Engineered RNA ligase variants

Abstract

The present disclosure relates to modified RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the modified RNA ligase polypeptides. The disclosure also provides methods of using the modified RNA ligase polypeptides or compositions thereof for molecular biology, diagnostic, and other purposes. In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution as provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or a reference sequence corresponding to SEQ ID NO:14.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 476,912, filed December 22, 2022, which is incorporated herein by reference.

[0002] Reference to a sequence listing, table, or computer program The sequence listing submitted concurrently herewith under the file name CX9-231WO1_ST26.xml was created on December 22, 2023, has a file size of 1,145,043 bytes, is a part of the specification, and is incorporated herein by reference.

[0003] The present disclosure provides modified RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the modified RNA ligase polypeptides. The present disclosure also provides methods of using the recombinant RNA ligases or compositions thereof, including for the synthesis of polynucleotides, as molecular biology tools, and for other purposes. [Background technology]

[0004] 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 essential proteins in biological processes. RNA ligases find applications in target sequence profiling, quantification of various RNA species, detection of specific RNA mutations, and polynucleotide synthesis. Known RNA ligases are classified into two groups based on their substrate preference. Single-stranded RNA ligases, also known as RNA ligase 1 or RNA ligase I, can join two RNA or DNA fragments without a hybridizing template. Double-stranded RNA ligases, also known as RNA ligase 2 or RNA ligase II, preferentially join nicks in the RNA duplex. RNA ligase type 1 proteins have been identified in fungi, baculoviruses, archaea, and thermostable archaeal viruses. RNA ligase type 2 enzymes have been identified in vibriophage KVP40, baculoviruses and entomopoxviruses, some parasitic worms, and archaeal species.

[0005] A typical RNA ligase is derived from bacteriophage T4. T4 RNA ligase 1 ligates 5'-phosphoryl nucleic acid donors and 3'-hydroxyl nucleic acid acceptors by connecting them through a phosphodiester bond in an ATP-dependent reaction. Substrates for T4 RNA ligase 1 include ssRNA, ssDNA, and dinucleoside pyrophosphates. T4 RNA ligase 1 is used for ligating ssRNA to DNA, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), ligating oligonucleotide adapters to cDNA, or single-stranded primer extension products for PCR, oligonucleotide synthesis, and various 5' nucleotide modifications of nucleic acids. T4 RNA ligase 2 ligates adjacent 5' phosphates at the ends of RNA and DNA strands to the 3' OH of the RNA strand in the context of nicked dsRNA or RNA / DNA hybrids. T4 RNA ligase 2 shows high homology to DNA ligase and mRNA capping enzyme and is used to seal nicks in dsRNA and dsRNA / DNA hybrids. T4 RNA ligase can act on RNA and DNA, but RNA is the preferred substrate, and DNA is a less efficient acceptor in ligation reactions.

[0006] While T4 RNA ligase has become a useful tool for labeling, circularizing, and ligating RNA and RNA / DNA, there is a need for ligases that ligate polynucleotide substrates more easily and efficiently, including improved ligation of polynucleotides containing nucleotide analogs such as modified sugar residues and non-standard internucleotide linkages. Summary of the Invention

[0007] The present disclosure provides modified RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the modified RNA ligase polypeptides. The present disclosure also provides methods of using the modified RNA ligase polypeptides and compositions thereof for nucleic acid synthesis, diagnostic assays, and other purposes.

[0008]

[0014] In one aspect, the disclosure provides a modified RNA ligase, or functional fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, or to a reference sequence corresponding to the even-numbered SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0009] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204, wherein the amino acid sequence comprises one or more substitutions.

[0010] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14 or to a reference sequence corresponding to SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14 or to a reference sequence corresponding to SEQ ID NO: 14.

[0011] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204, and the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

[0012] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582, wherein the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or to a reference sequence corresponding to SEQ ID NO:14.

[0013] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:24-128, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-128, wherein the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or to a reference sequence corresponding to SEQ ID NO:14.

[0014] In some embodiments, the amino acid sequence of the modified RNA ligase comprises the amino acid sequences at amino acid positions 2, 14, 15, 18, 33, 39, 63, 69, 81, 83, 85, 86, 89, 95, 116, 117, 119, 138, 142, 144, 149, 154, 156, 159, 171, 175, 177, 181, 185, 186, 195, 202, 212, 214, 224, 226, 230, 247, 256, 257, 260, 266, 275, 280, 283, 285, 288, 291, 296, 303, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0015] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 95 or 177, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0016] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0017] In some embodiments, the amino acid sequence of the modified RNA ligase comprises amino acid position(s) 177 / 260 / 307 / 345, 171 / 175 / 183 / 280 / 283 / 303, 95 / 159 / 177 / 260 / 288, 175 / 183 / 247 / 280 / 283 / 296, 177 / 307, 81 / 171 / 183 / 335, 175 / 183, 81 / 175 / 183 / 280 / 296 / 335 / 339, 81 / 183 / 247 / 266, 95 / 177, 171 / 280 / 283 / 296 / 303, 85 / 177 / 260 / 288 / 333 / 345, 177 / 291 / 307 / 333, 81 / 171 / 175 / 183 / 316 / 337, 81 / 171 / 266 / 280 / 283 / 335 / 339, 177, 95 / 159 / 177 / 260 / 280 / 288 / 306 / 345, 171 / 175 / 335, 177 / 260 / 30 / 333, 14 / 95 / 177 / 288 / 307, 95 / 177 / 345, 95 / 159 / 177 / 260 / 285 / 288, 266 / 296, 81 / 283 / 335 / 337, 95 / 159 / 177 / 260 / 342 / 345, 63 / 171 / 175 / 183 / 266 / 280 / 296 / 316, 280 / 335 / 337, 81 / 171 / 280, 81 / 171 / 175 / 296 / 316, 171 / 175 / 316 / 335, 171 / 280 / 283 / 296 / 316, 171 / 283 / 335, 63 / 171 / 175 / 183 / 266 / 283 / 3 03 / 316, 171 / 247 / 266 / 283 / 296 / 303 / 316 / 335, 63 / 81 / 171 / 175 / 183 / 266 / 280 / 283 / 337, 81 / 171 / 303 / 316 / 335, 288 / 307, 14 / 95 / 159 / 177 / 345, 81 / 171 / 175 / 183 / 247 / 266 / 280 / 283, 81 / 247 / 266, 280, 85 / 177 / 260, 63 / 183, 175 / 183 / 247 / 280 / 283 / 316, 291, 247, 171 / 280 / 283, 81 / 171 / 175 / 280 / 316, 333, 171 / 296 / 316, 81 / 171, or 280 / 316 / 335, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0018] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution as provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0019] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions of an RNA ligase variant provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or a reference sequence corresponding to SEQ ID NO:14.

[0020] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0021] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582.

[0022] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204, wherein the amino acid sequence comprises one or more substitutions.

[0023] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582, wherein the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or to a reference sequence corresponding to an even-numbered SEQ ID NO:42 or 204.

[0024] In some embodiments, the amino acid sequence of the modified RNA ligase is , 285, 288, 291, 296, 303, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0025] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 95 or 177, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0026] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0027] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or to a reference sequence corresponding to SEQ ID NO:42, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or to a reference sequence corresponding to SEQ ID NO:42.

[0028] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:130-478, or to a reference sequence corresponding to an even-numbered SEQ ID NO:130-478, and the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to a reference sequence corresponding to SEQ ID NO:42.

[0029] In some embodiments, the amino acid sequence of the modified RNA ligase comprises amino acid position(s) 149 / 156 / 195, 15 / 149 / 186 / 224 / 317, 15 / 156 / 195 / 224 / 226 / 317 / 331, 15 / 195 / 317 / 331, 149 / 224 / 331, 15 / 195 / 224 / 226 / 317 / 331, 15 / 33 / 86 / 149 / 154 / 195 / 317, 15 / 86 / 154 / 156 / 186 / 195 / 224 / 226 / 331, 15 / 149 / 224 / 317 / 331, 15 / 154 / 156 / 186 / 317 / 33 1, 15 / 33 / 149 / 224 / 226 / 331, 15 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 186 / 224 / 331, 195 / 224 / 331, 15 / 86 / 156 / 186 / 195 / 226 / 317 / 331, 15 / 195 / 22 / 331 , 156 / 186 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 224 / 226, 33 / 156 / 186 / 195 / 331, 15 / 86 / 149 / 154 / 156 / 317 / 331, 15 / 186 / 317 / 331, 15 / 86 / 149 / 154 / 195 / 33 1, 15 / 33 / 149 / 154 / 156 / 226, 15 / 149 / 154 / 186 / 317 / 331, 195 / 331, 15 / 86 / 156 / 186 / 195 / 331, 149 / 156 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 186 / 317 / 331, 15 / 154 / 186 / 195, 15 / 86 / 195 / 224 / 226, 230, 15 / 33 / 156 / 331, 15 / 33 / 156 / 195 / 224 / 226, 33 / 149 / 156 / 317, 15 / 33 / 186 / 195, 334, 15 / 154 / 156 / 224 / 317 , 15 / 149 / 186 / 331, 15 / 86 / 149 / 186 / 195 / 317, 15 / 33 / 154 / 195, 15 / 156 / 186 / 331, 15 / 149 / 186 / 317, 15 / 33 / 156 / 186 / 214 / 224 / 331, 15 / 86 / 186 / 224 / 22 6 / 317, 15 / 154 / 331, 15 / 149 / 154 / 226, 15 / 33 / 195 / 226, 15 / 224 / 317 / 331, 15 / 86 / 186 / 195 / 317, 226 / 317 / 331, 330, 89, 15 / 149 / 186, 156 / 195 / 331, 156,15 / 86 / 156 / 186 / 331, 275, 15 / 33 / 186 / 224, 15 / 186 / 317, 15 / 33 / 186 / 226 / 317, 15 / 149 / 154 / 156 / 331, 15 / 154 / 226 / 317, 15 / 86 / 156 / 186 / 195 / 317, 257, 15 / 154 / 156 / 186, 33 / 186 / 224 , 335, 15 / 86 / 156 / 186 / 195 / 224 / 331, 154 / 156 / 317, 39, 2, 15 / 224 / 226 / 331, 15 / 33 / 149 / 224, 89 / 307, 15 / 154 / 156 / 317, 15 / 186 / 331, 15 / 33 / 154 / 317, 15 / 33 / 195 / 224 / 317, 15 / 156 / 22 4 / 226, 154 / 186 / 331, 185, 15 / 18 / 149 / 186 / 195, 86 / 149 / 154 / 156, 15 / 33 / 149 / 331, 326, 202, 119, 212, 15 / 33 / 156 / 317, 280, 142, 15 / 149, 15 / 33 / 86 / 156 / 195, 315, 256, 15 / 33 / 156 / 1 At least one substitution or set of substitutions at residues 86, 138, 117, 116, 316, 69, 310, 317, 15 / 195 / 331, 15 / 33 / 331, 314, or 144, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42 or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0030] In some embodiments, the amino acid sequence of the modified RNA ligase is at amino acid position(s) 15 / 149 / 154 / 156 / 186 / 195 / 331, 149 / 154 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 154 / 186 / 195, 149 / 156 / 186 / 195 / 224 / 226 / 317, 15 / 149 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 226 / 331, 15 / 149 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 15 4 / 195 / 317, 15 / 149 / 154 / 195 / 224 / 317, 15 / 149 / 156 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 195 / 224 / 226 / 317 / 331, 149 / 195 / 224, 86 / 149 / 154 / 156 / 186 / 195 / 331, 15 / 33 / 149 / 156 / 195 / 224 / 317 / 331, 15 / 149 / 154 / 156 / 186 / 195 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 224 / 331, 15 / 149 / 154 / 156 / 195 / 224, 15 / 149 / 154 / 156 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 186 / 195 / 224 / 317, 33 / 149 / 156 / 186 / 224 / 331, 15 / 33 / 86 / 149 / 195 / 224 / 331, 15 / 86 / 149 / 154 / 156 / 186 / 195 / 331, 149 / 186 / 195 / 331, 15 / 154 / 156 / 186 / 226 / 331, 15 / 149 / 154 / 186 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 33 / 149 / 15 4 / 156 / 195 / 317 / 331, 15 / 33 / 154 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 149 / 186 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 149 / 154 / 156 / 224 / 2 26 / 317 / 331, 15 / 33 / 149 / 156 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 224 / 226 / 331, 154 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 224 / 317 / 331,15 / 149 / 154 / 156 / 186 / 195 / 224 / 226, 15 / 33 / 149 / 195 / 317 / 331, 15 / 149 / 154 / 224 / 331, 149 / 154 / 156 / 195 / 224 / 226 / 331, 15 / 149 / 154 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 195, 15 / 33 / 149 / 154 / 15 6 / 186 / 195, 15 / 33 / 86 / 149 / 154 / 195 / 224 / 317 / 331, or 149 / 186 / 195 / 224 / 226 / 331, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0031] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or to a reference sequence corresponding to SEQ ID NO:204, wherein the amino acid sequence comprises one or more substitutions.

[0032] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:480-582, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or relative to a reference sequence corresponding to SEQ ID NO:204.

[0033] In some embodiments, the amino acid sequence of the modified RNA ligase comprises amino acid position(s) 2 / 39 / 69 / 144 / 156 / 316 / 330, 2 / 39 / 69 / 119 / 185 / 316, 69 / 212 / 256 / 330, 39 / 119 / 256 / 316 / 326 / 330, 69 / 116 / 119 / 185 / 330, 2 / 39 / 119 / 144 / 156 / 159 / 202 / 256 / 316 / 326 / 330, 81 / 183 / 185 / 230, 2 / 39 / 69 / 116 / 144 / 156 / 185, 2 / 14 / 39 / 69 / 116 / 119 / 159 / 185 / 256 / 326, 2 / 116 / 119 / 316, 116 / 119 / 144 / 185 / 314 / 316, 39 / 69 / 116 / 144 / 181 / 185 / 256 / 330, 2 / 39 / 69 / 119 / 144 / 156 / 185 / 314, 14 / 39 / 69 / 144 / 156 / 21 2 / 256 / 314 / 316 / 326 / 330, 39 / 69 / 144 / 156 / 185 / 316 / 330, 156 / 185 / 334 / 335, 39 / 63 / 156 / 230 / 257 / 275 / 330 / 335, 156 / 266 / 316 / 330 / 334 / 335, 39 / 63 / 156 / 183 / 185 / 230 / 330 / 334, 69 / 144 / 316, 116 / 119 / 156 / 202 / 212 / 326 / 330, 39 / 69 / 119 / 138 / 181 / 185 / 316 / 326, 39 / 81 / 156 / 230, 2 / 14 / 69 / 116 / 119 / 1 44 / 159 / 326 / 330, 39 / 156 / 334 / 345, 230 / 266 / 334, 2 / 116 / 144 / 156 / 159, 156 / 183 / 185 / 230, 2 / 14 / 116 / 316 / 326 / 330, 39 / 63 / 156 / 230 / 316 / 345, 69 / 13 8 / 144 / 159 / 185 / 202 / 316, 39 / 156 / 257 / 266 / 316 / 334, 156 / 257 / 275 / 334, 183, 89 / 156 / 185 / 230 / 316 / 345, 2 / 14 / 119, 144 / 156 / 316 / 330, 39 / 81 / 185 / 3 16 / 334, 89 / 230 / 266 / 345, 39 / 156 / 230 / 275 / 316, 39 / 156 / 230 / 266 / 334 / 335, 156 / 185 / 275 / 316 / 330, 119 / 156 / 202, 230 / 257 / 275 / 316 / 330 / 345, 230,At least one substitution or set of substitutions at 81 / 156 / 330 / 335 / 345, 156 / 183 / 230 / 266, 39 / 185, 39 / 81 / 183 / 275 / 316 / 334 / 345, 156 / 230 / 257 / 316, 39 / 116 / 156, or 183 / 185 / 257 / 275 / 316 / 330 / 334, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 204 or relative to a reference sequence corresponding to SEQ ID NO: 204.

[0034] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution as provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0035] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions of an RNA ligase variant provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0036] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0037] In some embodiments, the modified RNA ligase comprises an amino acid sequence comprising residues 12-346 of an even-numbered SEQ ID NO: 14-582, or an amino acid sequence comprising an even-numbered SEQ ID NO: 14-582. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0038] In some embodiments, the modified RNA ligase comprises an amino acid sequence comprising residues 12-346 of SEQ ID NO: 42 or 204, or an amino acid sequence comprising SEQ ID NO: 42 or 204. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

[0039] In some embodiments, the modified RNA ligase has RNA ligase 2 activity. In some embodiments, the modified RNA ligase has RNA ligase 2 activity and at least one improved property compared to a reference RNA ligase.

[0040] In some embodiments, the improved property of the modified RNA ligase is selected from: i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased activity toward polynucleotide substrates containing phosphorothioate internucleotide linkages, vi) increased activity toward polynucleotide substrates containing 2'-modifications, vii) increased substrate tolerance, or any combination of i), ii), iii), iv), v), vi), and vii), relative to a reference RNA ligase. In some embodiments, the improved property of the modified RNA ligase is relative to a reference RNA ligase having a sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or a sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0041] In some embodiments, the modified RNA ligase is purified. In some further embodiments, the modified RNA ligase is provided in solution, as a lyophilizate, or immobilized on a substrate, such as a solid substrate, a porous substrate, a membrane, or a particle.

[0042] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding the modified RNA ligase disclosed herein.

[0043] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO: 14 and the even-numbered SEQ ID NOs: 24-582, wherein the amino acid sequence comprises a polynucleotide sequence encoding a modified RNA ligase that contains one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0044] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204.

[0045] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

[0046] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to a reference sequence corresponding to SEQ ID NO:42, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42.

[0047] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or to a reference sequence corresponding to SEQ ID NO:204, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or to a reference sequence corresponding to SEQ ID NO:204.

[0048] In some embodiments, a recombinant polynucleotide comprising a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 13, 41, or 203, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 13, 41, or 203, wherein the recombinant polynucleotide encodes an RNA ligase.

[0049] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1038 of an odd-numbered SEQ ID NO:23-581, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO:23-581, and the recombinant polynucleotide encodes an RNA ligase.

[0050] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide encoding the modified RNA ligase is codon-optimized for expression in an organism or cell type thereof, such as a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

[0051] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1038 of SEQ ID NO: 13, 41, or 203, or a polynucleotide sequence comprising SEQ ID NO: 13, 41, or 203.

[0052] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1038 of an odd-numbered SEQ ID NO: 23-581, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 23-581.

[0053] In a further aspect, the present disclosure provides an expression vector comprising at least one recombinant polynucleotide provided herein encoding a modified RNA ligase. In some embodiments, the recombinant polynucleotide of the expression vector is operably linked to a regulatory sequence. In some embodiments, the regulatory sequence comprises a promoter, particularly a heterologous promoter.

[0054] In another aspect, the present disclosure also provides a host cell comprising at least one expression vector provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell. In some preferred embodiments, the host cell is a bacterial cell, such as E. coli or B. subtilis.

[0055] In a further aspect, the disclosure provides methods of producing a modified RNA ligase polypeptide, the methods comprising culturing a host cell described herein under suitable culture conditions such that at least one modified RNA ligase is produced. In some embodiments, the methods further comprise recovering or isolating the modified RNA ligase from the culture and / or the host cell. In some embodiments, the methods further comprise purifying the modified RNA ligase.

[0056] In another aspect, the present disclosure provides compositions comprising at least one modified RNA ligase disclosed herein. In some embodiments, the compositions comprise at least one buffer. In some embodiments, the compositions further comprise one or more polynucleotide substrates, such as a nucleotide substrate (e.g., ATP or dATP) and a synthetic polynucleotide, particularly a modified polynucleotide.

[0057] In a further aspect, the disclosure provides methods of ligating at least a first polynucleotide strand and a second polynucleotide strand, the method comprising contacting the first polynucleotide strand and the second polynucleotide strand with a modified RNA ligase described herein in the presence of a nucleotide substrate under conditions suitable for ligation of the first polynucleotide strand to the second polynucleotide strand, wherein the first polynucleotide strand comprises a ligatable 5' end and the second polynucleotide strand comprises a 3' end that is ligatable to the 5' end of the first polynucleotide strand. In some embodiments, the first polynucleotide strand and / or the second polynucleotide strand comprises RNA or a mixture of RNA and DNA.

[0058] In some embodiments, the method further includes a third polynucleotide strand, wherein the first polynucleotide strand and the second polynucleotide strand hybridize adjacent to each other on the third polynucleotide strand, positioning the 5' end of the first polynucleotide strand adjacent to the 3' end of the second polynucleotide strand to form a nick. In some embodiments, the third polynucleotide strand is contiguous with the first polynucleotide strand or the second polynucleotide strand. In some embodiments, the third polynucleotide strand is contiguous with the first polynucleotide strand and the second polynucleotide strand to form a single, continuous polynucleotide substrate.

[0059] In some embodiments, the 3'-terminal region of the second polynucleotide strand that hybridizes to the third polynucleotide strand is at least 4, 6, 8, or more base pairs in length. In some embodiments, the 5'-terminal region of the first polynucleotide strand that hybridizes to the third polynucleotide strand is at least 4, 6, 8, or more base pairs in length.

[0060] In some embodiments of the method, the third polynucleotide strand comprises a splint or bridge polynucleotide, and the 5' terminal sequence of the first polynucleotide strand and the 3' terminal sequence of the second polynucleotide strand hybridize adjacent to each other on the splint or bridge polynucleotide, positioning the 5' end of the first polynucleotide strand adjacent to the 3' end of the second polynucleotide strand.

[0061] In some embodiments, the first and third polynucleotide strands hybridize to each other to form a first double-stranded polynucleotide fragment, and the second polynucleotide strand hybridizes to a fourth polynucleotide strand to form a second double-stranded fragment, and the first and second double-stranded fragments have complementary ends that can base-pair to form a substrate for a modified RNA ligase.

[0062] In some embodiments, modified RNA ligases are used in methods for synthesizing RNA or DNA / RNA polynucleotides by ligating shorter RNA or DNA / RNA oligonucleotides. In some embodiments, modified RNA ligases are used to ligate the 3'OH of RNA to the 5' phosphate of DNA or RNA. In some embodiments, modified RNA ligases are used to ligate the 3'OH of RNA to the 5' phosphate of DNA in double-stranded format NGS RNA library construction. In some embodiments, modified RNA ligases are used in methods for preparing RNA circles. In some embodiments, modified RNA ligases are used to repair nicks in dsRNA or dsRNA / DNA.

[0063] In a further aspect, the present disclosure also provides kits comprising at least one modified RNA ligase disclosed herein. In some embodiments, the kits further comprise one or more of a buffer, a nucleotide substrate (e.g., ATP or dATP), and / or one or more polynucleotide substrates. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present disclosure provides modified RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the modified RNA ligase polypeptides. The disclosure also provides methods of using the modified RNA ligase polypeptides and compositions thereof for molecular biology, diagnostic, and other purposes. In some embodiments, the modified RNA ligase polypeptides exhibit, among other things, increased activity, increased stability, increased product yield, increased thermostability, and / or increased activity toward polynucleotides containing one or more modified nucleotides or nucleotide analogs.

[0065] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art. These techniques are well known and described in numerous publications and references well known to those of ordinary skill in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis.

[0066] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, exemplary methods and materials are described herein. It is understood that the present invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art. Accordingly, the terms defined immediately below are more fully described by reference to the entire application.

[0067] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0068] As used herein, the term "comprising" and its cognates are used in an inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0069] It should also be understood that when the term "comprising" and its cognates are used in describing an embodiment, the embodiment may also be described using the phrase "consisting essentially of" or "consisting of."

[0070] Furthermore, numerical ranges are intended to be inclusive of the numbers defining that range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Also, every maximum (or minimum) numerical limitation disclosed herein is intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0071] As used herein, the term "about" refers to a tolerance for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a particular value range. In other cases, "about" means within 1, 2, 3, or 4 standard deviations of a particular value.

[0072] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the present invention that can be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole.

[0073] The "EC" number refers to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0074] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0075] "NCBI" refers to the National Center for Biological Information and the sequence databases it provides.

[0076] "Protein," "polypeptide," and "peptide" are used interchangeably to refer to polymers of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (such as glycosylation or phosphorylation).

[0077] "Amino acid(s)" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: Alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), 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 a three-letter abbreviation is used, an amino acid is referred to as having an alpha carbon (C) unless it is explicitly preceded by "L" or "D" or is otherwise clear from the context in which the abbreviation is used. α ) can be in either the L- or D-configuration with respect to the α-carbon. For example, "Ala" designates alanine without specifying the configuration of the α-carbon, while "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively. When single-letter abbreviations are used, uppercase letters designate amino acids in the L-configuration around the α-carbon, and lowercase letters designate amino acids in the D-configuration around the α-carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When a polypeptide sequence is represented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is represented in the amino (N) to carboxy (C) direction, according to common convention.

[0078] "Fusion protein" and "chimeric protein" and "chimera" refer to a hybrid protein created by the joining of two or more polynucleotides that originally encoded separate proteins. In some embodiments, fusion proteins are produced by recombinant techniques (e.g., molecular biology techniques known in the art).

[0079] "RNA ligase" refers to an enzyme that covalently joins the 5'-phosphoryl end of RNA or DNA to the 3'-hydroxyl end of RNA or DNA. Two families of RNA ligases are known to exist in nature. RNA ligase 1 catalyzes the covalent joining of a single-stranded 5'-phosphoryl end of RNA or DNA to a single-stranded 3-hydroxyl end of RNA or DNA. RNA ligase 2 also catalyzes the covalent joining of a 3'-hydroxyl end of RNA to 5'-phosphorylated RNA or DNA, but prefers double-stranded substrates. In some embodiments, RNA ligases include enzymes classified under EC 6.5.1.3.

[0080] Without being bound by any theory of operation, it is understood that while some DNA ligases can act on either DNA or RNA as a 3'-hydroxyl strand substrate, RNA ligase 2 acting at a duplex nick preferentially acts on the 3'-hydroxyl strand of RNA but cannot recognize whether the 5-phosphoryl strand is DNA or RNA. Furthermore, in some embodiments, the 3'-hydroxyl-bearing strand can contain 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 influenced by the 3'-hydroxyl strand, specifically the two terminal ribonucleotides on the 3'-hydroxyl side of the nick (see, e.g., 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.

[0081] As used herein, the terms "polynucleotide," "nucleic acid," or "oligonucleotide" refer to a polymer containing at least two nucleotides, where the nucleotides are either deoxyribonucleotides or ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used to genetically encode nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless otherwise specified, abbreviated nucleosides can be either ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated individually or collectively as either ribonucleosides or 2'-deoxyribonucleosides. When a polynucleotide, nucleic acid, or oligonucleotide sequence is represented as a string of single-letter abbreviations, the sequence is represented in the 5' to 3' direction according to common convention, and the phosphate is not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid. A polynucleotide or nucleic acid may be single-stranded, double-stranded, or contain both single-stranded and double-stranded regions.

