' end capable of being linked to the 5' end of the first polynucleotide.

92. The method of claim 91, further comprising a third polynucleotide chain, wherein the first polynucleotide chain and the second polynucleotide chain hybridize adjacently to each other on the third polynucleotide chain to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain.

93. The method of claim 92, wherein the third polynucleotide chain is continuous with the first polynucleotide chain or the second polynucleotide chain.

94. The method of claim 92, wherein the third polynucleotide chain is continuous with the first polynucleotide chain and the second polynucleotide chain to form a single continuous polynucleotide ligase substrate.

95. The method of claim 92, wherein the third polynucleotide chain comprises a splint or bridging polynucleotide, wherein the 5' end sequence of the first polynucleotide chain and the 3' end sequence of the second polynucleotide chain hybridize adjacently to each other on the splint or bridging polynucleotide to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain.

96. The method of claim 92, wherein the first polynucleotide chain and the third polynucleotide chain hybridize with each other to form a first double-stranded polynucleotide fragment, and the second polynucleotide chain hybridizes with a fourth polynucleotide chain to form a second double-stranded fragment, wherein the first double-stranded fragment and the second double-stranded fragment have complementary ends capable of base pairing to form a substrate of the engineered RNA ligase.

97. The method of any one of claims 91 to 96, wherein the first polynucleotide chain and / or the second polynucleotide chain comprises RNA or a mixture of RNA and DNA.

98. The method of any one of claims 91 to 97, wherein the 3' end of the second polynucleotide chain is 3'-OH.

99. The method of any one of claims 91 to 98, wherein the 5' end of the first polynucleotide chain is a 5'-phosphate ester.

100. The method of any one of claims 91 to 99, wherein the nucleoside internucleotide bond between the first polynucleotide chain and the second polynucleotide chain comprises a phosphate ester bond.

101. The method of any one of claims 91 to 99, wherein the first polynucleotide chain and / or the second polynucleotide chain comprises one or more nucleotide analogs, wherein the nucleotide analogs comprise modified nucleobases or nucleobase analogs, modified nucleoside sugar residues, modified internucleotide bonds, modified 5' phosphate groups, ligand conjugations, reverse nucleotides, debased nucleotides, or any combination thereof.

102. The method of claim 101, wherein the modified 5'-terminal phosphate group is a phosphate ester analog.

103. The method of claim 102, wherein the phosphate ester analog is a thiophosphate or a monomethyl phosphate.

104. The method of any one of claims 101 to 103, wherein the modified nucleoside sugar residue is 2'-O-alkyl, 2'-halogenated, β-D-ribose-LNA, or α-L-ribose-LNA (locked nucleic acid).

105. The method of any one of claims 91 to 104, wherein the nucleobase analog is xanthine, hypoxanthine, inosine, 7-methylguanine, 2,6-diaminopurine, 5-methylcytosine, 5-hydroxycytosine, 5-bromocytosine, 5-iodocytosine, 2-thiothymidine, 5-fluorouracil, 5-bromouracil, 8-bromoguanine, 8-aminoguanine, or 8-aza-7-deazoguanine.

106. The method of any one of claims 91 to 105, wherein the internucleotide bond is a thiophosphate, phosphate acetate, aminophosphate, methylphosphonate, or phosphonocarboxylic acid ester.

107. The method of any one of claims 91 to 106, wherein the ligand comprises lipids, carbohydrates, or peptides.

108. The method of any one of claims 91 to 107, wherein the nucleotide substrate is ATP or dATP.

109. A kit comprising at least one RNA ligase as claimed in any one of claims 1 to 73.

110. The kit of claim 109, further comprising one or more of a buffer, a nucleotide substrate, a polynucleotide substrate of the ligase, and / or a linker enhancer.

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