[0082] In some embodiments, the terms "polynucleotide," "nucleic acid," and "oligonucleotide" encompass polynucleotide or nucleic acid or oligonucleotide analogs, including, inter alia, nucleosides linked together via other than standard phosphodiester bonds, such as phosphoramidate, phosphorothioate, non-standard bonds such as amide bonds; nucleosides containing modified and / or synthetic nucleobases, such as inosine, xanthine, hypoxanthine, etc.; nucleosides containing modified sugar residues, such as 2'-O-alkyl, 2'-halo, 2,3-dideoxy, 2'-halo-2'deoxy, β-D-riboLNA, α-L-ribo-LNA (locked nucleic acid); and / or 5'-phosphate analogs including, inter alia, phosphorothioate, phosphoacetate, phosphoramidate, monomethylphosphate, methylphosphonate, or phosphonocarboxylate.

[0083] "Duplex" 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 pairs with T or U, C pairs with G), are arranged antiparallel in the 5' to 3' direction, and are held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).

[0084] As used herein, the term "complementary" is used to describe the structural relationship between nucleotide bases that can form base pairs with each other. For example, purine nucleotide bases present in a polynucleotide that are complementary to pyrimidine nucleotide bases on the polynucleotide can base pair with each other by forming hydrogen bonds. Complementary nucleotide bases can base pair with each other through Watson-Crick base pairing or any other method to form a stable duplex or other nucleic acid structure.

[0085] "Watson / Crick base pairing" refers to a specific pairing pattern of nucleobases and analogs that bind through sequence-specific hydrogen bonds, e.g., A pairs with T or U, G pairs with C, etc.

[0086] "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 a duplex, triplex, or quaternary structure. Annealing or hybridization occurs through Watson-Crick base-pairing interactions, but may also be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing. In some embodiments, a nucleobase polymer that anneals or hybridizes with another nucleobase polymer is a single nucleobase polymer, while in other embodiments, the nucleobase polymers are distinct nucleobase polymers.

[0087] "Modified," "recombinant," "non-naturally occurring," and "variant," when used with respect to a cell, polynucleotide, or polypeptide, refer to material that has been modified in a way that does not occur in nature or that corresponds to the natural or native form of that material, or to material that is identical but produced or derived from synthetic material and / or by manipulation using recombinant techniques or that corresponds to the natural or native form of that material.

[0088] "Wild-type" and "native" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is one that is present in an organism, can be isolated from a natural source, and has not been intentionally modified by human manipulation.

[0089] "Coding sequence" and its synonym "encoding" refer to that portion of a nucleic acid (such as a gene) that codes for the amino acid sequence of a protein.

[0090] "Percent (%) sequence identity" refers to a comparison between polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence to optimally align the two sequences. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where either the same nucleic acid base or amino acid residue occurs or where the nucleic acid base or amino acid residue aligns with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be carried out as is well known in the art, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection.Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available via the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence that match or meet some positive threshold score "T" when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for a pair of matching residues; always greater than 0) and "N" (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount "X" from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary sequence alignments and percent sequence identity determinations can utilize the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0091] A "reference sequence" refers to a defined sequence used as a 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. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length of the nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each contain (1) similar sequences (i.e., portions of the complete sequence) between the two sequences and (2) additionally mismatched sequences between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, where the reference sequence may have one or more alterations in the primary sequence. For example, the phrase "a reference sequence corresponding to SEQ ID NO: 14 with a lysine at the residue corresponding to X14" (or "a reference sequence corresponding to SEQ ID NO: 14 with a lysine at the residue corresponding to position 14") refers to a reference sequence in which the corresponding residue at position X14 of SEQ ID NO: 14 (e.g., alanine) has been changed to lysine.

[0092] A "comparison window" refers to a conceptual segment of contiguous nucleotide positions or amino acid residues within which a sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15-20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window can contain no more than 20 percent additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and optionally includes a window of 30, 40, 50, 100, or longer.

[0093] When used in relation to the numbering of a given amino acid or polynucleotide sequence, "corresponding to," "referring to," and "relative to" refer to the numbering of residues in a specified reference sequence when comparing a given amino acid or polynucleotide sequence to the reference sequence. In other words, the residue numbers or residue positions in a given polymer are specified relative to the reference sequence, rather than the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of a modified RNA ligase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, despite the presence of gaps, the numbering of residues in a given amino acid or polynucleotide sequence is relative to the reference sequence to which it is aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0094] "Mutation" refers to a change in a nucleic acid sequence. In some embodiments, a mutation results in a change in the encoded polypeptide sequence (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation comprises a substitution, resulting in a different amino acid. In some alternative embodiments, a mutation comprises an addition, resulting in an amino acid being added (e.g., inserted) to the original polypeptide sequence. In some further embodiments, a mutation comprises a deletion, resulting in an amino acid being deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0095] "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 amino acid residue at the corresponding position in a reference sequence. The amino acid position of an 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, a "residue difference at position X14 compared to SEQ ID NO:14" (or a "residue difference at position 14 compared to SEQ ID NO:14") refers to the amino acid residue difference at the polypeptide position corresponding to position 14 of SEQ ID NO:14. Thus, if a reference polypeptide of SEQ ID NO:14 has an alanine at position 14, then a "residue difference at position X14 compared to SEQ ID NO:14" refers to an amino acid substitution of any residue other than alanine at the polypeptide position corresponding to position 14 of SEQ ID NO:14. In some cases herein, a specific amino acid residue difference at a position is designated as "XnY," where "Xn" designates the corresponding residue and position in the reference polypeptide (as described above), and "Y" is the single-letter identifier of the amino acid found in the modified polypeptide (i.e., the residue that differs from the reference polypeptide). In some instances (e.g., in the Tables of Examples), the disclosure also provides specific amino acid differences designated with the conventional designation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the modified polypeptide. In some embodiments, the amino acid difference, e.g., substitution, is designated with the abbreviation "nB," without the residue identifier in the reference sequence. In some cases, the amino acid residue difference or substitution may be a deletion and may be designated with "-." In some embodiments, the phrase "amino acid residue nB" indicates the presence of an amino acid residue in the modified polypeptide, which may or may not be a substitution in the context of the reference polypeptide or amino acid sequence.

[0096] In some cases, polypeptides of the present disclosure can contain one or more amino acid residue differences relative to a reference sequence, as indicated by a list of designated positions at which the residue difference occurs relative to the reference sequence. In some embodiments, when multiple amino acids can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X39Q / X39S, X39Q / S, or 39Q / S). The present disclosure includes modified polypeptide sequences that contain one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0097] "Set of amino acid substitutions" and "set of substitutions" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, a set of substitutions includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a set of substitutions refers to a set of amino acid substitutions present in any of the variant RNA ligase polypeptides listed in any of the tables in the Examples. In these sets of substitutions, individual substitutions are separated by a semicolon (";", e.g., M95V;V177P) or a slash (" / ", e.g., M95V / V177P or 95V / 177P).

[0098] "Conservative amino acid substitution" refers to the substitution of one residue with a different residue having a similar side chain, and typically involves substituting an amino acid in a polypeptide with an amino acid within the same or a 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.

[0099] A "non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions use amino acids between, rather than within, a defined group and may affect (a) the structure of the peptide backbone in the area of ​​substitution (e.g., glycine for proline), (b) the charge or hydrophobicity, and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid, an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0100] "Deletion" refers to modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removing 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, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids comprising the reference polypeptide, while maintaining enzymatic activity and / or maintaining improved properties of the modified RNA ligase. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous. As noted above, deletions are indicated by "-" and can occur within a set of substitutions.

[0101] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids from a reference polypeptide. The insertion may be in an internal portion of the polypeptide or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins known in the art. An insertion may be a contiguous segment of amino acids, separated by one or more amino acids in a native polypeptide.

[0102] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion and / or an internal deletion, but whose remaining amino acid sequence is identical to the corresponding positions in a sequence to which it is compared (e.g., a full-length modified RNA ligase of the invention) and which retains substantially all of the activity of the full-length polypeptide.

[0103] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant RNA ligase polypeptides may be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, the recombinant RNA ligase polypeptides provided herein are isolated polypeptides.

[0104] "Substantially pure polypeptide" or "purified" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant on a molar or weight basis than any other individual macromolecular species in the composition); generally, a composition is substantially purified when the species of interest constitutes at least about 50 percent of the macromolecular species present on a molar or weight percent basis. Generally, a substantially pure RNA ligase composition comprises 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 the total macromolecular species present in the composition on a molar or weight percent basis. In some embodiments, the species of interest is purified to essential homogeneity (i.e., contaminating species can no longer be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated recombinant RNA ligase polypeptide is a substantially pure polypeptide composition.

[0105] "Improved enzymatic properties" refers to a modified RNA ligase polypeptide that has any improved enzymatic property compared to a reference RNA ligase polypeptide, such as a wild-type RNA ligase polypeptide (e.g., the RNA ligase polypeptide sequence of SEQ ID NO: 2 or 14) or another modified RNA ligase polypeptide. Improved properties include, but are not limited to, properties such as increased enzymatic activity, increased product yield, increased protein expression, increased thermal activity, increased thermostability, increased stability, increased substrate specificity and / or affinity, increased substrate range, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved solvent stability, increased solubility, and increased inhibitor resistance or tolerance. Exemplary improved properties are provided in the Examples.

[0106] "Increased enzymatic activity" and "enhanced catalytic activity" refer to an improvement in the properties of a modified RNA ligase polypeptide, which may be expressed in terms of an increase in specific activity (e.g., product produced / time / weight of protein) and / or the rate of substrate-to-product conversion (e.g., the rate of conversion of starting substrate to product in a specified time using a specified amount of RNA ligase) compared to a reference RNA ligase enzyme (e.g., a wild-type RNA ligase and / or another modified RNA ligase). Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max , or k cat Any property associated with enzymatic activity can be affected, including classical enzymatic properties of the enzyme, and these changes can result in increased enzymatic activity. The improvement in enzymatic activity can be from about 1.1-fold that of the corresponding wild-type enzyme to about 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more that of the naturally occurring RNA ligase or another modified RNA ligase from which the RNA ligase polypeptide is derived.

[0107] " Hybridization stringency " refers to the hybridization conditions, such as the washing conditions, in nucleic acid hybridization. Generally, hybridization reaction is carried out under lower stringency conditions, followed by washing under various 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 "moderately stringent hybridization" refers to the conditions that allow target DNA to bind to the complementary nucleic acid that has about 60% identity with target DNA, preferably about 75% identity, about 85% identity, and has more than about 90% identity with target polynucleotide. Exemplary moderately stringent conditions are equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to hybridization at a temperature higher than the thermal melting temperature, T, determined under solution conditions for the particular polynucleotide sequence. mto about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it is not stable under the high stringency conditions contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition includes hybridizing under conditions equivalent to hybridizing in 5x SSC containing 0.1% (w:v) SDS at 65°C and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, and moderately stringent conditions, are described in the references cited above.

[0108] "Codon-optimized" refers to changing the codons in a polynucleotide encoding a protein to those preferentially used in a particular organism, allowing the encoded protein to be expressed more efficiently in that organism. While the genetic code is degenerate, with most amino acids represented by multiple codons known as "synonyms" or "synonymous" codons, it is well known that codon usage by a particular organism is not random but is biased toward certain codon triplets. This codon usage bias can be higher in accordance with specific genes, genes of 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 embodiments, a polynucleotide encoding an RNA ligase enzyme is codon-optimized for optimal production from the host organism selected for expression.

[0109] As used herein, the term "control sequences" refers to all components necessary or advantageous for expression of a polynucleotide and / or polypeptide of the present disclosure. Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. In some embodiments, control sequences include a promoter, and transcriptional and translational stop signals. In some embodiments, control sequences are provided with linkers for the purpose of introducing specific restriction enzyme recognition sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0110] "Operably linked" or "operably linked" refers to a configuration in which control sequences are appropriately positioned (i.e., in functional relationship) with a polynucleotide of interest so that the control sequences direct or control expression of the polynucleotide of interest and, if appropriate, the encoded polypeptide of interest.

[0111] "Promoter" or "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence includes transcriptional control sequences that mediate expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides that are either homologous or heterologous to the host cell.

[0112] "Suitable reaction conditions" or "suitable conditions" refer to conditions in an enzyme conversion reaction solution (e.g., ranges of enzyme load, substrate load, temperature, pH, buffer, co-solvent, etc.) that allow an RNA ligase polypeptide of the disclosure to convert a polynucleotide substrate into a desired ligated product polynucleotide. Exemplary "suitable reaction conditions" are provided herein (see Examples).

[0113] In an enzymatic conversion process, the "product" refers to a compound or molecule that results from the action of an RNA ligase polypeptide on a substrate.

[0114] "Culturing" refers to the growth of a population of cells under appropriate conditions using any suitable medium (eg, liquid, gel, or solid).

[0115] A "vector" is a recombinant construct for introducing a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector operably linked to suitable control sequences capable of effecting expression of the polynucleotide or polypeptide encoded in the polynucleotide in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a polynucleotide (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0116] "Expression" includes all steps involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0117] "Produce" refers to the production of proteins and / or other compounds by a cell. The term "expression" is intended to encompass all steps involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0118] "Heterologous" or "recombinant" refers to the 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 not related in nature.

[0119] "Host cell" and "host strain" refer to suitable hosts for expression vectors containing polynucleotides provided herein (e.g., polynucleotide sequences encoding at least one RNA ligase variant). In some embodiments, host cells are prokaryotic or eukaryotic cells, and their progeny, that have been transformed or transfected with vectors constructed using recombinant DNA techniques, as known in the art.

[0120] "Analog" in the context of a polypeptide refers to a polypeptide that has greater than 70% but less than 100% sequence identity to a reference polypeptide (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, analogs include unnatural amino acid residues, including, but not limited to, homoarginine, ornithine, and norvaline, as well as natural amino acids. In some embodiments, analogs also include one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues. Modified RNA ligase polypeptides

[0121] In one aspect, the present disclosure provides RNA ligases, including modified RNA ligase polypeptide variants. In some embodiments, the RNA ligases and modified RNA ligase polypeptide variants are useful for preparing polynucleotides from short oligonucleotides and ligating polynucleotide substrates, such as for diagnostic and other purposes. The modified RNA ligase variants can be used in solution as well as in immobilized embodiments. In some embodiments, the modified RNA ligases can be prepared and used as non-fusion or fusion polypeptides.

[0122] In some embodiments, the modified RNA ligase, or functional fragment thereof, comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, or to a reference sequence corresponding to the even-numbered SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, wherein the amino acid sequence comprises one or more substitutions.

[0123] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204, wherein the amino acid sequence comprises one or more substitutions.

[0124] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14 or to a reference sequence corresponding to SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14 or to a reference sequence corresponding to SEQ ID NO: 14.

[0125] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204, and the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

[0126] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582, wherein the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or to a reference sequence corresponding to SEQ ID NO:14.

[0127] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:24-128, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-128, wherein the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or to a reference sequence corresponding to SEQ ID NO:14.

[0128] In some embodiments, the amino acid sequence of the modified RNA ligase comprises the amino acid sequences at amino acid positions 2, 14, 15, 18, 33, 39, 63, 69, 81, 83, 85, 86, 89, 95, 116, 117, 119, 138, 142, 144, 149, 154, 156, 159, 171, 175, 177, 181, 185, 186, 195, 202, 212, 214, 224, 226, 230, 247, 256, 257, 260, 266, 275, 280, 283, 285, 288, 291, 296, 303, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0129] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution 2G / V / E, 14K, 15L / E / M / P / Y, 18I, 33L, 39Q / S, 63V, 69D, 81Y, 85L, 86G, 89H / I / M / W, 95V, 116N, 117D, 119T, 138R, 142R, 144W, 149R / T, 154C, 156A / L / N / Q / T, 159F, 171E, 175K, 177P, 181D, 183V, 185G / K, 186D / R, 195G, 202Y, 212I, 214I, 224Y, 226I, 230D, 231G, 232G, 233G, 234G, 235G, 236G, 237G, 238G, 239G, 240G, 241G, 242G, 243G, 244G, 245G, 246G, 247G, 248G, 249G, 250G, 251G, 252G, 253G, 254G, 255G, 256G, 257G, 258G, 259G, 260G, 261G, 262G, 263G, 264G, 265G, 266G, 267G, 268G, 269G, 270G, 271G, 27 47K, 256A, 257L, 260S, 266L, 275N, 280G / N, 283I, 285K, 288E, 291P, 296K, 303Q, 306L, 307E / Q, 310K, 314W, 315S / T, 316L, 317A, 326R, 330M / R, 331R / W, 333V, 334R, 335D / H, 337L, 339P, 342I, or 345V, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0130] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution H2G / V / E, A14K, K15L / E / M / P / Y, S18I, G33L, H39Q / S, I63V, T69D, H81Y, M85L, K86G, K89H / I / M / W, M95V, G116N, G117D, Q119 T, S138R, K142R, Y144W, V149R / T, A154C, G156A / L / N / Q / T, L159F, A171E, Q175K, V177 P, K181D, I183V, N185G / K, Y186D / R, D195G, H202Y, T212I, V214I, N224Y, K226I, N230 D, A247K, K256A, V257L, T260S, V266L, T275N, S280G / N, L283I, H285K, S288E, A291P, R296K, K303Q, I306L, N307E / Q, A310K, H314W, D315S / T, M316L, L317A, K326R, T330M / R, I331R / W, I333V, Q334R, N335D / H, I337L, S339P, H342I, or L345V, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0131] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 95 or 177, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0132] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 95V or 177P, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0133] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution M95V or V177P, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0134] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 149, 154, 156, 186, 195, 224, or 226, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0135] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 15Y, 149R, 154C, 156T, 186R, 195G, 224Y, or 226I, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0136] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution K15Y, V149R / T, A154C, G156A / L / N / Q / T, Y186D / R, D195G, N224Y, or K226I, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0137] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0138] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 15L / E / M / P / Y, 95V, 149R / T, 154C, 156A / L / N / Q / T, 177P, 186D / R, 195G, 224Y, or 226I, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0139] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 15Y, 95V, 149R, 154C, 156T, 177P, 186R, 195G, 224Y, or 226I, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0140] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution K15Y, M95V, V149R, A154C, G156T, V177P, Y186R, D195G, N224Y, or K226I, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0141] In some embodiments, the amino acid sequence of the modified RNA ligase comprises amino acid position(s) 177 / 260 / 307 / 345, 171 / 175 / 183 / 280 / 283 / 303, 95 / 159 / 177 / 260 / 288, 175 / 183 / 247 / 280 / 283 / 296, 177 / 307, 81 / 171 / 183 / 335, 175 / 183, 81 / 175 / 183 / 280 / 296 / 335 / 339, 81 / 183 / 247 / 266, 95 / 177, 171 / 280 / 283 / 296 / 303, 85 / 177 / 260 / 288 / 333 / 345, 177 / 291 / 307 / 333, 81 / 171 / 175 / 183 / 316 / 337, 81 / 171 / 266 / 280 / 283 / 335 / 339, 177, 95 / 159 / 177 / 260 / 280 / 288 / 306 / 345, 171 / 175 / 335, 177 / 260 / 30 / 333, 14 / 95 / 177 / 288 / 307, 95 / 177 / 345, 95 / 159 / 177 / 260 / 285 / 288, 266 / 296, 81 / 283 / 335 / 337, 95 / 159 / 177 / 260 / 342 / 345, 63 / 171 / 175 / 183 / 266 / 280 / 296 / 316, 280 / 335 / 337, 81 / 171 / 280, 81 / 171 / 175 / 296 / 316, 171 / 175 / 316 / 335, 171 / 280 / 283 / 296 / 316, 171 / 283 / 335, 63 / 171 / 175 / 183 / 266 / 283 / 3 03 / 316, 171 / 247 / 266 / 283 / 296 / 303 / 316 / 335, 63 / 81 / 171 / 175 / 183 / 266 / 280 / 283 / 337, 81 / 171 / 303 / 316 / 335, 288 / 307, 14 / 95 / 159 / 177 / 345, 81 / 171 / 175 / 183 / 247 / 266 / 280 / 283, 81 / 247 / 266, 280, 85 / 177 / 260, 63 / 183, 175 / 183 / 247 / 280 / 283 / 316, 291, 247, 171 / 280 / 283, 81 / 171 / 175 / 280 / 316, 333, 171 / 296 / 316, 81 / 171, or 280 / 316 / 335, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0142] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions 177P / 260S / 307E / 345V, 171E / 175K / 183V / 280N / 283I / 303Q, 95V / 159F / 177P / 260S / 288E, 175K / 183V / 247K / 280N / 283I / 296K, 177P / 307E, 81Y / 171E / 183V / 335D, 175K / 183V, 81Y / 175K / 183V / 280N / 296K / 335D / 339P, 81Y / 183V / 247K / 266L, 95V / 177P, 171E / 280N / 283I / 296K / 303Q, 85L / 177P / 260S / 288E / 333V / 345V, 177 P / 291P / 307E / 333V, 81Y / 171E / 175K / 183V / 316L / 337L, 81Y / 171E / 266L / 28 0N / 283I / 335D / 339P, 177P, 95V / 159F / 177P / 260S / 280N / 288E / 306L / 345V, 171E / 175K / 335D, 177P / 260S / 306L / 333V, 14K / 95V / 177P / 288E / 307E, 95V / 1 77P / 345V, 95V / 159F / 177P / 260S / 285K / 288E, 266L / 296K, 81Y / 283I / 335D / 337L, 95V / 159F / 177P / 260S / 342I / 345V, 63V / 171E / 175K / 183V / 266L / 280N / 296K / 316L, 280N / 335D / 337L, 81Y / 171E / 280N, 81Y / 171E / 175K / 296K / 316 L, 171E / 175K / 316L / 335D, 171E / 280N / 283I / 296K / 316L, 171E / 283I / 335D, 6 3V / 171E / 175K / 183V / 266L / 283I / 303Q / 316L, 171E / 247K / 266L / 283I / 296K / 303Q / 316L / 335D, 63V / 81Y / 171E / 175K / 183V / 266L / 280N / 283I / 337L, 81Y / 171E / 303Q / 316L / 335D, 288E / 307E, 14K / 95V / 159F / 177P / 345V, 81Y / 171E / 175K / 183V / 247K / 266L / 280N / 283I, 81Y / 247K / 266L, 280N, 85L / 177P / 260S,63V / 183V, 175K / 183V / 247K / 280N / 283I / 316L, 291P, 247K, 171E / 280N / 283I, 81Y / 171E / 175K / 280N / 316L, 333V, 171E / 296K / 316L, 81Y / 171E, or 280N / 316L / 335D, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0143] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions V177P / T260S / N307E / L345V, A171E / Q175K / I183V / S280N / L283I / K303Q, M95V / L159F / V177P / T260S / S288E, Q175K / I183V / A247K / S280N / L283I / R296K, V177P / N307E, H81Y / A171E / I183V / N335D, Q175K / I183V, H81Y / Q175K / I183V / S280N / R2 96K / N335D / S339P, H81Y / I183V / A247K / V266L, M95V / V177P, A171E / S280N / L283I / R296K / K303Q, M85L / V177P / T260S / S288E / I333V / L345V, V177P / A2 91P / N307E / I333V, H81Y / A171E / Q175K / I183V / M316L / I337L, H81Y / A171E / V266L / S280N / L283I / N335D / S339P, V177P, M95V / L159F / V177P / T260S / S2 80N / S288E / I306L / L345V, A171E / Q175K / N335D, V177P / T260S / I306L / I33 3V, A14K / M95V / V177P / S288E / N307E, M95V / V177P / L345V, M95V / L159F / V17 7P / T260S / H285K / S288E, V266L / R296K, H81Y / L283I / N335D / I337L, M95V / L159F / V177P / T260S / H342I / L345V, I63V / A171E / Q175K / I183V / V266L / S28 0N / R296K / M316L, S280N / N335D / I337L, H81Y / A171E / S280N, H81Y / A171E / Q175K / R296K / M316L, A171E / Q175K / M316L / N335D, A171E / S280N / L283I / R2 96K / M316L, A171E / L283I / N335D, I63V / A171E / Q175K / I183V / V266L / L283 I / K303Q / M316L, A171E / A247K / V266L / L283I / R296K / K303Q / M316L / N335D,I63V / H81Y / A171E / Q175K / I183V / V266L / S280N / L283I / I337L, H81Y / A171E / K303Q / M316L / N335D, S288E / N307E, A14K / M95V / L159F / V177P / L345V, H81Y / A171E / Q175K / I183V / A247K / V266L / S280N / L283I, H81Y / A247K / V266L, S280N, M85L / V177P / T260S, I63V / I183V, Q175K / I183V / A247K / S280N / L283I / M316L, A291P, A247K, A171E / S280N / L283I, H81Y / A171E / Q175K / S280N / M316L, I333V, A171E / R296K / M316L, H81Y / A171E, or S280N / M316L / N335D, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0144] In some embodiments, the amino acid sequence of the modified RNA ligase is at amino acid positions 95 / 149 / 156 / 177 / 195, 15 / 95 / 149 / 177 / 186 / 224 / 317, 15 / 95 / 156 / 177 / 195 / 224 / 226 / 317 / 331, 15 / 95 / 177 / 195 / 317 / 331, 95 / 149 / 177 / 224 / 331, 15 / 95 / 177 / 195 / 224 / 226 / 317 / 331, 15 / 33 / 86 / 95 / 149 / 154 / 177 / 195 / 317, 15 / 86 / 95 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 331, 15 / 95 / 149 / 177 / 224 / 317 / 331, 15 / 95 / 154 / 156 / 177 / 186 / 317 / 331, 15 / 33 / 95 / 149 / 177 / 224 / 226 / 331, 15 / 95 / 177 / 186 / 224 / 226 / 317 / 331, 1 5 / 33 / 95 / 149 / 177 / 186 / 224 / 331, 95 / 177 / 195 / 224 / 331, 15 / 86 / 95 / 156 / 177 / 186 / 195 / 226 / 317 / 331, 15 / 95 / 177 / 195 / 226 / 331, 95 / 156 / 177 / 186 / 224 / 226 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 224 / 226, 33 / 95 / 156 / 177 / 186 / 195 / 331, 15 / 86 / 95 / 149 / 154 / 156 / 177 / 317 / 331, 15 / 95 / 177 / 186 / 317 / 331, 15 / 86 / 95 / 149 / 154 / 177 / 195 / 331, 15 / 33 / 95 / 149 / 154 / 156 / 177 / 226, 15 / 95 / 149 / 154 / 177 / 186 / 317 / 331, 95 / 177 / 195 / 331, 15 / 86 / 95 / 156 / 177 / 186 / 195 / 331 , 95 / 149 / 156 / 177 / 224 / 226 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 186 / 317 / 331, 15 / 95 / 154 / 177 / 186 / 195, 15 / 86 / 95 / 177 / 195 / 224 / 226, 95 / 177 / 230, 15 / 33 / 95 / 156 / 177 / 331, 15 / 33 / 95 / 156 / 177 / 195 / 224 / 226, 33 / 95 / 149 / 156 / 177 / 317, 15 / 33 / 95 / 177 / 186 / 195, 95 / 177 / 334, 15 / 95 / 154 / 156 / 177 / 224 / 317,15 / 95 / 149 / 177 / 186 / 331、15 / 86 / 95 / 149 / 177 / 186 / 195 / 317、15 / 33 / 95 / 154 / 177 / 195、15 / 95 / 156 / 177 / 186 / 331、15 / 95 / 149 / 177 / 186 / 317、15 / 33 / 95 / 156 / 177 / 186 / 214 / 224 / 331、15 / 86 / 95 / 177 / 186 / 224 / 226 / 317、15 / 95 / 154 / 177 / 331、15 / 95 / 149 / 154 / 177 / 226、15 / 33 / 95 / 177 / 195 / 226、15 / 95 / 177 / 224 / 317 / 331、15 / 86 / 95 / 177 / 186 / 195 / 317、95 / 177 / 226 / 317 / 331、95 / 177 / 330、89 / 95 / 177、15 / 95 / 149 / 177 / 186、95 / 156 / 177 / 195 / 331、95 / 156 / 177、15 / 86 / 95 / 156 / 177 / 186 / 331、95 / 177 / 275、15 / 33 / 95 / 177 / 186 / 224、15 / 95 / 177 / 186 / 317、15 / 33 / 95 / 177 / 186 / 226 / 317、15 / 95 / 149 / 154 / 156 / 177 / 331、15 / 95 / 154 / 177 / 226 / 317、15 / 86 / 95 / 156 / 177 / 186 / 195 / 317、95 / 177 / 257、15 / 95 / 154 / 156 / 177 / 186、33 / 95 / 177 / 186 / 224、95 / 177 / 335、15 / 86 / 95 / 156 / 177 / 186 / 195 / 224 / 331、95 / 154 / 156 / 177 / 317、39 / 95 / 177、2 / 95 / 177、15 / 95 / 177 / 224 / 226 / 331、15 / 33 / 95 / 149 / 177 / 224、89 / 95 / 177 / 307、15 / 95 / 154 / 156 / 177 / 317、15 / 95 / 177 / 186 / 331、15 / 33 / 95 / 154 / 177 / 317、15 / 33 / 95 / 177 / 195 / 224 / 317、15 / 95 / 156 / 177 / 224 / 226、95 / 154 / 177 / 186 / 331、95 / 177 / 185、15 / 18 / 95 / 149 / 177 / 186 / 195、86 / 95 / 149 / 154 / 156 / 177、15 / 33 / 95 / 149 / 177 / 331、95 / 177 / 326、95 / 177 / 202、95 / 119 / 177、95 / 177 / 212、15 / 33 / 95 / 156 / 177 / 317, 95 / 177 / 280, 95 / 142 / 177, 15 / 95 / 149 / 177, 15 / 33 / 86 / 95 / 156 / 177 / 195, 95 / 177 / 315, 95 / 177 / 256, 15 / 33 / 95 / 156 / 177 / 186, 95 / 138 / 177, 95 / 117 / 177, 95 / 116 / 177, 95 / 177 / 316, 69 / 9 and at least one substitution at residues 12-346 of SEQ ID NO: 14, or 95 / 144 / 177, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or a reference sequence corresponding to SEQ ID NO: 14.

[0145] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one of the following set of substitutions: 95V / 149R / 156T / 177P / 195G, 15M / 95V / 149T / 177P / 186R / 224Y / 317A, 15P / 95V / 156T / 177P / 195G / 224Y / 226I / 317A / 331L, 15M / 95V / 177P / 195G / 317A / 331R, 95V / 149R / 177P / 224Y / 331R, 15E / 95V / 177P / 195G / 224Y / 226I / 317A / 331W, 15P / 33L / 86G / 95V / 149R / 154C / 177P / 195G / 317A, 15M / 86G / 95V / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 331L, 15P / 95V / 149R / 177P / 224Y / 317A / 331R, 15M / 95V / 1 54C / 156T / 177P / 186R / 317A / 331R, 15M / 33L / 95V / 149R / 177P / 224Y / 226I / 3 31W, 15E / 95V / 177P / 186R / 224Y / 226I / 317A / 331R, 15Y / 33L / 95V / 149R / 177 P / 186R / 224Y / 331R, 95V / 177P / 195G / 224Y / 331R, 15P / 86G / 95V / 156T / 177 P / 186R / 195G / 226I / 317A / 331L, 15M / 95V / 177P / 195G / 226I / 331W, 95V / 156 T / 177P / 186R / 224Y / 226I / 331R, 15Y / 95V / 149R / 154C / 156T / 177P / 224Y / 2 26I, 33L / 95V / 156T / 177P / 186R / 195G / 331L, 15P / 86G / 95V / 149R / 154C / 156 T / 177P / 317A / 331R, 15M / 95V / 177P / 186D / 317A / 331R, 15P / 86G / 95V / 149R / 154C / 177P / 195G / 331L, 15Y / 33L / 95V / 149T / 154C / 156T / 177P / 226I, 15P / 95V / 149R / 154C / 177P / 186R / 317A / 331L, 95V / 177P / 195G / 331R, 15M / 86G / 9 5V / 156T / 177P / 186R / 195G / 331W, 95V / 149R / 156T / 177P / 224Y / 226I / 331W,15P / 95V / 149R / 154C / 156T / 177P / 186R / 317A / 331L、15Y / 95V / 154C / 177P / 186D / 195G、15E / 86G / 95V / 177P / 195G / 224Y / 226I、95V / 177P / 230D、15P / 33L / 95V / 156T / 177P / 331R、15L / 33L / 95V / 156T / 177P / 195G / 224Y / 226I、33L / 95V / 149R / 156T / 177P / 317A、15E / 33L / 95V / 177P / 186R / 195G、95V / 177P / 334R、15E / 95V / 154C / 156T / 177P / 224Y / 317A、15Y / 95V / 149R / 177P / 186R / 331W、15Y / 86G / 95V / 149T / 177P / 186R / 195G / 317A、15P / 33L / 95V / 154C / 177P / 195G、15P / 95V / 154C / 156T / 177P / 186R / 317A / 331R、15M / 95V / 156T / 177P / 186R / 331R、15Y / 95V / 149T / 177P / 186D / 317A、15E / 33L / 95V / 156T / 177P / 186R / 214I / 224Y / 331W、15E / 86G / 95V / 177P / 186R / 224Y / 226I / 317A、15P / 95V / 154C / 177P / 331R、15Y / 95V / 149T / 154C / 177P / 226I、15Y / 33L / 95V / 177P / 195G / 226I、15M / 95V / 177P / 224Y / 317A / 331W、15E / 86G / 95V / 177P / 186R / 195G / 317A、95V / 177P / 226I / 317A / 331L、95V / 177P / 330R、89W / 95V / 177P、15P / 95V / 149R / 177P / 186D / 331L、15P / 95V / 149R / 177P / 186R、89M / 95V / 177P、95V / 156T / 177P / 195G / 331L、95V / 156A / 177P、15E / 86G / 95V / 156T / 177P / 186R / 331R、95V / 177P / 195G / 331W、95V / 177P / 275N、15P / 33L / 95V / 177P / 186R / 224Y、15E / 95V / 177P / 186R / 317A、15M / 33L / 95V / 177P / 186R / 226I / 317A、89G / 95V / 177P、15Y / 95V / 149R / 154C / 156T / 177P / 331W、15Y / 95V / 154C / 177P / 226I / 317A、1 5Y / 86G / 95V / 156T / 177P / 186R / 195G / 317A、95V / 177P / 257L、15Y / 95V / 154C / 156T / 177P / 186D、33L / 95V / 177P / 186R / 224Y、95V / 177P / 335H、15Y / 86G / 95 V / 156T / 177P / 186R / 195G / 224Y / 331W、95V / 154C / 156T / 177P / 317A、39Q / 95V / 177P、2G / 95V / 177P、15M / 95V / 177P / 224Y / 226I / 331R、15M / 33L / 95V / 149R / 177P / 224Y、89I / 95V / 177P / 307Q、15M / 95V / 154C / 156T / 177P / 317A、15E / 95 V / 177P / 186R / 331W、15P / 33L / 95V / 154C / 177P / 317A、89H / 95V / 177P、15Y / 3 3L / 95V / 177P / 195G / 224Y / 317A、15E / 95V / 156T / 177P / 224Y / 226I、95V / 154C / 177P / 186R / 331L、95V / 177P / 185G、15M / 18I / 95V / 149R / 177P / 186R / 195G、 86G / 95V / 149R / 154C / 156T / 177P、15P / 33L / 95V / 149R / 177P / 331L、95V / 177P / 326R、95V / 177P / 202Y、95V / 119T / 177P、95V / 177P / 212I、2V / 95V / 177P、15 E / 95V / 149R / 154C / 156T / 177P / 331L、15P / 33L / 95V / 156T / 177P / 317A、15Y / 9 5V / 154C / 156T / 177P / 317A, 95V / 156L / 177P, 95V / 177P / 335D, 95V / 156T / 177P, 2E / 95V / 177P, 95V / 177P / 280G, 95V / 142R / 177P, 15E / 95V / 149R / 177P, 95 V / 156Q / 177P、15E / 33L / 86G / 95V / 156T / 177P / 195G、95V / 177P / 315T、95V / 1 77P / 185K、39S / 95V / 177P、95V / 177P / 256A、15Y / 33L / 95V / 156T / 177P / 186D、95V / 138R / 177P, 95V / 117D / 177P, 95V / 116N / 177P, 95V / 177P / 316L, 15M / 95V / 149R / 177P, 69D / 95 V / 177P, 95V / 156N / 177P, 95V / 177P / 310K, 95V / 177P / 317A, 15Y / 95V / 177P / 195G / 331L, 15M / 33L / 95V / 177P / 331L, 95V / 177P / 330M, 95V / 177P / 314W, 95V / 144W / 177P, 15Y / 95V / 149R / 177P, or 95V / 177P / 315S, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0146] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions provided in Table 6.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0147] In some embodiments, the amino acid sequence of the modified RNA ligase is at amino acid positions 15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 331, 95 / 149 / 154 / 177 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 95 / 149 / 154 / 177 / 186 / 195, 95 / 149 / 156 / 177 / 186 / 195 / 224 / 226 / 317, 15 / 95 / 149 / 156 / 177 / 195 / 224 / 226, 15 / 33 / 86 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 226 / 331, 15 / 95 / 149 / 156 / 177 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 95 / 149 / 154 / 177 / 195 / 317, 15 / 95 / 149 / 154 / 177 / 195 / 224 / 317, 15 / 95 / 149 / 156 / 177 / 186 / 224 / 226 / 31 7 / 331, 15 / 33 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 317 / 331, 95 / 149 / 177 / 195 / 224, 86 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 331, 15 / 33 / 95 / 149 / 156 / 177 / 195 / 224 / 317 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 317 / 331, 15 / 33 / 95 / 149 / 154 / 156 / 177 / 186 / 224 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 195 / 22 4, 15 / 95 / 149 / 154 / 156 / 177 / 195 / 224 / 226, 15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 317, 33 / 95 / 149 / 156 / 177 / 186 / 224 / 331, 15 / 33 / 86 / 95 / 149 / 177 / 195 / 224 / 331, 15 / 86 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 331, 95 / 149 / 177 / 186 / 195 / 331, 15 / 95 / 154 / 156 / 177 / 186 / 226 / 331, 15 / 95 / 149 / 154 / 177 / 186 / 195 / 224 / 226, 15 / 95 / 149 / 154 / 156 / 177 / 195 / 224 / 331, 15 / 33 / 95 / 149 / 154 / 156 / 177 / 195 / 317 / 331, 15 / 33 / 95 / 154 / 156 / 177 / 186 / 195 / 224 / 317 / 331,15 / 95 / 149 / 177 / 186 / 195 / 224 / 331, 15 / 33 / 95 / 149 / 154 / 156 / 177 / 195 / 224 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 224 / 226 / 317 / 331, 15 / 33 / 95 / 149 / 156 / 177 / 195 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 186 / 224 / 226 / 331, 95 / 154 / 156 / 177 / 195 / 224 / 226, 15 / 33 / 86 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 317 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226, 15 / 33 / 95 / 149 / 177 / 195 / 317 / 3 31, 15 / 95 / 149 / 154 / 177 / 224 / 331, 95 / 149 / 154 / 156 / 177 / 195 / 224 / 226 / 331, 15 / 95 / 149 / 154 / 177 / 195 / 331, 15 / 95 / 149 / 154 / 156 / 177 / 186 / 195, 15 / 33 / 95 / 149 / 154 / 156 / 177 / 186 / 195, 15 / 33 / 86 / 95 / 149 / 154 / 177 / 195 / 224 / 317 / 331, or 95 / 149 / 177 / 186 / 195 / 224 / 226 / 331, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0148] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one of the following substitutions: 15M / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 331R, 95V / 149R / 154C / 177P / 186D / 195G / 224Y / 317A / 331R, 15M / 33L / 95V / 149T / 154C / 177P / 186R / 195G, 95V / 149R / 156T / 177P / 186R / 195G / 224Y / 226I / 317A, 15Y / 95V / 149R / 156T / 177P ... 4Y / 226I, 15Y / 33L / 86G / 95V / 149T / 154C / 156T / 177P / 186R / 195G / 226I / 3 31W, 15Y / 95V / 149R / 156T / 177P / 186R / 195G / 224Y / 317A / 331W, 15M / 33L / 9 5V / 149T / 154C / 177P / 195G / 317A, 15M / 95V / 149R / 154C / 177P / 195G / 224Y / 317A, 15Y / 95V / 149R / 156T / 177P / 186D / 224Y / 226I / 317A / 331R, 15M / 95V / 149T / 154C / 156T / 177P / 186D / 195G / 331L, 15Y / 33L / 95V / 149R / 154C / 15 6T / 177P / 186R / 195G / 224Y / 226I / 317A / 331R, 95V / 149R / 177P / 195G / 224Y , 86G / 95V / 149T / 154C / 156T / 177P / 186R / 195G / 331L, 15P / 33L / 95V / 149R / 156T / 177P / 195G / 224Y / 317A / 331L, 15P / 95V / 149T / 154C / 156T / 177P / 18 6D / 195G / 317A / 331L, 15Y / 33L / 95V / 149T / 154C / 156T / 177P / 186D / 224Y / 331L, 15P / 95V / 149R / 156T / 177P / 186R / 195G / 224Y / 317A / 331W, 15Y / 95V / 149R / 154C / 156T / 177P / 195G / 224Y, 15M / 95V / 149T / 154C / 156T / 177P / 19 5G / 224Y / 226I, 15E / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 317A,33L / 95V / 149T / 156T / 177P / 186R / 224Y / 331L、15Y / 33L / 86G / 95V / 149R / 177P / 195G / 224Y / 331W、15E / 86G / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 331W、95V / 149R / 177P / 186R / 195G / 331W、15E / 95V / 154C / 156T / 177P / 186D / 226I / 331R、15Y / 95V / 149T / 154C / 177P / 186R / 195G / 224Y / 226I、15P / 95V / 149R / 154C / 156T / 177P / 195G / 224Y / 331W、15E / 33L / 95V / 149R / 154C / 156T / 177P / 195G / 317A / 331L、15E / 33L / 95V / 154C / 156T / 177P / 186R / 195G / 224Y / 317A / 331L、15P / 95V / 149R / 177P / 186D / 195G / 224Y / 331L、15P / 33L / 95V / 149R / 154C / 156T / 177P / 195G / 224Y / 331W、15P / 95V / 149T / 154C / 156T / 177P / 224Y / 226I / 317A / 331W、15P / 33L / 95V / 149T / 156T / 177P / 195G / 331W、15P / 95V / 149T / 154C / 156T / 177P / 186D / 224Y / 226I / 331L、95V / 154C / 156T / 177P / 195G / 224Y / 226I、15Y / 33L / 86G / 95V / 149R / 154C / 156T / 177P / 186D / 195G / 224Y / 317A / 331W、15Y / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I、15P / 33L / 95V / 149R / 177P / 195G / 317A / 331L、15E / 95V / 149T / 154C / 177P / 224Y / 331R、95V / 149T / 154C / 156T / 177P / 195G / 224Y / 226I / 331R、15M / 95V / 149R / 154C / 177P / 195G / 331L、15Y / 95V / 149R / 154C / 156T / 177P / 186R / 195G、15E / 95V / 149T / 154C / 156T / 177P / 186R / 195G / 331R、15M / 33L / 95V / 149T / 154C / 156T / 177P / 186R / 195G, 15P / 33L / 86G / 95V / 149R / 154C / 177P / 195G / 224Y / 317A / 331R, or 95V / 149R / 177P / 186D / 195G / 224Y / 226I / 331R, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0149] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions provided in Tables 7.1, 8.1, and 9.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0150] In some embodiments, the amino acid sequence of the modified RNA ligase is at amino acid positions 2 / 15 / 39 / 69 / 95 / 144 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 316 / 330, 2 / 15 / 39 / 69 / 95 / 119 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 316, 15 / 69 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 212 / 224 / 226 / 256 / 330, 15 / 39 / 95 / 119 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 316, 5 / 224 / 226 / 256 / 316 / 326 / 330, 15 / 69 / 95 / 116 / 119 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 330, 2 / 15 / 39 / 95 / 119 / 144 / 149 / 154 / 156 / 159 / 177 / 186 / 195 / 202 / 224 / 226 / 256 / 316 / 326 / 330, 15 / 81 / 95 / 149 / 154 / 156 / 177 / 183 / 185 / 186 / 195 / 224 / 226 / 230, 2 / 15 / 39 / 69 / 95 / 116 / 144 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226, 2 / 14 / 15 / 39 / 69 / 95 / 116 / 119 / 149 / 154 / 156 / 159 / 177 / 185 / 186 / 195 / 224 / 226 / 256 / 326, 2 / 15 / 95 / 116 / 119 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 316, 15 / 95 / 116 / 119 / 144 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 314 / 316, 15 / 39 / 69 / 95 / 116 / 144 / 149 / 154 / 156 / 177 / 181 / 185 / 186 / 195 / 224 / 226 / 256 / 330, 2 / 15 / 39 / 69 / 95 / 119 / 144 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 314, 14 / 15 / 39 / 69 / 95 / 144 / 149 / 154 / 156 / 177 / 186 / 195 / 212 / 224 / 226 / 256 / 314 / 316 / 326 / 330, 15 / 39 / 69 / 95 / 144 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 316 / 330,15 / 95 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 334 / 335、15 / 39 / 63 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 257 / 275 / 330 / 335、15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 266 / 316 / 330 / 334 / 335、15 / 39 / 63 / 95 / 149 / 154 / 156 / 177 / 183 / 185 / 186 / 195 / 224 / 226 / 230 / 330 / 334、15 / 69 / 95 / 144 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 316、15 / 95 / 116 / 119 / 149 / 154 / 156 / 177 / 186 / 195 / 202 / 212 / 224 / 226 / 326 / 330、15 / 39 / 69 / 95 / 119 / 138 / 149 / 154 / 156 / 177 / 181 / 185 / 186 / 195 / 224 / 226 / 316 / 326、15 / 39 / 81 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230、2 / 14 / 15 / 69 / 95 / 116 / 119 / 144 / 149 / 154 / 156 / 159 / 177 / 186 / 195 / 224 / 226 / 326 / 330、15 / 39 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 334 / 345、15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 266 / 334、2 / 15 / 95 / 116 / 144 / 149 / 154 / 156 / 159 / 177 / 186 / 195 / 224 / 226、15 / 95 / 149 / 154 / 156 / 177 / 183 / 185 / 186 / 195 / 224 / 226 / 230、2 / 14 / 15 / 95 / 116 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 316 / 326 / 330、15 / 39 / 63 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 316 / 345、15 / 69 / 95 / 138 / 144 / 149 / 154 / 156 / 159 / 177 / 185 / 186 / 195 / 202 / 224 / 226 / 316、15 / 39 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 257 / 266 / 316 / 334、15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 257 / 275 / 334、15 / 95 / 149 / 154 / 156 / 177 / 183 / 186 / 195 / 224 / 226、15 / 89 / 95 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 230 / 316 / 345、2 / 14 / 15 / 95 / 119 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226、15 / 95 / 144 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 316 / 330、15 / 39 / 81 / 95 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 316 / 334、15 / 89 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 266 / 345、15 / 39 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 275 / 316、15 / 39 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 266 / 334 / 335、15 / 95 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226 / 275 / 316 / 330、15 / 95 / 119 / 149 / 154 / 156 / 177 / 186 / 195 / 202 / 224 / 226、15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 257 / 275 / 316 / 330 / 345、15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230、15 / 81 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 330 / 335 / 345、15 / 95 / 149 / 154 / 156 / 177 / 183 / 186 / 195 / 224 / 226 / 230 / 266、15 / 39 / 95 / 149 / 154 / 156 / 177 / 185 / 186 / 195 / 224 / 226、15 / 39 / 81 / 95 / 149 / 154 / 156 / 177 / 183 / 186 / 195 / 224 / 226 / 275 / 316 / 334 / 345、15 / 95 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226 / 230 / 257 / 316、15 / 39 / 95 / 116 / 149 / 154 / 156 / 177 / 186 / 195 / 224 / 226、or at least one set of substitutions at 15 / 95 / 149 / 154 / 156 / 177 / 183 / 185 / 186 / 195 / 224 / 226 / 257 / 275 / 316 / 330 / 334, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14 or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0151] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one of the following substitutions: 2E / 15Y / 39S / 69D / 95V / 144W / 149R / 154C / 156L / 177P / 186R / 195G / 224Y / 226I / 316L / 330M, 2V / 15Y / 39S / 69D / 95V / 119T / 149R / 154C / 156T / 177P / 185K / 186R / 195G / 224Y / 226I / 316L, 15Y / 69D / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 212I / 224Y / 226I / 316L / 330M, 2V / 15Y / 39S / 69D / 95V / 119T / 149R / 154C / 156T / 177P / 186R / 195G / 212I / 224Y / 226I / 316L, 15Y / 69D / 95V ... 4Y / 226I / 256A / 330M, 15Y / 39S / 95V / 119T / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 256A / 316L / 326R / 330M, 15Y / 69D / 95V / 116N / 119T / 149R / 154C / 156T / 177P / 185K / 186R / 195G / 224Y / 226I / 330M, 2E / 15Y / 39S / 95V / 119T / 144W / 149R / 154C / 156N / 159F / 177P / 186R / 195G / 202Y / 224Y / 226I / 256A / 316L / 326R / 330M, 15Y / 81Y / 95V / 149R / 154C / 156T / 177P / 183V / 185 G / 186R / 195G / 224Y / 226I / 230D, 2V / 15Y / 39S / 69D / 95V / 116N / 144W / 149R / 154C / 156L / 177P / 185K / 186R / 195G / 224Y / 226I, 2E / 14K / 15Y / 39S / 69D / 9 5V / 116N / 119T / 149R / 154C / 156T / 159F / 177P / 185K / 186R / 195G / 224Y / 226 I / 256A / 326R, 2E / 15Y / 95V / 116N / 119T / 149R / 154C / 156T / 177P / 186R / 19 5G / 224Y / 226I / 316L, 15Y / 95V / 116N / 119T / 144W / 149R / 154C / 156T / 177P / 185K / 186R / 195G / 224Y / 226I / 314W / 316L, 15Y / 39S / 69D / 95V / 116N / 144W / 149R / 154C / 156T / 177P / 181D / 185K / 186R / 195G / 224Y / 226I / 256A / 330M,2E / 15Y / 39S / 69D / 95V / 119T / 144W / 149R / 154C / 156L / 177P / 185K / 186R / 195G / 224Y / 226I / 314W, 14K / 15Y / 39S / 69D / 95V / 144W / 149R / 154C / 156N / 177P / 186R / 195G / 212I / 224Y / 226I / 256A / 314W / 316L / 326R / 330M、15Y / 39S / 69 D / 95V / 144W / 149R / 154C / 156L / 177P / 185K / 186R / 195G / 224Y / 226I / 316L / 3 30M、15Y / 95V / 149R / 154C / 156Q / 177P / 185G / 186R / 195G / 224Y / 226I / 334R / 335H、15Y / 39Q / 63V / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226I / 230D / 257L / 275N / 330R / 335H、15Y / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226I / 266L / 316L / 330R / 334R / 335H、15Y / 39Q / 63V / 95V / 149R / 154C / 156Q / 177P / 183V / 185G / 186R / 195G / 224Y / 226I / 230D / 330R / 334R、15Y / 69 D / 95V / 144W / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 316L、15Y / 95 V / 116N / 119T / 149R / 154C / 156L / 177P / 186R / 195G / 202Y / 212I / 224Y / 226I / 326R / 330M、15Y / 39S / 69D / 95V / 119T / 138R / 149R / 154C / 156T / 177P / 181D / 185K / 186R / 195G / 224Y / 226I / 316L / 326R、15Y / 39Q / 81Y / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226I / 230D、2V / 14K / 15Y / 69D / 95V / 116N / 119 T / 144W / 149R / 154C / 156T / 159F / 177P / 186R / 195G / 224Y / 226I / 326R / 330M、 15Y / 39Q / 95V / 149R / 154C / 156A / 177P / 186R / 195G / 224Y / 226I / 334R / 345V、15Y / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 230D / 266L / 334R, 2V / 15Y / 95V / 116N / 144W / 149R / 154C / 156L / 159F / 177P / 186R / 195G / 224Y / 226I, 15Y / 95V / 149R / 154C / 156A / 177P / 183V / 185G / 186R / 195G / 224Y / 226I / 230D, 2E / 14K / 15Y / 95V / 116N / 149R / 154C / 156T / 177P / 186 R / 195G / 224Y / 226I / 316L / 326R / 330M、15Y / 39Q / 63V / 95V / 149R / 154C / 156A / 177P / 186R / 195G / 224Y / 226I / 230D / 316L / 345V、15Y / 69D / 95V / 13 8R / 144W / 149R / 154C / 156T / 159F / 177P / 185K / 186R / 195G / 202Y / 224Y / 2 26I / 316L、15Y / 39Q / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226 I / 257L / 266L / 316L / 334R, 15Y / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226I / 257L / 275N / 334R, 15Y / 95V / 149R / 154C / 156T / 177P / 183V / 186R / 195G / 224Y / 226I, 15Y / 89W / 95V / 149R / 154C / 156A / 177P / 185G / 186R / 195G / 224Y / 226I / 230D / 316L / 345V, 2E / 14K / 15Y / 95V / 119T / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I、15Y / 95V / 144W / 149R / 154C / 1 56L / 177P / 186R / 195G / 224Y / 226I / 316L / 330M、15Y / 39Q / 81Y / 95V / 149R / 154C / 156T / 177P / 185G / 186R / 195G / 224Y / 226I / 316L / 334R、15Y / 89H / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 230D / 266L / 345V、15Y / 39Q / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226I / 230D / 2 75N / 316L, 15Y / 39Q / 95V / 149R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226 I / 230D / 266L / 334R / 335H, 15Y / 95V / 149R / 154C / 156Q / 177P / 185G / 186 R / 195G / 224Y / 226I / 275N / 316L / 330R, 15Y / 95V / 119T / 149R / 154C / 156N / 177P / 186R / 195G / 202Y / 224Y / 226I, 15Y / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 230D / 257L / 275N / 316L / 330R / 345V, 15Y / 95V / 149R / 154C / 156T / 177P / 186R / 195G / 224Y / 226I / 230D, 15Y / 81Y / 95V / 14 9R / 154C / 156Q / 177P / 186R / 195G / 224Y / 226I / 330R / 335H / 345V, 15Y / 95 V / 149R / 154C / 156A / 177P / 183V / 186R / 195G / 224Y / 226I / 230D / 266L, 1 5Y / 39S / 95V / 149R / 154C / 156T / 177P / 185K / 186R / 195G / 224Y / 226I, 15Y / 39Q / 81Y / 95V / 149R / 154C / 156T / 177P / 183V / 186R / 195G / 224Y / 226I / 2 75N / 316L / 334R / 345V, 15Y / 95V / 149R / 154C / 156A / 177P / 186R / 195G / 22 4Y / 226I / 230D / 257L / 316L, 15Y / 39S / 95V / 116N / 149R / 154C / 156N / 177P / 186R / 195G / 224Y / 226I, or 15Y / 95V / 149R / 154C / 156T / 177P / 183V / 185G / 186R / 195G / 224Y / 226I / 257L / 275N / 316L / 330R / 334R, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to a reference sequence corresponding to SEQ ID NO: 14.

[0152] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions provided in Tables 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to a reference sequence corresponding to SEQ ID NO:14.

[0153] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at an amino acid position provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or a reference sequence corresponding to SEQ ID NO:14.

[0154] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0155] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions at an amino acid position(s) set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or a reference sequence corresponding to SEQ ID NO:14.

[0156] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions of an RNA ligase variant set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or a reference sequence corresponding to SEQ ID NO:14.

[0157] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0158] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582.

[0159] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204, wherein the amino acid sequence comprises one or more substitutions.

[0160] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582, wherein the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or to a reference sequence corresponding to an even-numbered SEQ ID NO:42 or 204.

[0161] In some embodiments, the amino acid sequence of the modified RNA ligase is , 285, 288, 291, 296, 303, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0162] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residues 2G / V / E, 14K, 15E / L / M / P / Y, 18I, 33L, 39Q / S, 63V, 69D, 81Y, 85L, 86G, 89H / I / M / W, 95V, 116N, 117D, 119T, 138R, 142R, 144W, 149R / T, 154C, 156A / L / N / Q / T, 159F, 171E, 175K, 177P, 181D, 183V, 185G / K, 186D / R, 195G, 202Y, 212I, 214I, 224Y, 226I, 230D, 247K, 250K, 251K, 252K, 253K, 254K, 255K, 256K, 257K, 258K, 259K, 260K, 261K, 262K, 263K, 264K, 265K, 266K, 267K, 268K, 269K, 270K, 271K, 272K, 273K, 274K, 275K, 276K, 277K, 278K, 279K, 280K, 281K, 282K, 283K, 284K, 285K, 286K, 287K, 288K, 289 , 256A, 257L, 260S, 266L, 275N, 280G / N, 283I, 285K, 288E, 291P, 296K, 303Q, 306L, 307E / Q, 310K, 314W, 315S / T, 316L, 317A, 326R, 330M / R, 331R / W, 333V, 334R, 335D / H, 337L, 339P, 342I, or 345V, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or relative to a reference sequence corresponding to SEQ ID NO:42 or 204.

[0163] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 95 or 177, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0164] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one amino acid residue 95V or 177P, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0165] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0166] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 15Y, 95V, 149R, 154C, 156T, 177P, 186R, 195G, 224Y, or 226I, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0167] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or to a reference sequence corresponding to SEQ ID NO:42, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or to a reference sequence corresponding to SEQ ID NO:42.

[0168] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:130-478, or to a reference sequence corresponding to an even-numbered SEQ ID NO:130-478, and the amino acid sequence comprises one or more substitutions to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to a reference sequence corresponding to SEQ ID NO:42.

[0169] In some embodiments, the amino acid sequence of the modified RNA ligase comprises amino acid position(s) 149 / 156 / 195, 15 / 149 / 186 / 224 / 317, 15 / 156 / 195 / 224 / 226 / 317 / 331, 15 / 195 / 317 / 331, 149 / 224 / 331, 15 / 195 / 224 / 226 / 317 / 331, 15 / 33 / 86 / 149 / 154 / 195 / 317, 15 / 86 / 154 / 156 / 186 / 195 / 224 / 226 / 331, 15 / 149 / 224 / 317 / 331, 15 / 154 / 156 / 186 / 317 / 33 1, 15 / 33 / 149 / 224 / 226 / 331, 15 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 186 / 224 / 331, 195 / 224 / 331, 15 / 86 / 156 / 186 / 195 / 226 / 317 / 331, 15 / 195 / 22 / 331 , 156 / 186 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 224 / 226, 33 / 156 / 186 / 195 / 331, 15 / 86 / 149 / 154 / 156 / 317 / 331, 15 / 186 / 317 / 331, 15 / 86 / 149 / 154 / 195 / 33 1, 15 / 33 / 149 / 154 / 156 / 226, 15 / 149 / 154 / 186 / 317 / 331, 195 / 331, 15 / 86 / 156 / 186 / 195 / 331, 149 / 156 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 186 / 317 / 331, 15 / 154 / 186 / 195, 15 / 86 / 195 / 224 / 226, 230, 15 / 33 / 156 / 331, 15 / 33 / 156 / 195 / 224 / 226, 33 / 149 / 156 / 317, 15 / 33 / 186 / 195, 334, 15 / 154 / 156 / 224 / 317 , 15 / 149 / 186 / 331, 15 / 86 / 149 / 186 / 195 / 317, 15 / 33 / 154 / 195, 15 / 156 / 186 / 331, 15 / 149 / 186 / 317, 15 / 33 / 156 / 186 / 214 / 224 / 331, 15 / 86 / 186 / 224 / 22 6 / 317, 15 / 154 / 331, 15 / 149 / 154 / 226, 15 / 33 / 195 / 226, 15 / 224 / 317 / 331, 15 / 86 / 186 / 195 / 317, 226 / 317 / 331, 330, 89, 15 / 149 / 186, 156 / 195 / 331, 156,15 / 86 / 156 / 186 / 331, 275, 15 / 33 / 186 / 224, 15 / 186 / 317, 15 / 33 / 186 / 226 / 317, 15 / 149 / 154 / 156 / 331, 15 / 154 / 226 / 317, 15 / 86 / 156 / 186 / 195 / 317, 257, 15 / 154 / 156 / 186, 33 / 186 / 2 24, 335, 15 / 86 / 156 / 186 / 195 / 224 / 331, 154 / 156 / 317, 39, 2, 15 / 224 / 226 / 331, 15 / 33 / 149 / 224, 89 / 307, 15 / 154 / 156 / 317, 15 / 186 / 331, 15 / 33 / 154 / 317, 15 / 33 / 195 / 224 / 317, 15 / 15 6 / 224 / 226, 154 / 186 / 331, 185, 15 / 18 / 149 / 186 / 195, 86 / 149 / 154 / 156, 15 / 33 / 149 / 331, 326, 202, 119, 212, 15 / 33 / 156 / 317, 280, 142, 15 / 149, 15 / 33 / 86 / 156 / 195, 315, 256, 15 / 33 / 156 / 186, 138, 117, 116, 316, 69, 310, 317, 15 / 195 / 331, 15 / 33 / 331, 314, or 144, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42 or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0170] In some embodiments, the amino acid sequence of the modified RNA ligase comprises a substitution or set of substitutions 149R / 156T / 195G, 15M / 149T / 186R / 224Y / 317A, 15P / 156T / 195G / 224Y / 226I / 317A / 331L, 15M / 195G / 317A / 331R, 149R / 224Y / 331R, 15E / 195G / 224Y / 226I / 317A / 331W, 15P / 33L / 86G / 149R / 154C / 195G / 317A, 15M / 86G / 154C / 156T / 186R / 195G / 224Y / 226I / 33 1L, 15P / 149R / 224Y / 317A / 331R, 15M / 154C / 156T / 186R / 317A / 331R, 15M / 33 L / 149R / 224Y / 226I / 331W, 15E / 186R / 224Y / 226I / 317A / 331R, 15Y / 33L / 149 R / 186R / 224Y / 331R, 195G / 224Y / 331R, 15P / 86G / 156T / 186R / 195G / 226I / 31 7A / 331L, 15M / 195G / 226 / 331W, 156T / 186R / 224Y / 226I / 331R, 15Y / 149R / 154 C / 156T / 224Y / 226I, 33L / 156T / 186R / 195G / 331L, 15P / 86G / 149R / 154C / 156 T / 317A / 331R, 15M / 186D / 317A / 331R, 15P / 86G / 149R / 154C / 195G / 331L, 15Y / 33L / 149T / 154C / 156T / 226I, 15P / 149R / 154C / 186R / 317A / 331L, 195G / 331 R, 15M / 86G / 156T / 186R / 195G / 331W, 149R / 156T / 224Y / 226I / 331W, 15P / 149R / 154C / 156T / 186R / 317A / 331L, 15Y / 154C / 186D / 195G, 15E / 86G / 195G / 224Y / 226I, 230D, 15P / 33L / 156T / 331R, 15L / 33L / 156T / 195G / 224Y / 226I, 33L / 14 9R / 156T / 317A, 15E / 33L / 186R / 195G, 334R, 15E / 154C / 156T / 224Y / 317A, 15 Y / 149R / 186R / 331W, 15Y / 86G / 149T / 186R / 195G / 317A, 15P / 33L / 154C / 195G,15P / 154C / 156T / 186R / 317A / 331R、15M / 156T / 186R / 331R、15Y / 149T / 186D / 317A、15E / 33L / 156T / 186R / 214I / 224Y / 331W、15E / 86G / 186R / 224Y / 226I / 3 17A、15P / 154C / 331R、15Y / 149T / 154C / 226I、15Y / 33L / 195G / 226I、15M / 224 Y / 317A / 331W、15E / 86G / 186R / 195G / 317A、226I / 317A / 331L、330R、89W、15P / 149R / 186D / 331L、15P / 149R / 186R、89M、156T / 195G / 331L、156A、15E / 86G / 1 56T / 186R / 331R、195G / 331W、275N、15P / 33L / 186R / 224Y、15E / 186R / 317A、1 5M / 33L / 186R / 226I / 317A、89G、15Y / 149R / 154C / 156T / 331W、15Y / 154C / 226 I / 317A、15Y / 86G / 156T / 186R / 195G / 317A、257L、15Y / 154C / 156T / 186D、33L / 186R / 224Y、335H、15Y / 86G / 156T / 186R / 195G / 224Y / 331W、154C / 156T / 317A 、39Q、2G、15M / 224Y / 226I / 331R、15M / 33L / 149R / 224Y、89I / 307Q、15M / 154C / 156T / 317A、15E / 186R / 331W、15P / 33L / 154C / 317A、89H、15Y / 33L / 195G / 22 4Y / 317A、15E / 156T / 224Y / 226I、154C / 186R / 331L、185G、15M / 18I / 149R / 186 R / 195G、86G / 149R / 154C / 156T、15P / 33L / 149R / 331L、326R、202Y、119T、212 I、2V、15E / 149R / 154C / 156T / 331L、15P / 33L / 156T / 317A、15Y / 154C / 156T / 3 17A, 156L, 335D, 156T, 2E, 280G, 142R, 15E / 149R, 156Q, 15E / 33L / 86G / 156T / 195G, 315T, 185K, 39S, 256A, 15Y / 33L / 156T / 186D, 138R, 117D, 116N, 316L15M / 149R, 69D, 156N, 310K, 317A, 15Y / 195G / 331L, 15M / 33L / 331L, 330M, 314W, 144W, 15Y / 149R, or 315S, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0171] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions: V149R / G156T / D195G, K15M / V149T / Y186R / N224Y / L317A, K15P / G156T / D195G / N224Y / K226I / L317A / I331L, K15M / D195G / L317A / I331R, V149R / N224Y / I331R, K15E / D195G / N224Y / K226I / L317A / I331W, K15P / G33L / K86G / V149R / A154C / D195G / L3 17A, K15M / K86G / A154C / G156T / Y186R / D195G / N224Y / K226I / I331L, K15P / V 149R / N224Y / L317A / I331R, K15M / A154C / G156T / Y186R / L317A / I331R, K15M / G33L / V149R / N224Y / K226I / I331W, K15E / Y186R / N224Y / K226I / L317A / I33 1R, K15Y / G33L / V149R / Y186R / N224Y / I331R, D195G / N224Y / I331R, K15P / K8 6G / G156T / Y186R / D195G / K226I / L317A / I331L, K15M / D195G / K226I / I331W, G156T / Y186R / N224Y / K226I / I331R, K15Y / V149R / A154C / G156T / N224Y / K22 6I, G33L / G156T / Y186R / D195G / I331L, K15P / K86G / V149R / A154C / G156T / L3 17A / I331R, K15M / Y186D / L317A / I331R, K15P / K86G / V149R / A154C / D195G / I 331L, K15Y / G33L / V149T / A154C / G156T / K226I, K15P / V149R / A154C / Y186R / L317A / I331L, D195G / I331R, K15M / K86G / G156T / Y186R / D195G / I331W, V149 R / G156T / N224Y / K226I / I331W, K15P / V149R / A154C / G156T / Y186R / L317A / I 331L, K15Y / A154C / Y186D / D195G, K15E / K86G / D195G / N224Y / K226I, N230D,K15P / G33L / G156T / I331R、K15L / G33L / G156T / D195G / N224Y / K226I、G33L / V149R / G156T / L317A、K15E / G33L / Y186R / D195G、Q334R、K15E / A154C / G156T / N224Y / L317A、K15Y / V149R / Y186R / I331W、K15Y / K86G / V149T / Y186R / D195G / L317A、K15P / G33L / A154C / D195G、K15P / A154C / G156T / Y186R / L317A / I331R、K15M / G156T / Y186R / I331R、K15Y / V149T / Y186D / L317A、K15E / G33L / G156T / Y186R / V214I / N224Y / I331W、K15E / K86G / Y186R / N224Y / K226I / L317A、K15P / A154C / I331R、K15Y / V149T / A154C / K226I、K15Y / G33L / D195G / K226I、K15M / N224Y / L317A / I331W、K15E / K86G / Y186R / D195G / L317A、K226I / L317A / I331L、T330R、K89W、K15P / V149R / Y186D / I331L、K15P / V149R / Y186R、K89M、G156T / D195G / I331L、G156A、K15E / K86G / G156T / Y186R / I331R、D195G / I331W、T275N、K15P / G33L / Y186R / N224Y、K15E / Y186R / L317A、K15M / G33L / Y186R / K226I / L317A、K89G、K15Y / V149R / A154C / G156T / I331W、K15Y / A154C / K226I / L317A、K15Y / K86G / G156T / Y186R / D195G / L317A、V257L、K15Y / A154C / G156T / Y186D、G33L / Y186R / N224Y、N335H、K15Y / K86G / G156T / Y186R / D195G / N224Y / I331W、A154C / G156T / L317A、H39Q、H2G、K15M / N224Y / K226I / I331R、K15M / G33L / V149R / N224Y、K89I / N307Q、K15M / A154C / G156T / L317A、K15E / Y186R / I331W、K15P / G33L / A154C / L317A, K89H, K15Y / G33L / D195G / N224Y / L317A, K15E / G156T / N224Y / K2 26I, A154C / Y186R / I331L, N185G, K15M / S18I / V149R / Y186R / D195G, K86G / V149R / A154C / G1 56T, K15P / G33L / V149R / I331L, K326R, H202Y, Q119T, T212I, H2V, K15E / V149R / A154C / G156 T / I331L, K15P / G33L / G156T / L317A, K15Y / A154C / G156T / L317A, G156L, N335D, G156T, H2E, S280G, K142R, K15E / V149R, G156Q, K15E / G33L / K86G / G156T / D195G, D315T, N185K, H39S, K 256A, K15Y / G33L / G156T / Y186D, S138R, G117D, G116N, M316L, K15M / V149R, T69D, G156N, A3 10K, L317A, K15Y / D195G / I331L, K15M / G33L / I331L, T330M, H314W, Y144W, K15Y / V149R, or D315S, and the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0172] In some embodiments, the amino acid sequence of the modified RNA ligase is at amino acid positions 15 / 149 / 154 / 156 / 186 / 195 / 331, 149 / 154 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 154 / 186 / 195, 149 / 156 / 186 / 195 / 224 / 226 / 317, 15 / 149 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 226 / 331, 15 / 149 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 154 / 19 5 / 317, 15 / 149 / 154 / 195 / 224 / 317, 15 / 149 / 156 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 195 / 224 / 226 / 317 / 331, 149 / 195 / 224, 86 / 149 / 154 / 156 / 186 / 195 / 331, 15 / 33 / 149 / 156 / 195 / 224 / 317 / 331, 15 / 149 / 154 / 156 / 186 / 195 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 224 / 331, 15 / 149 / 154 / 156 / 19 5 / 224, 15 / 149 / 154 / 156 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 186 / 195 / 224 / 317, 33 / 149 / 156 / 186 / 224 / 331, 15 / 33 / 86 / 149 / 195 / 224 / 331, 15 / 86 / 149 / 15 4 / 156 / 186 / 195 / 331, 149 / 186 / 195 / 331, 15 / 154 / 156 / 186 / 226 / 331, 15 / 149 / 154 / 186 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 317 / 331, 15 / 33 / 154 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 149 / 186 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 149 / 154 / 156 / 224 / 2 26 / 317 / 331, 15 / 33 / 149 / 156 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 224 / 226 / 331, 154 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 224 / 317 / 331,15 / 149 / 154 / 156 / 186 / 195 / 224 / 226, 15 / 33 / 149 / 195 / 317 / 331, 15 / 149 / 154 / 224 / 331, 149 / 154 / 156 / 195 / 224 / 226 / 331, 15 / 149 / 154 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 195, 15 / 33 / 149 / 154 / 156 / 186 / 195, 15 / 33 / 86 / 149 / 154 / 195 / 224 / 317 / 331, or 149 / 186 / 195 / 224 / 226 / 331, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0173] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one of the following substitutions: 15M / 149R / 154C / 156T / 186R / 195G / 331R, 149R / 154C / 186D / 195G / 224Y / 317A / 331R, 15M / 33L / 149T / 154C / 186R / 195G, 149R / 156T / 186R / 195G / 224Y / 226I / 317A, 15Y / 149R / 156T / 195G / 224Y / 226I, 15Y / 33L / 86G / 149T / 154C / 156T / 186R / 195G / 226I / 3 31W, 15Y / 149R / 156T / 186R / 195G / 224Y / 317A / 331W, 15M / 33L / 149T / 154C / 1 95G / 317A, 15M / 149R / 154C / 195G / 224Y / 317A, 15Y / 149R / 156T / 186D / 224Y / 226I / 317A / 331R, 15M / 149T / 154C / 156T / 186D / 195G / 331L, 15Y / 33L / 149R / 154C / 156T / 186R / 195G / 224Y / 226I / 317A / 331R, 149R / 195G / 224Y, 86G / 149T / 154C / 156T / 186R / 195G / 331L, 15P / 33L / 149R / 156T / 195G / 224Y / 317A / 331 L, 15P / 149T / 154C / 156T / 186D / 195G / 317A / 331L, 15Y / 33L / 149T / 154C / 156 T / 186D / 224Y / 331L, 15P / 149R / 156T / 186R / 195G / 224Y / 317A / 331W, 15Y / 14 9R / 154C / 156T / 195G / 224Y, 15M / 149T / 154C / 156T / 195G / 224Y / 226I, 15E / 14 9R / 154C / 156T / 186R / 195G / 224Y / 317A, 33L / 149T / 156T / 186R / 224Y / 331L, 15Y / 33L / 86G / 149R / 195G / 224Y / 331W, 15E / 86G / 149R / 154C / 156T / 186R / 19 5G / 331W, 149R / 186R / 195G / 331W, 15E / 154C / 156T / 186D / 226I / 331R, 15Y / 1 49T / 154C / 186R / 195G / 224Y / 226I, 15P / 149R / 154C / 156T / 195G / 224Y / 331W,15E / 33L / 149R / 154C / 156T / 195G / 317A / 331L, 15E / 33L / 154C / 156T / 186R / 195G / 224Y / 317A / 331L, 15P / 149R / 186D / 195G / 224Y / 331L, 15P / 33L / 149R / 154C / 156T / 195G / 224Y / 331W, 15P / 149T / 154C / 156T / 224Y / 226I / 317 A / 331W, 15P / 33L / 149T / 156T / 195G / 331W, 15P / 149T / 154C / 156T / 186D / 224Y / 226I / 331L, 154C / 156T / 195G / 2 24Y / 226I, 15Y / 33L / 86G / 149R / 154C / 156T / 186D / 195G / 224Y / 317A / 331W, 15Y / 149R / 154C / 156T / 186R / 195G / 224Y / 226I, 15P / 33L / 149R / 195G / 317A / 331L, 15E / 149T / 154C / 224Y / 331R, 149T / 154C / 156T / 195G / 224Y / 226 I / 331R, 15M / 149R / 154C / 195G / 331L, 15Y / 149R / 154C / 156T / 186R / 195G, 15E / 149T / 154C / 156T / 186R / 195G / 3 31R, 15M / 33L / 149T / 154C / 156T / 186R / 195G, 15P / 33L / 86G / 149R / 154C / 195G / 224Y / 317A / 331R, or 149R / 186D / 195G / 224Y / 226I / 331R, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or relative to a reference sequence corresponding to SEQ ID NO:42.

[0174] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one of the following substitutions: K15M / V149R / A154C / G156T / Y186R / D195G / I331R, V149R / A154C / Y186D / D195G / N224Y / L317A / I331R, K15M / G33L / V149T / A154C / Y186R / D195G, V149R / G156T / Y186R / D195G / N224Y / K226I / L317A, K15Y / V149R / G156T / D195G / N224Y / K226I, K15 Y / G33L / K86G / V149T / A154C / G156T / Y186R / D195G / K226I / I331W, K15Y / V 149R / G156T / Y186R / D195G / N224Y / L317A / I331W, K15M / G33L / V149T / A154 C / D195G / L317A, K15M / V149R / A154C / D195G / N224Y / L317A, K15Y / V149R / G156T / Y186D / N224Y / K226I / L317A / I331R, K15M / V149T / A154C / G156T / Y1 86D / D195G / I331L, K15Y / G33L / V149R / A154C / G156T / Y186R / D195G / N224 Y / K226I / L317A / I331R, V149R / D195G / N224Y, K86G / V149T / A154C / G156T / Y186R / D195G / I331L, K15P / G33L / V149R / G156T / D195G / N224Y / L317A / I 331L, K15P / V149T / A154C / G156T / Y186D / D195G / L317A / I331L, K15Y / G33L / V149T / A154C / G156T / Y186D / N224Y / I331L, K15P / V149R / G156T / Y186R / D195G / N224Y / L317A / I331W, K15Y / V149R / A154C / G156T / D195G / N224Y, K1 5M / V149T / A154C / G156T / D195G / N224Y / K226I, K15E / V149R / A154C / G156 T / Y186R / D195G / N224Y / L317A, G33L / V149T / G156T / Y186R / N224Y / I331L,K15Y / G33L / K86G / V149R / D195G / N224Y / I331W、K15E / K86G / V149R / A154C / G156T / Y186R / D195G / I331W、V149R / Y186R / D195G / I331W、K15E / A154C / G156T / Y186D / K226I / I331R、K15Y / V149T / A154C / Y186R / D195G / N224Y / K226I、K15P / V149R / A154C / G156T / D195G / N224Y / I331W、K15E / G33L / V149R / A154C / G156T / D195G / L317A / I331L、K15E / G33L / A154C / G156T / Y186R / D195G / N224Y / L317A / I331L、K15P / V149R / Y186D / D195G / N224Y / I331L、K15P / G33L / V149R / A154C / G156T / D195G / N224Y / I331W、K15P / V149T / A154C / G156T / N224Y / K226I / L317A / I331W、K15P / G33L / V149T / G156T / D195G / I331W、K15P / V149T / A154C / G156T / Y186D / N224Y / K226I / I331L、A154C / G156T / D195G / N224Y / K226I、K15Y / G33L / K86G / V149R / A154C / G156T / Y186D / D195G / N224Y / L317A / I331W、K15Y / V149R / A154C / G156T / Y186R / D195G / N224Y / K226I、K15P / G33L / V149R / D195G / L317A / I331L、K15E / V149T / A154C / N224Y / I331R、V149T / A154C / G156T / D195G / N224Y / K226I / I331R、K15M / V149R / A154C / D195G / I331L、K15Y / V149R / A154C / G156T / Y186R / D195G、K15E / V149T / A154C / G156T / Y186R / D195G / I331R、K15M / G33L / V149T / A154C / G156T / Y186R / D195G、K15P / G33L / K86G / V149R / A154C / D195G / N224Y / L317A / I331R、or V149R / Y186D / D195G / N224Y / K226I / I331R, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0175] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or to a reference sequence corresponding to SEQ ID NO:204, wherein the amino acid sequence comprises one or more substitutions.

[0176] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:480-582, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or relative to a reference sequence corresponding to SEQ ID NO:204.

[0177] In some embodiments, the amino acid sequence of the modified RNA ligase comprises amino acid position(s) 2 / 39 / 69 / 144 / 156 / 316 / 330, 2 / 39 / 69 / 119 / 185 / 316, 69 / 212 / 256 / 330, 39 / 119 / 256 / 316 / 326 / 330, 69 / 116 / 119 / 185 / 330, 2 / 39 / 119 / 144 / 156 / 159 / 202 / 256 / 316 / 326 / 330, 81 / 183 / 185 / 230, 2 / 39 / 69 / 116 / 144 / 156 / 185, 2 / 14 / 39 / 69 / 116 / 119 / 159 / 185 / 256 / 326, 2 / 116 / 119 / 316, 116 / 119 / 144 / 185 / 314 / 316, 39 / 69 / 116 / 144 / 181 / 185 / 256 / 330, 2 / 39 / 69 / 119 / 144 / 156 / 185 / 314, 14 / 39 / 69 / 144 / 156 / 21 2 / 256 / 314 / 316 / 326 / 330, 39 / 69 / 144 / 156 / 185 / 316 / 330, 156 / 185 / 334 / 335, 39 / 63 / 156 / 230 / 257 / 275 / 330 / 335, 156 / 266 / 316 / 330 / 334 / 335, 39 / 63 / 156 / 183 / 185 / 230 / 330 / 334, 69 / 144 / 316, 116 / 119 / 156 / 202 / 212 / 326 / 330, 39 / 69 / 119 / 138 / 181 / 185 / 316 / 326, 39 / 81 / 156 / 230, 2 / 14 / 69 / 116 / 119 / 1 44 / 159 / 326 / 330, 39 / 156 / 334 / 345, 230 / 266 / 334, 2 / 116 / 144 / 156 / 159, 156 / 183 / 185 / 230, 2 / 14 / 116 / 316 / 326 / 330, 39 / 63 / 156 / 230 / 316 / 345, 69 / 13 8 / 144 / 159 / 185 / 202 / 316, 39 / 156 / 257 / 266 / 316 / 334, 156 / 257 / 275 / 334, 183, 89 / 156 / 185 / 230 / 316 / 345, 2 / 14 / 119, 144 / 156 / 316 / 330, 39 / 81 / 185 / 3 16 / 334, 89 / 230 / 266 / 345, 39 / 156 / 230 / 275 / 316, 39 / 156 / 230 / 266 / 334 / 335, 156 / 185 / 275 / 316 / 330, 119 / 156 / 202, 230 / 257 / 275 / 316 / 330 / 345, 230,At least one substitution or set of substitutions at 81 / 156 / 330 / 335 / 345, 156 / 183 / 230 / 266, 39 / 185, 39 / 81 / 183 / 275 / 316 / 334 / 345, 156 / 230 / 257 / 316, 39 / 116 / 156, or 183 / 185 / 257 / 275 / 316 / 330 / 334, where the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 204 or relative to a reference sequence corresponding to SEQ ID NO: 204.

[0178] In some embodiments, the amino acid sequence of the modified RNA ligase contains at least one substitution or set of substitutions: 2E / 39S / 69D / 144W / 156L / 316L / 330M, 2V / 39S / 69D / 119T / 185K / 316L, 69D / 212I / 256A / 330M, 39S / 119T / 256A / 316L / 326R / 330M, 69D / 116N / 119T / 185K / 330M, 2E / 39S / 119T / 144W / 156N / 159F / 202Y / 256A / 316L / 326R / 330M, 81Y / 183V / 185G / 230D, 2V / 39S / 69D / 116N / 144W / 156L / 185K, 2E / 14K / 39S / 69D / 116N / 119T / 159F / 1 85K / 256A / 326R, 2E / 116N / 119T / 316L, 116N / 119T / 144W / 185K / 314W / 316L, 3 9S / 69D / 116N / 144W / 181D / 185K / 256A / 330M, 2E / 39S / 69D / 119T / 144W / 156L / 185K / 314W, 14K / 39S / 69D / 144W / 156N / 212I / 256A / 314W / 316L / 326R / 330M, 39S / 69D / 144W / 156L / 185K / 316L / 330M, 156Q / 185G / 334R / 335H, 39Q / 63V / 1 56Q / 230D / 257L / 275N / 330R / 335H, 156Q / 266L / 316L / 330R / 334R / 335H, 39Q / 63V / 156Q / 183V / 185G / 230D / 330R / 334R, 69D / 144W / 316L, 116N / 119T / 156L / 202Y / 212I / 326R / 330M, 39S / 69D / 119T / 138R / 181D / 185K / 316L / 326R, 39Q / 81Y / 156Q / 230D, 2V / 14K / 69D / 116N / 119T / 144W / 159F / 326R / 330M, 39Q / 156 A / 334R / 345V, 230D / 266L / 334R, 2V / 116N / 144W / 156L / 159F, 156A / 183V / 185 G / 230D, 2E / 14K / 116N / 316L / 326R / 330M, 39Q / 63V / 156A / 230D / 316L / 345V, 69D / 138R / 144W / 159F / 185K / 202Y / 316L, 39Q / 156Q / 257L / 266L / 316L / 334R,156Q / 257L / 275N / 334R, 183V, 89W / 156A / 185G / 230D / 316L / 345V, 2E / 14K / 119T, 144W / 156L / 316L / 330M, 39Q / 81Y / 185G / 316L / 334R, 89H / 230D / 266 L / 345V, 39Q / 156Q / 230D / 275N / 316L, 39Q / 156Q / 230D / 266L / 334R / 335H, 156Q / 185G / 275N / 316L / 330R, 119T / 156N / 202Y, 230D / 257L / 275N / 316L / 3 30R / 345V, 230D, 81Y / 156Q / 330R / 335H / 345V, 156A / 183V / 230D / 266L, 39S / 185K, 39Q / 81Y / 183V / 275N / 316L / 334R / 345V, 156A / 230D / 257L / 316L, 39S / 116N / 156N, or 183V / 185G / 257L / 275N / 316L / 330R / 334R, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or relative to a reference sequence corresponding to SEQ ID NO:204.

[0179] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions H2E / H39S / T69D / Y144W / T156L / M316L / T330M, H2V / H39S / T69D / Q119T / N185K / M316L, T69D / T212I / K256A / T330M, H39S / Q119T / K256A / M316L / K326R / T330M, T69D / G116N / Q119T / N185K / T330M, H2E / H39S / Q119T / Y144W / T156N / L159F / H202Y / K2 56A / M316L / K326R / T330M, H81Y / I183V / N185G / N230D, H2V / H39S / T69D / G1 16N / Y144W / T156L / N185K, H2E / A14K / H39S / T69D / G116N / Q119T / L159F / N18 5K / K256A / K326R, H2E / G116N / Q119T / M316L, G116N / Q119T / Y144W / N185K / H 314W / M316L, H39S / T69D / G116N / Y144W / K181D / N185K / K256A / T330M, H2E / H 39S / T69D / Q119T / Y144W / T156L / N185K / H314W, A14K / H39S / T69D / Y144W / T 156N / T212I / K256A / H314W / M316L / K326R / T330M, H39S / T69D / Y144W / T156L / N185K / M316L / T330M, T156Q / N185G / Q334R / N335H, H39Q / I63V / T156Q / N23 0D / V257L / T275N / T330R / N335H, T156Q / V266L / M316L / T330R / Q334R / N335H , H39Q / I63V / T156Q / I183V / N185G / N230D / T330R / Q334R, T69D / Y144W / M31 6L, G116N / Q119T / T156L / H202Y / T212I / K326R / T330M, H39S / T69D / Q119T / S 138R / K181D / N185K / M316L / K326R, H39Q / H81Y / T156Q / N230D, H2V / A14K / T6 9D / G116N / Q119T / Y144W / L159F / K326R / T330M, H39Q / T156A / Q334R / L345V,N230D / V266L / Q334R, H2V / G116N / Y144W / T156L / L159F, T156A / I183V / N185G / N230D, H2E / A14K / G116N / M316L / K326R / T330M, H39Q / I63V / T156A / N230D / M316L / L345V, T69D / S138R / Y144W / L159F / N185K / H202Y / M316L, H39Q / T156Q / V257L / V266L / M316L / Q334R, T156Q / V257L / T275N / Q334R, I183V, K89W / T156A / N185G / N230D / M316L / L345V, H2E / A14K / Q119T, Y144W / T156L / M316L / T330M, H39Q / H81Y / N185G / M316L / Q334R, K89H / N230D / V266L / L345V, H39Q / T156Q / N230D / T275N / M316L, H39Q / T156Q / N230D / V266L / Q334R / N335H, T156Q / N185G / T275N / M316L / T330R, Q119T / T156N / H202Y, N230D / V25 7L / T275N / M316L / T330R / L345V, N230D, H81Y / T156Q / T330R / N335H / L345V, T156A / I183V / N230D / V266L, H39S / N185K, H 39Q / H81Y / I183V / T275N / M316L / Q334R / L345V, T156A / N230D / V257L / M316L, H39S / G116N / T156N, or I183V / N185G / V257L / T275N / M316L / T330R / Q334R, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 204, or relative to a reference sequence corresponding to SEQ ID NO: 204.

[0180] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution at an amino acid position provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0181] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution as provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0182] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions at an amino acid position provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0183] In some embodiments, the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions of an RNA ligase variant provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0184] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0185] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising residues 12-346 of a modified RNA ligase described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, or to a sequence of a modified RNA ligase described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1.

[0186] In some embodiments, the modified RNA ligase is selected from the group consisting of SEQ ID NOs: 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, 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, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 2 78, 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, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 40, 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, 40 2, 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, 568, 570, 572, 574, 576, 578, 580, or 582.

[0187] In some embodiments, the modified RNA ligase is selected from the group consisting of SEQ ID NOs: 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, 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, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 2 78, 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, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 40, 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, 40 2, 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, or 582.

[0188] In some embodiments, the modified RNA ligase comprises an amino acid sequence comprising residues 12-346 of an even-numbered SEQ ID NO:24-582, or an amino acid sequence comprising an even-numbered SEQ ID NO:24-582. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 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 modified RNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions.

[0189] In some embodiments, the modified RNA ligase is selected from the group consisting of SEQ ID NOs: 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, 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, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 2 78, 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, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 40, 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, 40 2, 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,In some embodiments, the amino acid sequence of the modified RNA ligase comprises residues 12-346 of 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, or 582. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 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 modified RNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions.

[0190] In some embodiments, the modified RNA ligase is selected from the group consisting of SEQ ID NOs: 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, 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, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 2 78, 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, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 40, 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, 40 2, 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,In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 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 modified RNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions.

[0191] In some embodiments, the modified RNA ligase comprises an amino acid sequence comprising residues 12-346 of SEQ ID NO: 42 or 204, or an amino acid sequence comprising SEQ ID NO: 42 or 204. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 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 modified RNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the modified RNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions.

[0192] In some of the foregoing embodiments, the modified RNA ligase polypeptide has 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence. In some embodiments, the modified RNA ligase polypeptide has 1, 2, 3, or 4 substitutions in the amino acid sequence. In some embodiments, the substitutions comprise non-conservative or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, guidance regarding non-conservative and conservative substitutions is provided by the variants disclosed herein.

[0193] In some embodiments, the modified RNA ligases of the present disclosure have RNA ligase 2 activity with improved or enhanced properties, particularly those described herein. In some embodiments, the modified RNA ligases have at least one improved or enhanced property compared to a reference RNA ligase. Exemplary improved properties are provided in the Examples.

[0194] In some embodiments, the modified RNA ligase has increased activity compared to a reference RNA ligase. In some embodiments, the modified RNA ligase has at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more activity compared to a reference RNA ligase. In some embodiments, the increased activity is with modified polynucleotide substrates, as discussed herein and in the Examples.

[0195] In some embodiments, the modified RNA ligase exhibits increased product yield compared to a reference RNA ligase. In some embodiments, the modified RNA ligase exhibits increased product yield compared to a reference RNA ligase at substrate concentrations of 10 μM to 400 μM, 20 μM to 350 μM, 50 μM to 300 μM, or 100 μM to 250 μM, preferably 200 to 400 μM. In some embodiments, the modified RNA ligase exhibits increased product yield compared to a reference RNA ligase at substrate concentrations of 10 μM, 20 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, or more.

[0196] In some embodiments, the modified RNA ligase has increased stability compared to a reference RNA ligase, hi some embodiments, the modified RNA ligase has increased thermal stability compared to a reference RNA ligase.

[0197] In some embodiments, the modified RNA ligase has increased activity on polynucleotide substrates containing phosphorothioate internucleotide linkages compared to a reference RNA ligase, hi some embodiments, the modified RNA ligase has increased activity or product yield on polynucleotide substrates containing 2'-modifications (e.g., 2'-O-methyl and / or 2'-fluoro) compared to a reference RNA ligase.

[0198] In some embodiments, the reference RNA ligase has a sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or a sequence corresponding to SEQ ID NO: 14, 42, or 204. In some embodiments, the reference RNA ligase has a sequence corresponding to residues 12-346 of SEQ ID NO: 14, or a sequence corresponding to SEQ ID NO: 14.

[0199] In some embodiments, the modified RNA ligase has one or more improved properties compared to a reference RNA ligase selected from: i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased activity toward polynucleotides containing phosphorothioate internucleotide linkages, vi) increased activity toward oligonucleotides containing 2'-modifications, vii) increased substrate tolerance, or any combination of i), ii), iii), iv), v), vi), and vii). In some embodiments, the reference RNA ligase has a sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or a sequence corresponding to SEQ ID NO: 14, 42, or 204. In some embodiments, the reference RNA ligase has a sequence corresponding to residues 12-346 of SEQ ID NO: 14, or a sequence corresponding to SEQ ID NO: 14.

[0200] In some embodiments, the present disclosure provides: residues 12 to 345 of SEQ ID NO:2; residues 12 to 343 of SEQ ID NO:4; residues 12 to 250 of SEQ ID NO:6; residues 12 to 346 of SEQ ID NO:8; residues 12 to 345 of SEQ ID NO:10; residues 12 to 345 of SEQ ID NO:12; residues 12 to 346 of SEQ ID NO: 14; residues 12 to 346 of SEQ ID NO: 16; residues 12 to 350 of SEQ ID NO:18; residues 12 to 350 of SEQ ID NO:20; or a modified RNA ligase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12-344 of SEQ ID NO:22.

[0201] In some embodiments, the modified RNA ligase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22.

[0202] In some embodiments, the modified RNA ligase residues 12 to 345 of SEQ ID NO:2; residues 12 to 343 of SEQ ID NO:4; residues 12 to 250 of SEQ ID NO:6; residues 12 to 346 of SEQ ID NO:8; residues 12 to 345 of SEQ ID NO:10; residues 12 to 345 of SEQ ID NO:12; residues 12 to 346 of SEQ ID NO: 14; residues 12 to 346 of SEQ ID NO: 16; residues 12 to 350 of SEQ ID NO:18; residues 12 to 350 of SEQ ID NO:20; or an amino acid sequence comprising residues 12 to 344 of SEQ ID NO:22.

[0203] In some embodiments, the modified RNA ligase comprises an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22.

[0204] In some embodiments, the modified RNA ligase is expressed as a fusion protein. In some embodiments, the modified RNA ligase described herein can be fused to various polypeptide sequences, including, by way of non-limiting example, polypeptide tags that can be used for detection and / or purification. In some embodiments, the modified RNA ligase fusion protein comprises a glycine-histidine or histidine tag (His tag). In some embodiments, the modified RNA ligase fusion protein comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the modified RNA ligase fusion protein comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N) terminus of the modified RNA ligase polypeptide. In some embodiments, the fusion is to the carboxy (C) terminus of the modified RNA ligase polypeptide.

[0205] In some embodiments, the modified RNA ligase polypeptides described herein are isolated compositions. In some embodiments, the modified RNA ligase polypeptides are purified, as described in more detail herein. In some embodiments, the modified RNA ligase is provided in solution, as a lyophilizate, or immobilized on a substrate, as described in more detail herein.

[0206] In some embodiments, the present disclosure further provides functional or biologically active fragments of the modified RNA ligase polypeptides described herein. Thus, for any embodiment of a modified RNA ligase described herein, a functional or biologically active fragment of the modified RNA ligase is provided herewith. In some embodiments, a functional or biologically active fragment of a modified RNA ligase comprises at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the RNA ligase polypeptide (i.e., the parent RNA ligase) from which it is derived. In some embodiments, a functional or biologically active fragment comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the RNA ligase. In some embodiments, the functional fragment is truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, fewer than 50, fewer than 55, fewer than 60, fewer than 65, or fewer than 70 amino acids.

[0207] In some embodiments, a functional fragment of a modified RNA ligase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the modified RNA ligase. In some embodiments, the functional fragment is truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, fewer than 50, fewer than 55, fewer than 60, fewer than 65, or fewer than 70 amino acids.

[0208] In some embodiments, functional or biologically active fragments of the modified RNA ligase polypeptides described herein include at least one mutation or set of mutations within the amino acid sequence of the modified RNA ligase described herein. Thus, in some embodiments, functional or biologically active fragments of the modified RNA ligase exhibit enhanced or improved properties relative to the mutation or set of mutations in the parent RNA ligase. Polynucleotides encoding modified polypeptides, expression vectors, and host cells

[0209] In another aspect, the present disclosure provides recombinant polynucleotides encoding the modified RNA ligases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to generate recombinant polynucleotide constructs capable of expressing the modified RNA ligase. In some embodiments, an expression construct comprising at least one heterologous polynucleotide encoding a modified RNA ligase polypeptide is introduced into a suitable host cell to express the corresponding RNA ligase polypeptide.

[0210] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides that can encode a polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, makes it possible to generate a vast number of nucleic acids, all of which encode the modified RNA ligases of the present disclosure. Thus, the present disclosure provides methods and compositions for generating all possible variations of producible polynucleotides that encode the modified RNA ligase polypeptides described herein by selecting combinations based on possible codon choices, and all such variations of polynucleotides are intended to be specifically disclosed with respect to any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1) and the accompanying Sequence Listing.

[0211] In some embodiments, codons are preferably optimized for utilization by a host cell selected for protein production. In some embodiments, preferred codons in bacterial cells are used for expression in bacterial cells. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, a codon-optimized polynucleotide encoding a modified RNA ligase polypeptide described herein comprises preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions within the full-length coding region.

[0212] Thus, in some embodiments, a recombinant polynucleotide of the disclosure encodes a modified RNA ligase polypeptide described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon-optimized.

[0213] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO: 14 and the even-numbered SEQ ID NOs: 24-582, wherein the amino acid sequence comprises a polynucleotide sequence encoding a modified RNA ligase that contains one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0214] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204.

[0215] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

[0216] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204, and wherein the amino acid sequence comprises a polynucleotide sequence encoding a modified RNA ligase that contains one or more substitutions relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

[0217] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582, and wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO:14.

[0218] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:24-128, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-128, and wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO:14.

[0219] In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence at amino acid positions 2, 14, 15, 18, 33, 39, 63, 69, 81, 83, 85, 86, 89, 95, 116, 117, 119, 138, 142, 144, 149, 154, 156, 159, 171, 175, 177, 181, 185, 186, 195, 202, 212, 214, 224, 226, 230, 247, 256, 257, 260, 266, 275, 280, 283, 285, 288, 291, and a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence containing at least one substitution in 296, 303, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0220] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution at amino acid position 95 or 177, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0221] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0222] In some embodiments, the recombinant polynucleotide comprises a sequence comprising an amino acid sequence at amino acid position(s) 177 / 260 / 307 / 345, 171 / 175 / 183 / 280 / 283 / 303, 95 / 159 / 177 / 260 / 288, 175 / 183 / 247 / 280 / 283 / 296, 177 / 307, 81 / 171 / 183 / 335, 175 / 183, 81 / 175 / 183 / 280 / 296 / 335 / 339, 81 / 183 / 247 / 266, 95 / 177, 171 / 280 / 283 / 296 / 303, 85 / 177 / 260 / 288 / 333 / 345, 177 / 2 91 / 307 / 333, 81 / 171 / 175 / 183 / 316 / 337, 81 / 171 / 266 / 280 / 283 / 335 / 339, 177, 95 / 159 / 177 / 260 / 280 / 288 / 306 / 345, 171 / 175 / 335, 177 / 260 / 30 / 333, 1 4 / 95 / 177 / 288 / 307, 95 / 177 / 345, 95 / 159 / 177 / 260 / 285 / 288, 266 / 296, 81 / 283 / 335 / 337, 95 / 159 / 177 / 260 / 342 / 345, 63 / 171 / 175 / 183 / 266 / 280 / 296 / 3 16, 280 / 335 / 337, 81 / 171 / 280, 81 / 171 / 175 / 296 / 316, 171 / 175 / 316 / 335, 171 / 280 / 283 / 296 / 316, 171 / 283 / 335, 63 / 171 / 175 / 183 / 266 / 283 / 303 / 316, 171 / 247 / 266 / 283 / 296 / 303 / 316 / 335, 63 / 81 / 171 / 175 / 183 / 266 / 280 / 283 / 337, 81 / 171 / 303 / 316 / 335, 288 / 307, 14 / 95 / 159 / 177 / 345, 81 / 171 / 175 / 18 and a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution or set of substitutions at amino acid positions 3 / 247 / 266 / 280 / 283, 81 / 247 / 266, 280, 85 / 177 / 260, 63 / 183, 175 / 183 / 247 / 280 / 283 / 316, 291, 247, 171 / 280 / 283, 81 / 171 / 175 / 280 / 316, 333, 171 / 296 / 316, 81 / 171, or 280 / 316 / 335, relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14.or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0223] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution at an amino acid position provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0224] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0225] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution or set of substitutions at an amino acid position set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0226] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution or set of substitutions of an RNA ligase variant set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO:14 or relative to a reference sequence corresponding to SEQ ID NO:14.

[0227] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0228] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:24-582.

[0229] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to a reference sequence corresponding to SEQ ID NO: 42 or 204, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204.

[0230] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs: 24-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO: 24-582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to the reference sequence corresponding to SEQ ID NO: 42 or 204.

[0231] In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence at amino acid positions 2, 14, 15, 18, 33, 39, 63, 69, 81, 83, 85, 86, 89, 95, 116, 117, 119, 138, 142, 144, 149, 154, 156, 159, 171, 175, 177, 181, 185, 186, 195, 202, 212, 214, 224, 226, 230, 247, 256, 257, 260, 266, 275, 280, 283, 285, 288, 291, 296, 303 , 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, containing at least one substitution, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution at amino acid position 95 or 177, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204.

[0234] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to a reference sequence corresponding to SEQ ID NO:42, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42.

[0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:130-478, or to a reference sequence corresponding to an even-numbered SEQ ID NO:130-478, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or relative to the reference sequence corresponding to SEQ ID NO:42.

[0236] In some embodiments, the recombinant polynucleotide is selected from the group consisting of amino acid positions 149 / 156 / 195, 15 / 149 / 186 / 224 / 317, 15 / 156 / 195 / 224 / 226 / 317 / 331, 15 / 195 / 317 / 331, 149 / 224 / 331, 15 / 195 / 224 / 226 / 317 / 331, 15 / 33 / 86 / 149 / 154 / 195 / 317, 15 / 86 / 154 / 156 / 186 / 195 / 224 / 226 / 331, 15 / 149 / 224 / 317 / 331, 15 / 154 / 156 / 186 / 317 / 331, 15 / 33 / 149 / 224 / 226 / 331, 15 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 186 / 224 / 331, 195 / 224 / 331, 15 / 86 / 156 / 186 / 195 / 226 / 317 / 331, 15 / 195 / 22 / 331, 156 / 1 86 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 224 / 226, 33 / 156 / 186 / 195 / 331, 15 / 86 / 149 / 154 / 156 / 317 / 331, 15 / 186 / 317 / 331, 15 / 86 / 149 / 154 / 195 / 331, 15 / 33 / 149 / 154 / 156 / 226, 15 / 149 / 154 / 186 / 317 / 331, 195 / 331, 15 / 86 / 156 / 186 / 195 / 331, 149 / 156 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 186 / 317 / 331, 15 / 1 54 / 186 / 195, 15 / 86 / 195 / 224 / 226, 230, 15 / 33 / 156 / 331, 15 / 33 / 156 / 195 / 224 / 226, 33 / 149 / 156 / 317, 15 / 33 / 186 / 195, 334, 15 / 154 / 156 / 224 / 317, 15 / 149 / 186 / 331, 15 / 86 / 149 / 186 / 195 / 317, 15 / 33 / 154 / 195, 15 / 156 / 186 / 331, 15 / 149 / 186 / 317, 15 / 33 / 156 / 186 / 214 / 224 / 331, 15 / 86 / 186 / 224 / 226 / 317, 15 / 154 / 331, 15 / 149 / 154 / 226, 15 / 33 / 195 / 226, 15 / 224 / 317 / 331, 15 / 86 / 186 / 195 / 317, 226 / 317 / 331, 330, 89, 15 / 149 / 186, 156 / 195 / 331, 156,15 / 86 / 156 / 186 / 331, 275, 15 / 33 / 186 / 224, 15 / 186 / 317, 15 / 33 / 186 / 226 / 317, 15 / 149 / 154 / 156 / 331, 15 / 154 / 226 / 317, 15 / 86 / 156 / 186 / 195 / 317, 257, 15 / 154 / 156 / 186, 33 / 186 / 224, 335, 15 / 8 6 / 156 / 186 / 195 / 224 / 331, 154 / 156 / 317, 39, 2, 15 / 224 / 226 / 331, 15 / 33 / 149 / 224, 89 / 307, 15 / 154 / 156 / 317, 15 / 186 / 331, 15 / 33 / 154 / 317, 15 / 33 / 195 / 224 / 317, 15 / 156 / 224 / 226, 154 / 186 / 331, 185, 15 / 18 / 149 / 186 / 195, 86 / 149 / 154 / 156, 15 / 33 / 149 / 331, 326, 202, 119, 212, 15 / 33 / 156 / 317, 280, 142, 15 / 149, 15 / 33 / 86 / 156 / 195, 315, 256, 15 / 33 / 156 / 186, 138, 117, 116, 316, 69, 310, 31 The present invention also includes a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence containing at least one substitution or set of substitutions at positions 7, 15 / 195 / 331, 15 / 33 / 331, 314, or 144, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to a reference sequence corresponding to SEQ ID NO: 42.

[0237] In some embodiments, the recombinant polynucleotide is selected from the group consisting of amino acid positions 15 / 33 / 149 / 154 / 186 / 195, 149 / 156 / 186 / 195 / 224 / 226 / 317, 15 / 149 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 226 / 331, 15 / 149 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 154 / 195 / 317, 15 / 149 / 154 / 195 / 224 / 317, 15 / 149 / 156 / 186 / 224 / 226 / 317 / 33 1, 15 / 33 / 149 / 154 / 156 / 186 / 195 / 224 / 226 / 317 / 331, 149 / 195 / 224, 86 / 149 / 154 / 156 / 186 / 195 / 331, 15 / 33 / 149 / 156 / 195 / 224 / 317 / 331, 15 / 149 / 154 / 156 / 186 / 195 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 224 / 331, 15 / 149 / 154 / 156 / 195 / 224, 15 / 149 / 154 / 156 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 186 / 195 / 224 / 317, 33 / 149 / 156 / 186 / 224 / 331, 15 / 33 / 86 / 149 / 195 / 224 / 331, 15 / 86 / 149 / 154 / 156 / 186 / 195 / 331, 149 / 186 / 195 / 331, 15 / 154 / 156 / 186 / 226 / 33 1, 15 / 149 / 154 / 186 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 317 / 331, 15 / 33 / 154 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 1 49 / 186 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 149 / 154 / 156 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 156 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 22 4 / 226 / 331, 154 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 149 / 154 / 156 / 186 / 195 / 224 / 226, 15 / 33 / 149 / 195 / 317 / 331,and a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution or set of substitutions at 15 / 149 / 154 / 224 / 331, 149 / 154 / 156 / 195 / 224 / 226 / 331, 15 / 149 / 154 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 195, 15 / 33 / 149 / 154 / 156 / 186 / 195, 15 / 33 / 86 / 149 / 154 / 195 / 224 / 317 / 331, or 149 / 186 / 195 / 224 / 226 / 331, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:42, or relative to a reference sequence corresponding to SEQ ID NO:42.

[0238] In some embodiments, the recombinant polynucleotide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or to a reference sequence corresponding to SEQ ID NO:204, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or to a reference sequence corresponding to SEQ ID NO:204.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA ligase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs:480-582, or to a reference sequence corresponding to an even-numbered SEQ ID NO:480-582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:204 or relative to the reference sequence corresponding to SEQ ID NO:204.

[0240] In some embodiments, the recombinant polynucleotide is selected from the group consisting of amino acid positions 2 / 39 / 69 / 144 / 156 / 316 / 330, 2 / 39 / 69 / 119 / 185 / 316, 69 / 212 / 256 / 330, 39 / 119 / 256 / 316 / 326 / 330, 69 / 116 / 119 / 185 / 330, 2 / 39 / 119 / 144 / 156 / 159 / 202 / 256 / 316 / 326 / 330, 81 / 183 / 185 / 230, 2 / 39 / 69 / 116 / 144 / 156 / 185, 2 / 14 / 39 / 69 / 116 / 119 / 159 / 185 / 256 / 3 26, 2 / 116 / 119 / 316, 116 / 119 / 144 / 185 / 314 / 316, 39 / 69 / 116 / 144 / 181 / 185 / 256 / 330, 2 / 39 / 69 / 119 / 144 / 156 / 185 / 314, 14 / 39 / 69 / 144 / 156 / 212 / 256 / 314 / 316 / 326 / 330, 39 / 69 / 144 / 156 / 185 / 316 / 330, 156 / 185 / 334 / 335, 39 / 63 / 156 / 230 / 257 / 275 / 330 / 335, 156 / 266 / 316 / 330 / 334 / 335, 39 / 63 / 156 / 183 / 185 / 230 / 330 / 334, 69 / 144 / 316, 116 / 119 / 156 / 202 / 212 / 326 / 330, 39 / 69 / 119 / 138 / 181 / 185 / 316 / 326, 39 / 81 / 156 / 230, 2 / 14 / 69 / 116 / 119 / 144 / 159 / 326 / 330, 39 / 156 / 334 / 345, 230 / 266 / 334, 2 / 116 / 144 / 156 / 159, 156 / 183 / 185 / 230, 2 / 14 / 116 / 316 / 326 / 330, 39 / 63 / 156 / 230 / 316 / 345, 69 / 138 / 144 / 159 / 185 / 202 / 316, 39 / 156 / 257 / 266 / 316 / 334, 156 / 257 / 275 / 334, 183, 89 / 156 / 185 / 230 / 316 / 345, 2 / 14 / 119, 144 / 156 / 316 / 330, 39 / 81 / 185 / 31 6 / 334, 89 / 230 / 266 / 345, 39 / 156 / 230 / 275 / 316, 39 / 156 / 230 / 266 / 334 / 335, 156 / 185 / 275 / 316 / 330, 119 / 156 / 202, 230 / 257 / 275 / 316 / 330 / 345, 230,and a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence containing at least one substitution or set of substitutions at 81 / 156 / 330 / 335 / 345, 156 / 183 / 230 / 266, 39 / 185, 39 / 81 / 183 / 275 / 316 / 334 / 345, 156 / 230 / 257 / 316, 39 / 116 / 156, or 183 / 185 / 257 / 275 / 316 / 330 / 334, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 346 of SEQ ID NO:204, or relative to a reference sequence corresponding to SEQ ID NO:204.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising substitutions at the amino acid positions provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising at least one substitution provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising at least one substitution or set of substitutions at an amino acid position provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, where the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising at least one substitution or set of substitutions of an RNA ligase variant provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to a reference sequence corresponding to SEQ ID NO: 42 or 204, as provided in each of the tables.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to a reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence comprising residues 12-346 of a modified RNA ligase set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, or to the sequence of a modified RNA ligase set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising residues 12-346 of an even-numbered SEQ ID NO:24-582, or an even-numbered SEQ ID NO:24-582, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising residues 12-346 of SEQ ID NO: 42 or 204, or an amino acid sequence comprising SEQ ID NO: 42 or 204, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1038 of an odd-numbered SEQ ID NO:13-581, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO:13-581, wherein the recombinant polynucleotide encodes an RNA ligase.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 13, 41, or 203, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 13, 41, or 203, wherein the recombinant polynucleotide encodes an RNA ligase.

[0251] In some embodiments, the recombinant polynucleotide is selected from the group consisting of SEQ ID NOs: 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, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 1, 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, 253, 255, 257, 259, 261, 263, 265, 267, 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, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 39, 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, 40 1, 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,and 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 more sequence identity to a reference sequence corresponding to nucleotide residues 34 to 1038 of 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, or 581, wherein the recombinant polynucleotide encodes an RNA ligase.

[0252] In some embodiments, the recombinant polynucleotide is selected from the group consisting of SEQ ID NOs: 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, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 1, 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, 253, 255, 257, 259, 261, 263, 265, 267, 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, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 39, 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, 40 1, 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, or 581, wherein the recombinant polynucleotide encodes an RNA ligase.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1038 of an odd-numbered SEQ ID NO: 13-581, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 13-581.

[0254] In some embodiments, the recombinant polynucleotide is selected from the group consisting of SEQ ID NOs: 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, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 1, 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, 253, 255, 257, 259, 261, 263, 265, 267, 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, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 39, 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, 40 1, 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,and a polynucleotide sequence comprising nucleotide residues 34 to 1038 of 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, or 581.

[0255] In some embodiments, the recombinant polynucleotide is selected from the group consisting of SEQ ID NOs: 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, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 1, 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, 253, 255, 257, 259, 261, 263, 265, 267, 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, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 39, 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, 40 1, 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, or 581.

[0256] In some embodiments, the recombinant polynucleotide comprises: residues 12 to 345 of SEQ ID NO:2; residues 12 to 343 of SEQ ID NO:4; residues 12 to 250 of SEQ ID NO:6; residues 12 to 346 of SEQ ID NO:8; residues 12 to 345 of SEQ ID NO:10; residues 12 to 345 of SEQ ID NO:12; residues 12 to 346 of SEQ ID NO: 14; residues 12 to 346 of SEQ ID NO: 16; residues 12 to 350 of SEQ ID NO:18; residues 12 to 350 of SEQ ID NO:20; or a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12 to 344 of SEQ ID NO:22.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22.

[0258] In some embodiments, the recombinant polynucleotide comprises: residues 12 to 345 of SEQ ID NO:2; residues 12 to 343 of SEQ ID NO:4; residues 12 to 250 of SEQ ID NO:6; residues 12 to 346 of SEQ ID NO:8; residues 12 to 345 of SEQ ID NO:10; residues 12 to 345 of SEQ ID NO:12; residues 12 to 346 of SEQ ID NO: 14; residues 12 to 346 of SEQ ID NO: 16; residues 12 to 350 of SEQ ID NO:18; residues 12 to 350 of SEQ ID NO:20; or a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising residues 12 to 344 of SEQ ID NO:22.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a modified RNA ligase comprising an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22.

[0260] In some embodiments, the present disclosure provides recombinant polynucleotides capable of hybridizing under high stringency conditions to a reference polynucleotide encoding a modified RNA ligase polypeptide described herein, e.g., a recombinant polynucleotide provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under high stringency conditions to a reference polynucleotide corresponding to nucleotide residues 34-1038 of SEQ ID NO: 13, 41, or 203, or a reference polynucleotide sequence corresponding to SEQ ID NO: 13, 41, or 203, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under high stringency conditions to a polynucleotide sequence corresponding to nucleotide residues 34-1038 of an odd-numbered SEQ ID NO: 23-581, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 23-581, or a reverse complement thereof.

[0261] In some embodiments, the disclosure provides recombinant polynucleotides capable of hybridizing under high stringency conditions to the reverse complement of a reference polynucleotide encoding a modified RNA ligase polypeptide described herein, wherein the recombinant polynucleotides that hybridize under stringent conditions encode an RNA ligase polypeptide comprising an amino acid sequence having one or more amino acid differences compared to SEQ ID NO: 14, 42, or 204 at a residue position selected from any of the positions set forth in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1. In some embodiments, a recombinant polynucleotide that hybridizes under high stringency conditions to the reverse complement of a reference polynucleotide encoding a modified RNA ligase polypeptide described herein encodes an RNA ligase polypeptide having an amino acid sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:24-582, or an amino acid sequence comprising an even-numbered SEQ ID NO:24-582, wherein the amino acid difference is relative to SEQ ID NO:14, 42, or 204.

[0262] In some embodiments, a recombinant polynucleotide that hybridizes under highly stringent conditions 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 more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 23, 41, or 203, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 23, 41, or 203, or its reverse complement. In some embodiments, recombinant polynucleotides that hybridize under highly stringent conditions comprise 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 more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1038 of an odd-numbered SEQ ID NO:23-581, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO:23-581, or its reverse complement.

[0263] In some additional embodiments, a polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence encoding a modified RNA ligase polypeptide that has at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reverse complement of a polynucleotide reference sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 23, 41, or 203, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 23, 41, or 203. In some embodiments, a polynucleotide that hybridizes under high stringency conditions comprises a polynucleotide sequence encoding a modified RNA ligase polypeptide that has at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reverse complement of a polynucleotide reference sequence corresponding to nucleotide residues 34-1038 of an odd-numbered SEQ ID NO:23-581, or a polynucleotide sequence that has at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polynucleotide reference sequence corresponding to an odd-numbered SEQ ID NO:23-581.

[0264] In some embodiments, a recombinant polynucleotide encoding any of the RNA ligases described herein is manipulated in various ways to facilitate expression of the RNA ligase polypeptide. In some embodiments, the recombinant polynucleotide encoding the RNA ligase comprises an expression vector in which one or more regulatory sequences are present to regulate expression of the RNA ligase polynucleotide and / or polypeptide. Depending on the expression vector used, it may be desirable or necessary to manipulate the isolated polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, regulatory sequences include, among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0265] In some embodiments, an appropriate promoter is selected based on the choice of host cell. For bacterial host cells, promoters suitable for directing transcription of the nucleic acid constructs of the present disclosure include promoters derived from the E. coli lactose operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 97:1111-1112, 2002). 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 filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (Aspergillus niger neutral α-amylase and Aspergillus Examples of promoters suitable for use in yeast host cells include, but are not limited to, the promoters derived from Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other promoters useful for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in insect cells include, but are not limited to, the polyhedrin, p10, ELT, OpIE2, hr5 / ie1 promoters, and the like.Exemplary promoters for use in mammalian cells include, but are not limited to, promoters from cytomegalovirus (CMV), chicken β-actin promoter fused to a CMV enhancer, simian vacuolar virus 40 (SV40), Homo sapiens phosphoglycerate kinase, β-actin, elongation factor 1a or glyceraldehyde-3-phosphate dehydrogenase, and chicken β-actin.

[0266] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the RNA ligase polypeptide. Any suitable terminator that functions in the selected host cell finds use in the present invention. In the case of bacterial expression, the transcription terminator can be a Rho-dependent terminator that relies on Rho transcription factors, or a Rho-independent terminator that does not require transcription factors, i.e., an endogenous terminator. 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 for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, 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 for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8(6):423-88). Exemplary terminators for mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), simian virus 40 (SV40), homo sapiens growth hormone (hGH), bovine growth hormone (BGH), and human or rabbit beta globulin.

[0267] In some embodiments, the control sequence is a suitable leader sequence, which is a non-translated region of an mRNA used for translation by the host cell. In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the RNA ligase polypeptide. Any suitable leader sequence that functions in the host cell of choice finds use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include, but are not limited to, the 5'-UTR elements present in orthopoxvirus mRNAs.

[0268] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of a nucleic acid sequence that, upon transcription, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that functions in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3'UTR sequences for mammalian host cells include, but are not limited to, the 3'-UTRs of α- and β-globin mRNAs, which contain several sequence elements that increase mRNA stability and translation.

[0269] In some embodiments, a 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 that directs the encoded polypeptide into the secretory pathway of a cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence essentially contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a selected host cell finds use in expressing an engineered polypeptide. Useful signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the genes encoding Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.Useful signal peptides for yeast host cells include, but are not limited to, those derived from the Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase genes. Useful signal peptides for insect and mammalian host cells include, but are not limited to, those derived from the immunoglobulin gamma (IgG) gene and signal peptides within human secreted proteins, such as the human beta-galactosidase polypeptide.

[0270] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source, including, but not limited to, the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both a signal peptide region and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0271] In some embodiments, regulatory sequences are also utilized. These sequences facilitate regulation of polypeptide expression relative to the growth of the host cell. 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 operator 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 alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0272] In another aspect, the present disclosure provides recombinant expression vectors comprising a recombinant polynucleotide encoding a modified RNA ligase polypeptide and, depending on the type of host into which it will be introduced, one or more expression control regions, such as a promoter and terminator, an origin of replication, etc. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce a recombinant expression vector that contains one or more convenient restriction enzyme recognition sites, allowing for the insertion or substitution of a nucleic acid sequence encoding an RNA ligase polypeptide. Alternatively, in some embodiments, a nucleic acid sequence of the present disclosure is expressed by inserting the nucleic acid sequence, or a nucleic acid construct comprising the sequence, into a vector suitable for expression. In some embodiments involving the creation of an expression vector, the coding sequence is placed into the vector such that it is operably linked to control sequences suitable for expression.

[0273] Recombinant expression vector can be any suitable vector (for example, plasmid or virus) that can be easily subjected to recombinant DNA procedures to cause the expression of RNA ligase polynucleotide sequence.The selection of vector usually depends on the compatibility of the vector with the host cell that the vector is introduced into.Vector can be linear plasmid or closed circular plasmid.

[0274] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some alternative embodiments, the vector is one that, upon introduction into a host cell, integrates into the genome and is replicated along with the chromosome into which it has been integrated. Furthermore, some embodiments utilize a single vector or plasmid, or two or more vectors or plasmids, and / or transposons, that together contain the total DNA to be introduced into the genome of the host cell.

[0275] In some embodiments, the recombinant polynucleotide can be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on a replication-defective viral vector (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0276] In some embodiments, expression vectors include one or more selectable markers to allow for the selection of transformed cells. A "selectable marker" is a gene the result of which confers biocide or viral resistance, heavy metal resistance, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0277] In another aspect, the disclosure provides a host cell comprising a recombinant polynucleotide encoding at least one modified RNA ligase polypeptide described herein, wherein the polynucleotide is operably linked to one or more control sequences for expression of the modified RNA ligase enzyme in the host cell. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide encoding a modified RNA ligase polypeptide described herein, wherein the polynucleotide is operably linked to one or more control sequences. Suitable host cells for use in expressing the polypeptides encoded by the expression vectors of the invention are known in the art and include, but are not limited to, bacterial cells such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various strains of Escherichia coli (e.g., W3110(ΔfhuA) and BL21).

[0278] In another aspect, the disclosure provides methods of producing a modified RNA ligase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the modified RNA ligase polypeptide under conditions suitable for expression of the polypeptide, such that the modified RNA ligase is produced. In some embodiments, the method further comprises isolating the RNA ligase polypeptide from the culture and / or the host cell. In some embodiments, the method further comprises purifying the expressed RNA ligase polypeptide as described herein.

[0279] In some embodiments, the RNA ligase polypeptide expressed in the host cells is recovered from the cells and / or culture medium using one or more known techniques for protein purification, including lysozyme or detergent treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others, such as those described herein.

[0280] Chromatographic techniques for separating / purifying RNA ligase polypeptides include, among others, reverse-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. The purification conditions for RNA ligase 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 techniques can be used to isolate RNA ligase. Purification by affinity chromatography can use antibodies that specifically bind to RNA ligase polypeptides. In some embodiments, an affinity tag, such as a His tag, can be introduced into the RNA ligase polypeptide for separation / purification purposes.

[0281] Suitable culture media and growth conditions for host cells are known in the art. Any suitable method for introducing a polynucleotide into a cell for expression of an RNA ligase polypeptide is contemplated to find use in the present invention. Suitable techniques include, but are not limited to, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0282] In some embodiments, recombinant polypeptides (e.g., RNA ligase enzyme variants) can be produced using any suitable method known in the art. For example, there are a variety of different mutagenesis techniques known to those of skill in the art. Additionally, mutagenesis kits are available from many commercial molecular biology suppliers. Methods are available for specific substitutions at predetermined amino acids (site-directed), specific or random mutations in local regions of a gene (region-directed), or random mutagenesis throughout the gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those of skill in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein modification are described in the following patents: U.S. Patent Nos. 6,117,679, 6,420,175, 6,376,246, 6,586,182, 7,747,391, 7,747,393, 7,783,428, and 8,383,346. After variants are produced, they can be screened for any desired properties (e.g., high or increased activity, low or decreased activity, increased thermal activity, increased stability, increased substrate range, increased inhibitor resistance or tolerance, increased salt tolerance, and / or pH stability, etc.). Exemplary methods are provided in the Examples.

[0283] In some embodiments, modified RNA ligase polypeptides having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or modified RNA ligase polypeptide to suitable mutagenesis and / or directed evolution methods known in the art, for example, as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767, and U.S. Pat. No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

[0284] Mutagenesis and directed evolution methods can be applied to polynucleotides encoding RNA ligases to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution method finds use in the present disclosure and is known in the art (e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406 ... No. 6,251,674, No. 6,265,201, No. 6,277,638, No. 6,287,861, No. 6,287,862, No. 6,291,242, No. 6,297,053 Specification, Specification No. 6,303,344, Specification No. 6,309,883, Specification No. 6,319,713, Specification No. 6,319,714, Specification No. 6,323,030, Specification No. 6,326,204, Specification No. 6,335,160 , 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, Specification No. 6,365,408, Specification No. 6,368,861, Specification No. 6,372,497, Specification No. 6,337,186, Specification No. 6,376,246, Specification No. 6,379,964, Specification No. 6,387,702, Specification No. 6 , 391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,4 Specification No. 20,175, Specification No. 6,423,542, Specification No. 6,426,224, Specification No. 6,436,675, Specification No. 6,444,468, Specification No. 6,455,253, Specification No. 6,479,652, Specification No. 6,482,Inventory No. 647, same as Inventory No. 6,483,011, same as Inventory No. 6,484,105, same as Inventory No. 6,489,146, same as Inventory No. 6,500,617, same as Inventory No. 6,500,639, same as Inventory No. 6,506,602, same as Inventory No. 6,506,603, same as Inventory No. 6, Inventory No. 518,065, same as Inventory No. 6,519,065, same as Inventory No. 6,521,453, same as Inventory No. 6,528,311, same as Inventory No. 6,537,746, same as Inventory No. 6,573,098, same as Inventory No. 6,576,467, same as Inventory No. 6,579,678. Same as Item No. 6,586,182, Item No. 6,602,986, Item No. 6,605,430, Item No. 6,613,514, Item No. 6,653,072, Item No. 6,686,515, Item No. 6,703,240, and Item No. 6,716,631. Inventory No. 6,825,001, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, and No. 7,058. Inventory No. 515, same as Inventory No. 7,105,297, same as Inventory No. 7,148,054, same as Inventory No. 7,220,566, same as Inventory No. 7,288,375, same as Inventory No. 7,384,387, same as Inventory No. 7,421,347, same as Inventory No. 7,430,477, same as Inventory No. 7, Inventory Nos. 462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, and 7,747,393. Same as Inventory No. 7,751,986, Same as Inventory No. 7,776,598, Same as Inventory No. 7,783,428, Same as Inventory No. 7,795,030, Same as Inventory No. 7,853,410, Same as Inventory No. 7,868,138, Same as Inventory No. 7,783,428, Same as Inventory No. 7,873,477 Inventory No. 7,873,499, Inventory No. 7,904,249, Inventory No. 7,957,912, Inventory No. 7,981,614, Inventory No. 8,014,961, Inventory No. 8,029,988, Inventory No. 8,048,674, Inventory No. 8,058Inventory No. 001, same as Inventory No. 8,076,138, same as Inventory No. 8,108,150, same as Inventory No. 8,170,806, same as Inventory No. 8,224,580, same as Inventory No. 8,377,681, same as Inventory No. 8,383,346, same as Inventory No. 8,457,903, same as Inventory No. 8,504,4 Inventory No. 98, same as Inventory No. 8,589,085, same as Inventory No. 8,762,066, same as Inventory No. 8,768,871, same as Inventory No. 9,593,326, same as Inventory No. 9,665,694, same as Inventory No. 9,684,771, PCT and Miwaiwai Taishinko; Ling et al., Anal. Biochem., 1997, 254(2): 157-78; Dale et al., Meth. Mol. Biol., 1996, 57: 369-74; Smith, Ann. Rev. Genet., 1985, 19: 423-462; Botstein et al. al., Science, 1985, 229: 1193-1201; Carter, Biochem. J., 1986, 237: 1-7; Kramer et al., Cell, 1984, 38: 879-887; Wells et al., Gene, 1985, 34: 315-323; Minshull et al. al.,Curr.Op.Chem.Biol.,1999,3:284-290;Christians et al., Nat. Biotechnol., 1999, 17: 259-264; Crameri et al., Nature, 1998, 391: 288-291; Crameri, et al., Nat. Biotechnol., 1997, 15: 436-438; Zhang et al. al.,Proc.Nat.Acad.Sci.USA,1997,94:4504-4509;Crameri et al.,Nat.Biotechnol.,1996,14:315-319;Stemmer,Nature,1994,366:389-391;Stemmer,Proc.Nat.Acad.Sci.USA,1994,91:10747-10751; EP3049973; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767; WO2009 / 152336; and WO2015 / 048573 (all of which are incorporated herein by reference).

[0285] In some embodiments, clones obtained after mutagenesis treatment are screened by exposing the enzyme preparation to defined treatment or assay conditions (e.g., temperature, pH, type of ligase substrate (e.g., DNA, RNA, secondary structure, etc.), input substrate concentration, nucleotide cofactor, etc.) and measuring enzyme activity after treatment or other suitable assay conditions. Clones containing polynucleotides encoding the polypeptides of interest are then isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in host cells. Measurement of enzyme activity from expression libraries can be performed using any suitable method known in the art and described in the Examples.

[0286] In some embodiments, in the case of modified polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase synthesis methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then joined (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired contiguous sequence (see, e.g., 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 classical phosphoramidite method (see, e.g., Beaucage et al., Tet. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), which is commonly practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized (eg, in an automatic DNA synthesizer), purified, annealed, ligated and cloned in suitable vectors.

[0287] In some embodiments, a method of preparing a modified RNA ligase polypeptide can include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequences of any variant described herein, and (b) expressing the RNA ligase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or more (e.g., up to 3, 4, 5, or up to 10) deletions, insertions, and / or substitutions of amino acid residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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. In some embodiments, the substitutions are conservative or non-conservative.

[0288] In some embodiments, any of the modified RNA ligase polypeptides expressed in the host cells are recovered and / or purified from the cells and / or culture medium using one or more known techniques for protein purification, including lysozyme or detergent treatment, such as those described herein, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others. Composition of RNA ligase

[0289] In a further aspect, the present disclosure provides compositions of the RNA ligases disclosed herein. In some embodiments, the compositions comprise at least one modified RNA ligase polypeptide described herein. In some embodiments, the modified 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 comprising the modified RNA ligase polypeptides suitable for a variety of applications and uses.

[0290] In some embodiments, the composition comprises at least one modified RNA ligase described herein, for example, the composition comprises at least one modified RNA ligase provided in Tables 4.1, 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1.

[0291] In some embodiments, the composition comprises an RNA ligase provided in Table 4.1. In some embodiments, the composition comprises an RNA ligase comprising an amino acid sequence comprising residues 12-345 of SEQ ID NO:2; residues 12-343 of SEQ ID NO:4; residues 12-250 of SEQ ID NO:6; residues 12-346 of SEQ ID NO:8; residues 12-345 of SEQ ID NO:10; residues 12-345 of SEQ ID NO:12; residues 12-346 of SEQ ID NO:14; residues 12-346 of SEQ ID NO:16; residues 12-350 of SEQ ID NO:18; residues 12-350 of SEQ ID NO:20; or residues 12-344 of SEQ ID NO:22. In some embodiments, the composition comprises an RNA ligase comprising an amino acid sequence comprising SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22.

[0292] 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 substrates, e.g., polynucleotide substrates comprising modified nucleotides.

[0293] In some embodiments, the composition further comprises an additive or ligation enhancer, such as one or more polyethylene glycols (e.g., PEG6000, PEG8000, etc.) or other molecular crowding reagents, including bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000), among others.

[0294] In some embodiments, the modified RNA ligase described herein is provided in solution, as a lyophilizate, or immobilized on a substrate. In some embodiments, the substrate is a solid substrate, a porous substrate, a membrane, or a particle. The enzyme can be entrapped in a matrix or membrane. In some embodiments, the matrix includes polymeric materials such as calcium alginate, agar, κ-carrageenan, polyacrylamide, agarose or its derivatives (e.g., cross-linked agarose), and collagen, or solid matrices such as activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is ​​a particle, membrane, or fiber. Types of membranes include nylon, cellulose, polysulfone, or polyacrylate, among others.

[0295] 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 covalently immobilized to the support material. Support materials include inorganic materials such as alumina, silica, porous glass, ceramic, diatomaceous earth, clay, and bentonite, among others, or organic materials such as cellulose (CMC, DEAE cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion exchange resins such as Amberlite, Sephadex, and Dowex.

[0296] Uses of modified RNA ligase polypeptides and kits In another aspect, the disclosure provides for the use of modified RNA ligases for polynucleotide synthesis, RNA repair, or other molecular biology applications.

[0297] In some embodiments, modified RNA ligases are used to ligate polynucleotides and oligonucleotides. In some embodiments, modified RNA ligases are used to synthesize polynucleotides from shorter oligonucleotides. In some embodiments, a method of ligating at least a first polynucleotide strand and a second polynucleotide strand comprises contacting the first polynucleotide strand and the second polynucleotide strand with a modified RNA ligase described herein in the presence of a nucleotide substrate under conditions suitable for ligation of the first polynucleotide strand to the second polynucleotide strand, wherein the first polynucleotide strand comprises a ligatable 5' end and the second polynucleotide strand comprises a 3' end that can be ligated to the 5' end of the first polynucleotide strand.

[0298] In some embodiments, the second polynucleotide strand comprising the ligatable 3' end 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 strand is a 3'-OH. In some embodiments of the method, the 5' end of the first polynucleotide strand is a 5'-phosphate. In some embodiments of the method, the internucleotide linkages in the first polynucleotide strand and / or the second polynucleotide strand comprise a phosphate linkage.

[0299] In some embodiments, the method further includes a third polynucleotide strand, wherein the first polynucleotide strand and the second polynucleotide strand hybridize adjacent to each other on the third polynucleotide strand, with the 5' end of the first polynucleotide strand positioned adjacent to the 3' end of the second polynucleotide strand, e.g., forming a nick. In some embodiments of the method, the third polynucleotide strand is contiguous with the first polynucleotide strand or the second polynucleotide strand. In some embodiments of the method, the third polynucleotide strand is contiguous with the first polynucleotide strand and the second polynucleotide strand to form a single, continuous polynucleotide ligase substrate. In some embodiments, the third polynucleotide strand is RNA, DNA, or a mixture of RNA and DNA.

[0300] In some embodiments of the method, the third polynucleotide comprises a splint or bridge polynucleotide, and the 5' terminal sequence of the first polynucleotide strand and the 3' terminal sequence of the second polynucleotide strand hybridize adjacent to each other on the splint or bridge polynucleotide, positioning the 5' end of the first polynucleotide strand adjacent to the 3' end of the second polynucleotide strand. In some embodiments, the splint or bridge polynucleotide is RNA, DNA, or a mixture of RNA and DNA.

[0301] In some embodiments, the first polynucleotide strand hybridizes to a third polynucleotide strand to form a first double-stranded fragment, and the second polynucleotide strand hybridizes to a fourth polynucleotide strand to form a second double-stranded fragment, and the first and second double-stranded fragments can base-pair to form a substrate for an engineered RNA ligase. In some embodiments, the first double-stranded fragment and the second double-stranded fragment have complementary overhangs or complementary single-stranded ends (also called cohesive or sticky ends) that can base-pair to form a double-stranded nick between the first double-stranded fragment and the second double-stranded fragment, which serves as a substrate for an engineered RNA ligase.

[0302] In some embodiments, the complementary ends (e.g., sticky or cohesive ends) are long enough to allow the double-stranded fragments to base-pair and form a suitable substrate for the modified RNA ligase. In some embodiments, the complementary single-stranded ends comprise at least 1-50, 2-40, 3-35, 4-30, 5-25, 6-20, or 8-15 or more nucleotides in length. In some embodiments, the complementary single-stranded ends are 1-10, 2-8, or 4-6 nucleotides in length. In some embodiments, the complementary single-stranded ends are 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more nucleotides in length.

[0303] In some embodiments, the length of the double-stranded region or complementary portion of each double-stranded fragment is 4 to 50, 6 to 45, 8 to 40, 10 to 35, 12 to 30, or 14 to 25 nucleotides, hi 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.

[0304] In some embodiments of the method, the ligase substrate comprises at least two, three, four, five, or six or more double-stranded fragments, each having a complementary end that can base-pair with at least one other double-stranded fragment that also has a complementary end to form a substrate for RNA ligase. In some embodiments, the double-stranded fragments have complementary or cohesive ends, for example, at the 5' and 3' ends, such that the double-stranded fragments can be ligated to form concatemers. In some embodiments, when the number of double-stranded fragments is three, at least one of the double-stranded fragments has complementary ends at the 5' and 3' ends of the fragment, and the double-stranded fragment can base-pair with two other different double-stranded fragments to form a substrate that can be ligated to a product comprising three double-stranded fragments. It should be understood that when three or more double-stranded fragments are ligated, the complementary ends of the double-stranded fragments can be designed to ligate all of the different double-stranded fragments.

[0305] In some embodiments of the method, the first polynucleotide strand and / or the second polynucleotide strand comprises one or more modified nucleotides or nucleotide analogs, wherein the modified nucleotides or nucleotide analogs comprise a nucleobase analog or modified nucleobase, a modified nucleoside sugar residue, a modified internucleotide linkage, a modified 5'-terminal phosphate group, and / or a modified 3'-terminal hydroxyl group.

[0306] In some embodiments, the polynucleotide substrate comprises a modified 5'-terminal 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 phosphorothioate, phosphoramidate, monomethyl phosphate, methyl phosphonate, vinyl phosphonate, or phosphonocarboxylate.

[0307] In some embodiments of the method, the polynucleotide substrate comprises one or more modified nucleoside sugar residues. In some embodiments of the method, the first polynucleotide and / or the second polynucleotide comprises one or more modified nucleoside sugar residues. In some embodiments, the modified nucleoside sugar residue is a 2'-O-alkyl, 2'-halo, β-D-riboLNA, or α-L-ribo-LNA (e.g., a locked nucleic acid). In some embodiments, the modified nucleoside sugar residue is, inter alia, a 2'-O-methyl, 2'-O-ethyl, or 2'-O-propyl group. In some embodiments, the modified nucleoside sugar residue is 2'-fluoro, 2'-bromo, or 2'-chloro, preferably 2'-fluoro. In some embodiments, the sugar residue is modified with a conjugate, e.g., a targeting moiety, e.g., GalNac, or a lipid moiety.

[0308] In some embodiments of the method, the polynucleotide substrate comprises one or more modified nucleotide residues having a modified nucleobase or nucleobase analog. In some embodiments of the method, the first polynucleotide and / or the second polynucleotide comprises one or more modified nucleotide residues having a modified nucleobase or nucleobase analog. In some embodiments, the nucleobase analog is, inter alia, 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-deazaguanine. In some embodiments, the nucleobase is modified with a conjugate, e.g., a targeting moiety, e.g., GalNac, and a lipid moiety.

[0309] In some embodiments of the method, the polynucleotide substrate comprises one or more modified or non-standard internucleotide linkages, i.e., internucleotide linkages other than phosphate linkages. In some embodiments, the first polynucleotide and / or the second polynucleotide comprise one or more modified or non-standard internucleotide linkages. In some embodiments, the internucleotide linkages are phosphorothioate, phosphoacetate, phosphoramidate, methylphosphonate, or phosphonocarboxylate. In some embodiments, at least 1%, 2%, 5%, 10%, or more of the internucleotide linkages are non-standard internucleotide linkages. In some embodiments, the non-standard internucleotide linkages are present in 1, 2, 3, 4, or 5 nucleotides at the 5' and / or 3' ends of the polynucleotide substrate.

[0310] In some embodiments of this method, the polynucleotide substrate comprises a modified 3'-hydroxyl group. In some embodiments, the 3' end of the polynucleotide substrate is attached to a matrix or surface, such as a solid matrix. In such cases, the polynucleotide substrate attached to the matrix has a ligatable 5' end. In some embodiments, the 3' end of the polynucleotide substrate is modified with an amine, halo, phosphate, phosphate analog, -O-alkyl, lipid moiety, or detectable label.

[0311] In some embodiments, the polynucleotide substrate comprises a C-4' modification, including, among others, a 4'-thio-C2' modification, a 4' / 5' aminoalkyl / C2' modification, a C4'-guanidino-C2' modification, and a C4'-O-Me / C2' modification (see, e.g., Gangopadhyay et al., RNA Biology, 2022, 19:1, 452-467).

[0312] In some embodiments, the method includes a nucleotide substrate used by the RNA ligase to catalyze the joining reaction. In some embodiments, the nucleotide substrate is ATP and / or dATP. Preferably, the nucleotide substrate is ATP. In some embodiments, the ligation reaction conditions include Mg +2 , buffers, and / or salts.

[0313] In some embodiments, the reaction conditions also include ligation enhancing reagents, including polyethylene glycols (e.g., PEG 6000 and PEG 8000) or other molecular crowding reagents, such as bovine serum albumin (BSA), dextran, and Ficoll, among others.

[0314] In some embodiments, the ligation reaction is carried out at an appropriate temperature and reaction time. In some embodiments, the ligation reaction temperature is 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 ligation reaction temperatures can be used, for example, a temperature at which stable hybrids form between polynucleotide substrates and a higher temperature to promote the subsequent completion of the ligation reaction.

[0315] In some embodiments, the ligation reaction time can be a time sufficient for ligation of the polynucleotide substrates. In some embodiments, the ligation reaction time is 0.5 to 72 hours or more. In some embodiments, the ligation reaction time is 1 to 72 hours, 2 to 48 hours, or 2 to 24 hours. In some embodiments, the ligation reaction time is 0.5, 1, 2, 4, 5, 12, 24, 48, or 72 hours or more.

[0316] In some embodiments, modified RNA ligases are used to ligate nicks or related nick structures (see, e.g., Cheng et al., Royal Soc Chem Adv., 2019, 9:8620-8627). In some embodiments, modified RNA ligases are used to synthesize RNA from shorter oligonucleotides, for example, by using splint nucleic acids (see, e.g., Stark et al., RNA, 2006, 12:2014-2019). In some embodiments, modified RNA ligases are used to ligate modified oligonucleotides, such as those provided in the examples. Other examples of modified oligonucleotide products that can be synthesized using short oligonucleotide substrates are described in, among others, patent publications WO22104366, WO22029209, WO22031847, WO20226960, US2022072024, US2021238606, US11286488, US2017305956, WO22212153, WO22192519, WO22125490, WO22072447, WO21257568, WO 21102373, WO21072395, WO21022108, US2022079971, US11034957, US2021332365, US11015201, US10995336, US11091759, US10889813, US10130651, US10513703, WO19232255, WO21108640, WO22147304, and WO21138537.

[0317] In a further aspect, the present disclosure provides kits comprising the modified RNA ligases described herein. In some embodiments, the kits further comprise one or more of a buffer, a nucleotide substrate (e.g., ATP or dATP), and / or one or more polynucleotide substrates. In some embodiments, the kits further comprise additives or ligation enhancers, including one or more polyethylene glycols (e.g., PEG 6000, PEG 8000, etc.) or other molecular crowding reagents (including bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000), among others). Example

[0318] The following examples, including experiments and results achieved, are provided for illustrative purposes only and should not be construed as limiting the invention.

[0319] In the experimental disclosure that follows, the following abbreviations apply where relevant: ppm (parts per million); M (mole); mM (millimole); uM and μM (micromolar); nM (nanomole); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); ul, uL, ml, and mL (microliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (minute); h and hr (hour); U (unit); OD (optical density); MW (molecular weight); rpm (revolutions per minute); rcf (relative centrifugal force); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); NGS (next generation sequencing); ds (double stranded); ss (single stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used laboratory E. coli strain, Coli Available from the Genetic Stock Center [CGSC], New Haven, CT); HTP (High Throughput); HPLC (High Pressure Liquid Chromatography); FPLC (Fast Protein Liquid Chromatography); ddH2O (Double Distilled Water); PBS (Phosphate Buffered Saline); BSA (Bovine Serum Albumin); DTT (Dithiothreitol); CAM (Chloramphenicol); CAT (Chloramphenicol Acetyltransferase); IPTG (Isopropyl β-D-1-thiogalactopyranoside); FIOPC (Fold Improvement from Positive Control); FIOP (Fold Improvement from Parent); LB (Luria-Bertani); TB (Terrific Broth).

[0320] Example 1 E. coli expression host containing recombinant RNA ligase 2 (Rnl2) gene To produce the enzyme, the RNA ligase 2 gene was cloned into one of two different expression vectors. In Examples 3-6, pCK110900 was used, operably linked to a lac promoter under the control of the lacl repressor (see, e.g., Figure 3 of U.S. Patent Application Publication No. 2006 / 0195947). The expression vector also contained a P15a origin of replication and a chloramphenicol resistance gene. In Examples 7-13, the pJV vector system was used (see, U.S. Patent No. 10,184,117). The resulting plasmids were transformed into E. coli W3110 using standard methods known in the art. Transformants were isolated by subjecting the cells to chloramphenicol selection, as known in the art (see, e.g., U.S. Patent No. 8,383,346 and WO2010 / 144103).

[0321] Example 2 Preparation of RNA ligase (Rnl2)-containing wet cell pellet E. coli cells containing the recombinant Rnl2-encoding gene from a monoclonal colony were inoculated into 180 μl of LB medium containing 1% glucose and 30 μg / mL chloramphenicol (CAM) in wells of a 96-well shallow-well microtiter plate. The plate was sealed with an O2-permeable seal, and the cultures were grown overnight at 30°C, 200 rpm, and 85% humidity. Next, 10 μl of each cell culture was transferred to a well of a 96-well deep-well plate containing 390 mL of TB and 30 μg / mL CAM. The deep-well plate was sealed with an O2-permeable seal and incubated at 30°C, 250 rpm, and 85% humidity until the OD600 reached 0.6-0.8. The cell culture was then induced with IPTG to a final concentration of 1 mM and incubated overnight under the same conditions used initially. The cells were then pelleted using centrifugation at 4,000 rpm for 10 minutes. The supernatant was discarded and the pellet was frozen at −80°C before lysis.

[0322] Example 3 E. coli shake-flake expression and purification of recombinant RNA ligase 2 (Rnl2) gene To determine the first-round backbone, a set of 10 ligase enzymes was selected based on their homology to SEQ ID NO:2. HTP cultures grown as described above were plated onto LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol and grown overnight at 37°C. One colony from each culture was transferred to 6 ml of LB broth containing 1% glucose and 30 μg / ml chloramphenicol. The cultures were grown at 30°C and 250 rpm for 18 hours and then subcultured at an approximately 1:50 dilution into 250 ml of Terrific Broth containing 30 μg / ml chloramphenicol to a final OD600 of approximately 0.05. The cultures were incubated at 30°C and 250 rpm for approximately 3 hours to an OD600 of 0.6-0.8, and then induced by the addition of IPTG to a final concentration of 1 mM. The induced cultures were incubated at 30°C and 250 rpm for 20 hours. After this incubation period, the cultures were centrifuged at 4,000 rpm for 10 minutes. The culture supernatant was discarded, and the pellet was resuspended in 35 ml of 50 mM Tris-HCl (pH 7.5). The cell suspension was cooled in an ice bath and lysed using a Microfluidizer cell disrupter (Microfluidics M-110L). The crude lysate was pelleted by centrifugation (16,000 rpm for 60 minutes at 4°C), and the supernatant was filtered through a 0.2 μm PES membrane to further clarify the lysate. These RNA Ligase 2 lysates were supplemented with 20 mM imidazole and 500 mM NaCl. The lysates were then purified using an AKTA Pure purification system and a 5 ml HisTrap FF column (GE Healthcare) using the run parameters shown in Table 1 below. The wash buffer contained 50 mM Tris-HCl (pH 8), 500 mM NaCl, 20 mM imidazole, and the elution buffer contained 50 mM Tris-HCl (pH 8), 500 mM NaCl, 300 mM imidazole. [Table 1]

[0323] The most concentrated fractions were identified by UV absorbance (A280) and pooled, and for buffer exchange, 3 ml of the eluate was dialyzed overnight against 1x ligase storage buffer (10 mM Tris-HCl (pH 7.5), 50 mM KCl, 35 mM ammonium sulfate, 50% glycerol) in a 3K Slide-A-Lyzer™ dialysis cassette (Thermo Fisher). The concentration of RNA Ligase 2 in the preparation was determined by absorbance at 280 nm.

[0324] Example 4 Improved qPCR amplification activity compared to SEQ ID NO: 2 The oligonucleotides in Table 2 were used to assay RNA ligase activity, with each oligonucleotide in the 5'-3' orientation. [Table 2]

[0325] The oligonucleotides in Table 2 have the following characteristics: Sense strand fragment 1 has 2-O-methyl on nucleotide residues 1-7 and 12-14, 2'-fluoro, 5'-OH termini, and 3'-OH termini on nucleotide residues 8-11, and a phosphorothioate internucleotide linkage between nucleotide residues 1-2. Sense strand fragment 2 has 2'-O-methyl, 5'-phosphate, and 3-OH termini on nucleotide residues 1-10. Sense fragment 3 has 2'-O-methyl, 5'-phosphate, and 3'-OH termini on nucleotide residues 1-3 and 7-12, and a GalNac moiety attached to the 2' position of each of nucleotide residues 4-6, as shown below. Sense fragment 4 has 2'-O-methyl, 5'-phosphate, and 3'-OH termini on nucleotide residues 1-13 and 17-18, and a GalNac attached to the 2' position of each of nucleotide residues 14-16, as shown below. Sense strand fragment 5 has 2'-O-methyl, 5'-phosphate, and 3'-OH termini on nucleotide residues 1-4. Antisense fragment 1 has 2'-O-methyl on nucleotide residues 1-3 and 5-12, 2'-fluoro, 5'-phosphate, and 3'-OH termini on nucleotide residue 4, and phosphorothioate internucleotide linkages between nucleotide residues 10-11 and 11-12. Antisense fragment 2 has 2'-O-methyl on nucleotide residues 1, 6, 8, and 9, 2'-fluoro, 5'-monomethylphosphate, and 3'-OH termini on nucleotide residues 2-5, 7, and 10, and phosphorothioate internucleotide linkages between residues 1-2, 2-3, and 3-4, respectively. Antisense fragment 3 has 2'-O-methyl, 5'-phosphate, and 3'-OH on nucleotide residues 1-6, and phosphorothioate internucleotide linkages between nucleotide residues 4-5 and 5-6, respectively. Antisense fragment 4 has 2'-O-methyl on nucleotide residues 1-3 and 5-6, 2'-fluoro on nucleotide residue 4, a 5'-phosphate terminus, and a 3'-OH terminus. The oligonucleotide combinations used in the RNA ligase reaction are shown in Table 3. [Table 3]

[0326] The GalNac residue is attached to the 2' position of the adenine nucleotide residue as follows: [ka]

[0327] The target products of each ligation reaction in Table 3 have the following sequences: product 1-UUGCCAAGCUUGGUCAUCUAGCAGCCGAAAGGCUGC (sequence number 589) and product 2-UAGAUGACCAAGCUUGGCAAGG (sequence number 590), each product having a modification in the substrate oligonucleotide, and product 1 and product 2 hybridize to each other to form an shRNA with a GalNac ligand.

[0328] To test the activity of the samples, 0.1 g / L of protein was assayed in four reactions containing 50 mM Tris-HCl buffer, pH 7.5, 2 mM MgCl2, 1 mM DTT, 0.4 mM ATP, and an equimolar mixture of the fragments (Reaction 1: sense strands 1, 4, and 5, and antisense strands 1-2; Reaction 2: sense strands 1-3, and antisense strands 1-2; Reaction 3: sense strands 1, 4, and 5, and antisense strands 2-4; or Reaction 4: sense strands 1-3, and antisense strands 2-4). After mixing, the reactions were incubated at 37°C for 2 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 10.0 mM, and the samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0329] The % conversion for SEQ ID NO:2 was calculated as follows:

number

[0330] To test for thermal stability, each sample was incubated at 1.5 g / L in 40 mM Tris-HCl (pH 7.5), 100 mM KCl, and 0.1 mM EDTA for 1 h at room temperature and 30–80°C. After incubation, each sample was tested for activity. 0.05 g / L of protein was assayed in a reaction containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl2, 1 mM DTT, 0.4 mM ATP, and an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1–3 and antisense strands 1–2) at 0.4 mM. After mixing, the reactions were incubated at 37°C for 2 h in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 10.0 mM, and the samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0331] Using a plot of % residual activity versus incubation temperature, the temperature at which 50% of the untested sample for SEQ ID NO:2 is achieved is calculated as follows:

number

[0332] Example 5 Improved RNA ligase 2 activity compared to SEQ ID NO: 14 SEQ ID NO: 14 was selected as the parent enzyme after screening for activity and stability in all four reaction ligation scenarios. A library of modified genes was generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP as described in Example 1, and cell paste was produced as described in Example 2.

[0333] To prepare the cells for lysis, 300 μl of 0.1 mg / mL lysozyme in 50 mM Tris-HCl buffer, pH 7.5, was added to the cell paste of each sample. After thorough resuspension, the cells were incubated in a PCR thermocycler at 40°C for 1 hour, followed by another incubation at 44°C for an additional hour. The samples were then centrifuged at 4000 rpm for 10 minutes at 4°C, and the clear supernatant was used for subsequent biocatalysis.

[0334] For screening, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1-3 and antisense strands 1-2). After mixing, the reactions were incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 18.3 mM, and samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0335] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0336] Activity relative to SEQ ID NO: 14 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 14 and is shown in Table 5.1. [Table 5-1-1] [Table 5-1-2]

[0337] Example 6 Improved RNA Ligase 2 activity compared to SEQ ID NO: 42 SEQ ID NO:42 was selected as the parent enzyme after screening variants as described in Example 5 above. A library of modified genes was generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP as described in Example 1, and cell paste was produced as described in Example 2.

[0338] To prepare the cells for lysis, 300 μl of 0.1 mg / mL lysozyme in 50 mM Tris-HCl buffer, pH 7.5, was added to the cell paste of each sample. After thorough resuspension, the cells were incubated at 45°C for 1 hour in a PCR thermocycler. Subsequently, the samples were centrifuged at 4000 rpm for 10 minutes at 4°C, and the clear supernatant was used for subsequent biocatalysis.

[0339] For screening, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1-3 and antisense strands 1-2). After mixing, the reactions were incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 18.3 mM, and samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0340] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0341] Activity relative to SEQ ID NO: 42 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 42 and is shown in Table 6.1. [Table 6-1-1] [Table 6-1-2] [Table 6-1-3] [Table 6-1-4] [Table 6-1-5] [Table 6-1-6] [Table 6-1-7]

[0342] Example 7 Improved RNA ligase 2 activity compared to SEQ ID NO: 128 SEQ ID NO:42 was recloned into the pJV vector to yield SEQ ID NO:128, which was further selected as the parent enzyme. A library of modified genes was generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP as described in Example 1, and cell paste was produced as described in Example 2.

[0343] To prepare the cells for lysis, 300 μl of 0.1 mg / mL lysozyme in 50 mM Tris-HCl buffer, pH 7.5, was added to the cell paste of each sample. After thorough resuspension, the cells were incubated at 45°C for 1 hour in a PCR thermocycler. Subsequently, the samples were centrifuged at 4000 rpm for 10 minutes at 4°C, and the clear supernatant was used for subsequent biocatalysis.

[0344] For screening, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1-3 and antisense strands 1-2). After mixing, the reactions were incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 18.3 mM, and samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0345] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0346] Activity relative to SEQ ID NO: 128 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 128 and is shown in Table 7.1. [Table 7-1-1] [Table 7-1-2] [Table 7-1-3] [Table 7-1-4]

[0347] Example 8 Improved RNA ligase 2 activity compared to SEQ ID NO: 128 SEQ ID NO: 128 was selected as the parent enzyme after screening variants as described in Example 5 above. The top variants were selected from Example 7 above and retested under different conditions. A library of modified genes was generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP as described in Example 1, and cell paste was produced as described in Example 2.

[0348] To prepare the cells for lysis, 300 μl of 0.1 mg / mL lysozyme in 50 mM Tris-HCl buffer, pH 7.5, was added to the cell paste of each sample. After thorough resuspension, the cells were incubated at 45°C for 1 hour in a PCR thermocycler. Subsequently, the samples were centrifuged at 4000 rpm for 10 minutes at 4°C, and the clear supernatant was used for subsequent biocatalysis.

[0349] For screening, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 1 (sense strands 1, 4, and 5, and antisense strands 1-2). After mixing, the reactions were incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 18.3 mM, and samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0350] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0351] Activity relative to SEQ ID NO: 128 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 128 and is shown in Table 8.1. [Table 8-1-1] [Table 8-1-2] [Table 8-1-3] [Table 8-1-4]

[0352] Example 9 Improved RNA ligase 2 activity compared to SEQ ID NO: 128 The same samples from Example 8 were tested again, this time using reaction 3. To screen, 20% (v / v) of the lysate was transferred to a reaction containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 3 (sense strands 1, 4, and 5, and antisense strands 2-4). The reactions were mixed and then incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 18.3 mM, and the samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0353] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0354] Activity relative to SEQ ID NO: 128 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 128 and is shown in Table 9.1. [Table 9-1-1] [Table 9-1-2] [Table 9-1-3]

[0355] Example 10 Improved RNA Ligase 2 activity compared to SEQ ID NO: 204 SEQ ID NO:204 was selected as the parent enzyme after screening variants as described in Examples 7-9 above. A library of modified genes was generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP as described in Example 1, and cell paste was produced as described in Example 2.

[0356] To prepare the cells for lysis, 300 μl of 0.1 mg / mL lysozyme in 50 mM Tris-HCl buffer, pH 7.5, was added to the cell paste of each sample. After thorough resuspension, the cells were incubated at 48°C for 1 hour in a PCR thermocycler. The samples were then centrifuged at 4000 rpm for 10 minutes at 4°C, and the clear supernatant was used for subsequent biocatalysis.

[0357] For screening, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1-3 and antisense strands 1-2). After mixing, the reactions were incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 20.0 mM, and samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0358] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0359] Activity relative to SEQ ID NO: 204 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 204 and is shown in Table 10.1. [Table 10-1-1] [Table 10-1-2] [Table 10-1-3] [Table 10-1-4] [Table 10-1-5] [Table 10-1-6]

[0360] Example 11 Improved RNA Ligase 2 activity compared to SEQ ID NO: 204 SEQ ID NO: 204 was selected as the parent enzyme after screening variants as described above in Examples 7-9. The top variants were selected from Example 10 above and retested under different conditions.

[0361] A library of modified genes was generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTPs as described in Example 1, and cell paste was produced as described in Example 2.

[0362] To prepare the cells for lysis, 300 μl of 0.1 mg / mL lysozyme in 50 mM Tris-HCl buffer, pH 7.5, was added to the cell paste of each sample. After thorough resuspension, the cells were incubated at 48°C for 1 hour in a PCR thermocycler. The samples were then centrifuged at 4000 rpm for 10 minutes at 4°C, and the clear supernatant was used for subsequent biocatalysis.

[0363] For screening, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 3 mM of an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1-3 and antisense strands 1-2). After mixing, the reactions were incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 20 mM, and samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0364] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0365] Activity relative to SEQ ID NO: 204 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 204 and is shown in Table 11.1. [Table 11-1-1] [Table 11-1-2] [Table 11-1-3] [Table 11-1-4] [Table 11-1-5]

[0366] Example 12 Improved RNA Ligase 2 activity compared to SEQ ID NO: 204 The same samples from Example 11 were tested again, this time with a higher fragment load. To screen, 20% (v / v) of the lysate was transferred to reactions containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl, 1 mM DTT, 0.4 mM ATP, and 0.4 mM of an equimolar mixture of the compromising fragments from reaction 2 (sense strands 1-3 and antisense strands 1-2). The reactions were mixed and then incubated at 37°C for 16 hours in a PCR thermocycler. EDTA was then added to each reaction to a final concentration of 20 mM, and the samples were analyzed by UPLC to quantify the concentration of the remaining fragments and the product yield.

[0367] Using peak areas from Analytical Method 13.1, the % conversion for each sample was calculated as indicated herein.

number

[0368] Activity relative to SEQ ID NO: 204 (FIOP) was calculated as the % conversion formed by the variant relative to the % conversion of SEQ ID NO: 204 and is shown in Table 12.1. [Table 12-1-1] [Table 12-1-2] [Table 12-1-3] [Table 12-1-4] [Table 12-1-5]

[0369] Example 13 Analytical detection of RNA oligomers by UPLC-UV The data described in Examples 4-12 were collected using the analytical methods shown in Table 13.1. The methods provided herein are useful for analyzing variants produced using the present disclosure. [Table 13-1]

[0370] Although the invention has been described with reference to specific embodiments, various modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, and equivalents may be substituted, thereby achieving the advantages of the invention without departing from the scope of the appended claims.

[0371] For all purposes, all publications and patent documents cited in this disclosure are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not an indication that any such document is pertinent prior art or an admission as to the contents or date thereof.

Claims

1. 1. A modified RNA ligase, or functional fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12-346 of SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, or to a reference sequence corresponding to the even-numbered SEQ ID NOs: 14 and the even-numbered SEQ ID NOs: 24-582, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or relative to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

2. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, 42, or 204, or to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204, wherein the amino acid sequence comprises one or more substitutions.

3. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO:

14.

4. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to the reference sequence corresponding to SEQ ID NO: 42 or 204, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO:

14.

5. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO:24-582, or to the reference sequence corresponding to an even-numbered SEQ ID NO:24-582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

6. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO:24-128, or to the reference sequence corresponding to an even-numbered SEQ ID NO:24-128, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

7. 10. The amino acid sequence of the modified RNA ligase, wherein the amino acid sequence at amino acid positions 2, 14, 15, 18, 33, 39, 63, 69, 81, 83, 85, 86, 89, 95, 116, 117, 119, 138, 142, 144, 149, 154, 156, 159, 171, 175, 177, 181, 185, 186, 195, 202, 212, 214, 224, 226, 230, 247, 256, 257, 260, 266, 275, 280, 283, 285, 288, 291, 292, 293, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 7. The modified RNA ligase of any one of claims 1 to 6, comprising at least one substitution at amino acid positions 12-346 of SEQ ID NO: 14 or at amino acid positions 6, 303, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

8. 10. The amino acid sequence of the modified RNA ligase may have at least one substitution or amino acid residues 2G / V / E, 14K, 15L / E / M / P / Y, 18I, 33L, 39Q / S, 63V, 69D, 81Y, 85L, 86G, 89H / I / M / W, 95V, 116N, 117D, 119T, 138R, 142R, 144W, 149R / T, 154C, 156A / L / N / Q / T, 159F, 171E, 175K, 177P, 181D, 183V, 185G / K, 186D / R, 195G, 202Y, 212I, 214I, 224Y, 226I, 230D, 247K, 256A, 257L, 26 8. The modified RNA ligase of any one of claims 1 to 7, wherein the modified RNA ligase comprises the amino acid sequences 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,

9. 8. The modified RNA ligase of any one of claims 1 to 7, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 95 or 177, or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

10. 10. The modified RNA ligase of any one of claims 1 to 7 and 9, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 95V or 177P, or a combination thereof, and the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

11. 8. The modified RNA ligase of any one of claims 1 to 7, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

12. 12. The modified RNA ligase of any one of claims 1 to 7 and 11, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 15L / E / M / P / Y, 95V, 149R / T, 154C, 156A / L / N / Q / T, 177P, 186D / R, 195G, 224Y, or 226I, or a combination thereof, and the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

13. 12. The modified RNA ligase of any one of claims 1 to 7 and 11, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 15Y, 95V, 149R, 154C, 156T, 177P, 186R, 195G, 224Y, or 226I, or a combination thereof, and the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

14. 177 / 260 / 307 / 345, 171 / 175 / 183 / 280 / 283 / 303, 95 / 159 / 177 / 260 / 288, 175 / 183 / 247 / 280 / 283 / 296, 177 / 307, 81 / 171 / 183 / 335, 175 / 183, 81 / 175 / 183 / 280 / 296 / 335 / 339, 81 / 183 / 247 / 266, 95 / 177, 171 / 280 / 283 / 296 / 303, 85 / 177 / 260 / 288 / 333 / 345, 177 / 291 / 307 / 333, 81 / 171 / 175 / 183 / 316 / 337, 81 / 171 / 266 / 280 / 283 / 335 / 339, 177, 95 / 159 / 177 / 260 / 280 / 288 / 306 / 345, 171 / 175 / 335, 177 / 260 / 30 / 333, 14 / 95 / 177 / 288 / 307, 95 / 177 / 345, 95 / 159 / 177 / 260 / 285 / 288, 266 / 296, 81 / 283 / 335 / 337, 95 / 159 / 177 / 260 / 342 / 345, 63 / 171 / 175 / 183 / 266 / 280 / 296 / 31 6, 280 / 335 / 337, 81 / 171 / 280, 81 / 171 / 175 / 296 / 316, 171 / 175 / 316 / 335, 171 / 280 / 283 / 296 / 316, 171 / 283 / 335, 63 / 171 / 175 / 183 / 266 / 283 / 303 / 316, 171 / 247 / 266 / 283 / 296 / 303 / 316 / 335, 63 / 81 / 171 / 175 / 183 / 266 / 280 / 283 / 337, 81 / 171 / 303 / 316 / 335, 288 / 307, 14 / 95 / 159 / 177 / 345, 81 / 171 / 175 / 18 3 / 247 / 266 / 280 / 283, 81 / 247 / 266, 280, 85 / 177 / 260, 63 / 183, 175 / 183 / 247 / 280 / 283 / 316, 291, 247, 171 / 280 / 283, 81 / 171 / 175 / 280 / 316, 333, 171 / 296 / 316, 81 / 171, or 280 / 316 / 335, wherein said amino acid positions are relative to said reference sequence corresponding to residues 12-346 of SEQ ID NO: 14 or relative to said reference sequence corresponding to SEQ ID NO: 14.The modified RNA ligase according to any one of claims 1 to 7.

15. 17. The amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions: 177P / 260S / 307E / 345V, 171E / 175K / 183V / 280N / 283I / 303Q, 95V / 159F / 177P / 260S / 288E, 175K / 183V / 247K / 280N / 283I / 296K, 177P / 307E, 81Y / 171E / 183V / 335D, 175K / 183V, 81Y / 175K / 183V / 280N / 296K / 335D / 339P, 81Y / 183V / 247K / 266L, 95V / 177P, 171E / 175K / 183V / 280N / 296K / 335D / 339P, 171E / 175K / 183V / 247K / 266L, 95V / 159F ... E / 280N / 283I / 296K / 303Q, 85L / 177P / 260S / 288E / 333V / 345V, 177P / 291P / 3 07E / 333V, 81Y / 171E / 175K / 183V / 316L / 337L, 81Y / 171E / 266L / 280N / 283I / 3 35D / 339P, 177P, 95V / 159F / 177P / 260S / 280N / 288E / 306L / 345V, 171E / 175K / 335D, 177P / 260S / 306L / 333V, 14K / 95V / 177P / 288E / 307E, 95V / 177P / 345V, 95V / 159F / 177P / 260S / 285K / 288E, 266L / 296K, 81Y / 283I / 335D / 337L, 95V / 159F / 177P / 260S / 342I / 345V, 63V / 171E / 175K / 183V / 266L / 280N / 296K / 316 L, 280N / 335D / 337L, 81Y / 171E / 280N, 81Y / 171E / 175K / 296K / 316L, 171E / 17 5K / 316L / 335D, 171E / 280N / 283I / 296K / 316L, 171E / 283I / 335D, 63V / 171E / 1 75K / 183V / 266L / 283I / 303Q / 316L, 171E / 247K / 266L / 283I / 296K / 303Q / 316 L / 335D, 63V / 81Y / 171E / 175K / 183V / 266L / 280N / 283I / 337L, 81Y / 171E / 303Q / 316L / 335D, 288E / 307E, 14K / 95V / 159F / 177P / 345V, 81Y / 171E / 175K / 183V / 247K / 266L / 280N / 283I, 81Y / 247K / 266L, 280N, 85L / 177P / 260S, 63V / 183V,15. The modified RNA ligase of any one of claims 1 to 7 and 14, comprising 175K / 183V / 247K / 280N / 283I / 316L, 291P, 247K, 171E / 280N / 283I, 81Y / 171E / 175K / 280N / 316L, 333V, 171E / 296K / 316L, 81Y / 171E, or 280N / 316L / 335D, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

16. 2. The modified RNA ligase of claim 1, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution as provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, and the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

17. 2. The modified RNA ligase of claim 1, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions of an RNA ligase variant provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, and the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

18. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or to the reference sequence corresponding to SEQ ID NO:42 or 204.

19. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:24-582, or to the reference sequence corresponding to an even-numbered SEQ ID NO:24-582.

20. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or to the reference sequence corresponding to SEQ ID NO: 42 or 204, wherein the amino acid sequence comprises one or more substitutions.

21. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NOs:24-582, or to the reference sequence corresponding to an even-numbered SEQ ID NO:24-582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or relative to the reference sequence corresponding to SEQ ID NO:42 or 204.

22. 10. The amino acid sequence of the modified RNA ligase, wherein the amino acid sequence at amino acid positions 2, 14, 15, 18, 33, 39, 63, 69, 81, 83, 85, 86, 89, 95, 116, 117, 119, 138, 142, 144, 149, 154, 156, 159, 171, 175, 177, 181, 185, 186, 195, 202, 212, 214, 224, 226, 230, 247, 256, 257, 260, 266, 275, 280, 283, 285, 288, 291, 296, 30 22. The modified RNA ligase of claim 20 or 21, comprising at least one substitution at amino acid positions 3, 306, 307, 310, 314, 315, 316, 317, 326, 330, 331, 333, 334, 335, 337, 339, 342, or 345, or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or relative to the reference sequence corresponding to SEQ ID NO:42 or 204.

23. 10. The amino acid sequence of the modified RNA ligase has at least one substitution or amino acid residues 2G / V / E, 14K, 15E / L / M / P / Y, 18I, 33L, 39Q / S, 63V, 69D, 81Y, 85L, 86G, 89H / I / M / W, 95V, 116N, 117D, 119T, 138R, 142 R, 144W, 149R / T, 154C, 156A / L / N / Q / T, 159F, 171E, 175K, 177P, 181D, 183V, 185G / K , 186D / R, 195G, 202Y, 212I, 214I, 224Y, 226I, 230D, 247K, 256A, 257L, 260S, 266L, 23. The modified RNA ligase of any one of claims 20-22, comprising 275N, 280G / N, 283I, 285K, 288E, 291P, 296K, 303Q, 306L, 307E / Q, 310K, 314W, 315S / T, 316L, 317A, 326R, 330M / R, 331R / W, 333V, 334R, 335D / H, 337L, 339P, 342I, or 345V, or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or relative to the reference sequence corresponding to SEQ ID NO:42 or 204.

24. 23. The modified RNA ligase of any one of claims 20-22, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 95 or 177, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to the reference sequence corresponding to SEQ ID NO: 42 or 204.

25. 25. The modified RNA ligase of any one of claims 20 to 22 and 24, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution or amino acid residue 95V or 177P, or a combination thereof, and the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42 or 204, or relative to the reference sequence corresponding to SEQ ID NO: 42 or 204.

26. 23. The modified RNA ligase of any one of claims 20-22, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution at amino acid position 15, 95, 149, 154, 156, 177, 186, 195, 224, or 226, or a combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO: 42 or 204, or relative to the reference sequence corresponding to SEQ ID NO: 42 or 204.

27. 27. The modified RNA ligase of any one of claims 20 to 22 and 26, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution 15Y, 95V, 149R, 154C, 156T, 177P, 186R, 195G, 224Y, or 226I, or a combination thereof, and the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42 or 204, or relative to the reference sequence corresponding to SEQ ID NO: 42 or 204.

28. 21. The modified RNA ligase of claim 20, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to the reference sequence corresponding to SEQ ID NO:42, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to the reference sequence corresponding to SEQ ID NO:

42.

29. 22. The modified RNA ligase of claim 21, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO:130-478, or to the reference sequence corresponding to an even-numbered SEQ ID NO:130-478, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or to the reference sequence corresponding to SEQ ID NO:

42.

30. The amino acid sequence of the modified RNA ligase may comprise at amino acid position(s) 149 / 156 / 195, 15 / 149 / 186 / 224 / 317, 15 / 156 / 195 / 224 / 226 / 317 / 331, 15 / 195 / 317 / 331, 149 / 224 / 331, 15 / 195 / 224 / 226 / 317 / 331, 15 / 33 / 86 / 149 / 154 / 195 / 317, 15 / 86 / 154 / 156 / 186 / 195 / 224 / 226 / 331, 15 / 149 / 224 / 317 / 331, 15 / 154 / 156 / 186 / 317 / 331, 15 / 3 3 / 149 / 224 / 226 / 331, 15 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 186 / 224 / 331, 195 / 224 / 331, 15 / 86 / 156 / 186 / 195 / 226 / 317 / 331, 15 / 195 / 22 / 331, 156 / 186 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 224 / 226, 33 / 156 / 186 / 195 / 331, 15 / 86 / 149 / 154 / 156 / 317 / 331, 15 / 186 / 317 / 331, 15 / 86 / 149 / 154 / 195 / 331, 15 / 33 / 149 / 154 / 156 / 226, 15 / 149 / 154 / 186 / 317 / 331, 195 / 331, 15 / 86 / 156 / 186 / 195 / 331, 149 / 156 / 224 / 226 / 331, 15 / 149 / 154 / 156 / 186 / 317 / 331, 15 / 154 / 186 / 195, 15 / 86 / 195 / 224 / 226, 230, 15 / 33 / 156 / 331, 15 / 33 / 156 / 195 / 224 / 226, 33 / 149 / 156 / 317, 15 / 33 / 186 / 195, 334, 15 / 154 / 156 / 224 / 317, 1 5 / 149 / 186 / 331, 15 / 86 / 149 / 186 / 195 / 317, 15 / 33 / 154 / 195, 15 / 156 / 186 / 331, 15 / 149 / 186 / 317, 15 / 33 / 156 / 186 / 214 / 224 / 331, 15 / 86 / 186 / 224 / 226 / 317, 15 / 154 / 331, 15 / 149 / 154 / 226, 15 / 33 / 195 / 226, 15 / 224 / 317 / 331, 15 / 86 / 186 / 195 / 317, 226 / 317 / 331, 330, 89, 15 / 149 / 186, 156 / 195 / 331, 156,15 / 86 / 156 / 186 / 331, 275, 15 / 33 / 186 / 224, 15 / 186 / 317, 15 / 33 / 186 / 226 / 317, 15 / 149 / 154 / 156 / 331, 15 / 154 / 226 / 317, 15 / 86 / 156 / 186 / 195 / 317, 257, 15 / 154 / 156 / 186, 33 / 186 / 224, 335, 1 5 / 86 / 156 / 186 / 195 / 224 / 331, 154 / 156 / 317, 39, 2, 15 / 224 / 226 / 331, 15 / 33 / 149 / 224, 89 / 307, 15 / 154 / 156 / 317, 15 / 186 / 331, 15 / 33 / 154 / 317, 15 / 33 / 195 / 224 / 317, 15 / 156 / 224 / 226, 154 / 1 86 / 331, 185, 15 / 18 / 149 / 186 / 195, 86 / 149 / 154 / 156, 15 / 33 / 149 / 331, 326, 202, 119, 212, 15 / 33 / 156 / 317, 280, 142, 15 / 149, 15 / 33 / 86 / 156 / 195, 315, 256, 15 / 33 / 156 / 186, 138, 117, 116, 31 30. The modified RNA ligase of claim 28 or 29, comprising a substitution or set of substitutions at positions 6, 69, 310, 317, 15 / 195 / 331, 15 / 33 / 331, 314, or 144, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO:42, or relative to the reference sequence corresponding to SEQ ID NO:

42.

31. The amino acid sequence of the modified RNA ligase may comprise a substitution or set of substitutions 149R / 156T / 195G, 15M / 149T / 186R / 224Y / 317A, 15P / 156T / 195G / 224Y / 226I / 317A / 331L, 15M / 195G / 317A / 331R, 149R / 224Y / 331R, 15E / 195G / 224Y / 226I / 317A / 331W, 15P / 33L / 86G / 149R / 154C / 195G / 317A, 15M / 86G / 154C / 156T / 186R / 195G / 224Y / 226I / 331L, 15P / 149R / 224Y / 317A / 331R, 15M / 154C / 156T / 186R / 317A / 331R, 15M / 33L / 149 R / 224Y / 226I / 331W, 15E / 186R / 224Y / 226I / 317A / 331R, 15Y / 33L / 149R / 186 R / 224Y / 331R, 195G / 224Y / 331R, 15P / 86G / 156T / 186R / 195G / 226I / 317A / 33 1L, 15M / 195G / 226 / 331W, 156T / 186R / 224Y / 226I / 331R, 15Y / 149R / 154C / 15 6T / 224Y / 226I, 33L / 156T / 186R / 195G / 331L, 15P / 86G / 149R / 154C / 156T / 3 17A / 331R, 15M / 186D / 317A / 331R, 15P / 86G / 149R / 154C / 195G / 331L, 15Y / 33 L / 149T / 154C / 156T / 226I, 15P / 149R / 154C / 186R / 317A / 331L, 195G / 331R, 1 5M / 86G / 156T / 186R / 195G / 331W, 149R / 156T / 224Y / 226I / 331W, 15P / 149R / 1 54C / 156T / 186R / 317A / 331L, 15Y / 154C / 186D / 195G, 15E / 86G / 195G / 224Y / 2 26I, 230D, 15P / 33L / 156T / 331R, 15L / 33L / 156T / 195G / 224Y / 226I, 33L / 149 R / 156T / 317A, 15E / 33L / 186R / 195G, 334R, 15E / 154C / 156T / 224Y / 317A, 15Y / 149R / 186R / 331W, 15Y / 86G / 149T / 186R / 195G / 317A, 15P / 33L / 154C / 195G,15P / 154C / 156T / 186R / 317A / 331R、15 M / 156T / 186R / 331R、15Y / 149T / 186D / 317A、15E / 33L / 156T / 186R / 214I / 224 Y / 331W、15E / 86G / 186R / 224Y / 226I / 3 17A、15P / 154C / 331R、15Y / 149T / 154C / 226I、15Y / 33L / 195G / 226I、15M / 224 Y / 317A / 331W、15E / 86G / 186R / 195G / 317A、226I / 317A / 331L、330R、89W、15P / 149R / 186D / 331L、15P / 149R / 186R、89 M、156T / 195G / 331L、156A、15E / 86G / 1 56T / 186R / 331R、195G / 331W、275N、15 P / 33L / 186R / 224Y、15E / 186R / 317A、1 5M / 33L / 186R / 226I / 317A、89G、15Y / 1 49R / 154C / 156T / 331W、15Y / 154C / 226 I / 317A、15Y / 86G / 156T / 186R / 195G / 3 17A、257L、15Y / 154C / 156T / 186D、33L / 186R / 224Y、335H、15Y / 86G / 156T / 186 R / 195G / 224Y / 331W、154C / 156T / 317A 、39Q、2G、15M / 224Y / 226I / 331R、15M / 33L / 149R / 224Y、89I / 307Q、15M / 154C / 156T / 317A、15E / 186R / 331W、15P / 33 L / 154C / 317A、89H、15Y / 33L / 195G / 22 4Y / 317A、15E / 156T / 224Y / 226I、154C / 186R / 331L、185G、15M / 18I / 149R / 186 R / 195G、86G / 149R / 154C / 156T、15P / 3 3L / 149R / 331L、326R、202Y、119T、212 I、2V、15E / 149R / 154C / 156T / 331L、15 P / 33L / 156T / 317A、15Y / 154C / 156T / 3 17A, 156L, 335D, 156T, 2E, 280G, 142R, 15E / 149R, 156Q, 15E / 33L / 86G / 156T / 195G、315T、185K、39S、256A、15Y / 33 L / 156T / 186D、138R、117D、116N、316L、31. The modified RNA ligase of any one of claims 28 to 30, comprising 15M / 149R, 69D, 156N, 310K, 317A, 15Y / 195G / 331L, 15M / 33L / 331L, 330M, 314W, 144W, 15Y / 149R, or 315S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO:42, or relative to the reference sequence corresponding to SEQ ID NO:

42.

32. The amino acid sequence of the modified RNA ligase may be selected from the group consisting of amino acid position(s) 15 / 149 / 154 / 156 / 186 / 195 / 331, 149 / 154 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 154 / 186 / 195, 149 / 156 / 186 / 195 / 224 / 226 / 317, 15 / 149 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 226 / 331, 15 / 149 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 33 / 149 / 154 / 195 / 317, 15 / 149 / 154 / 195 / 224 / 317, 15 / 149 / 156 / 186 / 224 / 226 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 195 / 224 / 226 / 317 / 331, 149 / 195 / 224, 86 / 149 / 154 / 1 56 / 186 / 195 / 331, 15 / 33 / 149 / 156 / 195 / 224 / 317 / 331, 15 / 149 / 154 / 156 / 186 / 195 / 317 / 331, 15 / 33 / 149 / 154 / 156 / 186 / 224 / 331, 15 / 149 / 154 / 156 / 195 / 224, 15 / 149 / 154 / 156 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 186 / 195 / 224 / 317, 33 / 149 / 156 / 186 / 224 / 331, 15 / 33 / 86 / 149 / 195 / 224 / 331, 15 / 86 / 149 / 154 / 156 / 186 / 195 / 331, 149 / 186 / 195 / 331, 15 / 154 / 156 / 186 / 226 / 331, 15 / 149 / 154 / 186 / 195 / 224 / 226, 15 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 317 / 331, 15 / 33 / 154 / 156 / 186 / 195 / 224 / 317 / 331, 15 / 149 / 186 / 195 / 224 / 331, 15 / 33 / 149 / 154 / 156 / 195 / 224 / 331, 15 / 149 / 154 / 156 / 224 / 22 6 / 317 / 331, 15 / 33 / 149 / 156 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 224 / 226 / 331, 154 / 156 / 195 / 224 / 226, 15 / 33 / 86 / 149 / 154 / 156 / 186 / 195 / 224 / 317 / 331,15 / 149 / 154 / 156 / 186 / 195 / 224 / 226, 15 / 33 / 149 / 195 / 317 / 331, 15 / 149 / 154 / 224 / 331, 149 / 154 / 156 / 195 / 224 / 226 / 331, 15 / 149 / 154 / 195 / 331, 15 / 149 / 154 / 156 / 186 / 195, 15 / 33 / 149 / 154 / 156 / 186 / 195, 15 / 30. The modified RNA ligase of claim 28 or 29, comprising at least one set of substitutions at 33 / 86 / 149 / 154 / 195 / 224 / 317 / 331, or 149 / 186 / 195 / 224 / 226 / 331, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO: 42, or relative to the reference sequence corresponding to SEQ ID NO:

42.

33. 15Y / 149R / 156T / 186R / 195G / 224Y / 317A / 331R, 149R / 154C / 186D / 195G / 224Y / 317A / 331R, 15M / 33L / 149T / 154C / 186R / 195G, 149R / 156T / 186R / 195G / 224Y / 226I / 317A, 15Y / 149R / 156T / 195G / 224Y / 226I, 15Y / 33L / 86G / 149T / 154C / 156T / 186R / 195G / 226I / 331W, 15Y / 33L / 86G / 149T / 154C / 156T / 186R / 195G / 226I / 331W, 15Y / 33L / 86G / 149T / 154C / 156T / 186R / 195G / 226I / 331W, 15Y / 33L / 86G / 149T / 154C / 156T / 186R / 195G / 224Y / 317A / 331R ... Y / 149R / 156T / 186R / 195G / 224Y / 317A / 331W, 15M / 33L / 149T / 154C / 195G / 3 17A, 15M / 149R / 154C / 195G / 224Y / 317A, 15Y / 149R / 156T / 186D / 224Y / 226I / 317A / 331R, 15M / 149T / 154C / 156T / 186D / 195G / 331L, 15Y / 33L / 149R / 154C / 156T / 186R / 195G / 224Y / 226I / 317A / 331R, 149R / 195G / 224Y, 86G / 149T / 154 C / 156T / 186R / 195G / 331L, 15P / 33L / 149R / 156T / 195G / 224Y / 317A / 331L, 1 5P / 149T / 154C / 156T / 186D / 195G / 317A / 331L, 15Y / 33L / 149T / 154C / 156T / 1 86D / 224Y / 331L, 15P / 149R / 156T / 186R / 195G / 224Y / 317A / 331W, 15Y / 149R / 154C / 156T / 195G / 224Y, 15M / 149T / 154C / 156T / 195G / 224Y / 226I, 15E / 149R / 154C / 156T / 186R / 195G / 224Y / 317A, 33L / 149T / 156T / 186R / 224Y / 331L, 1 5Y / 33L / 86G / 149R / 195G / 224Y / 331W, 15E / 86G / 149R / 154C / 156T / 186R / 195 G / 331W, 149R / 186R / 195G / 331W, 15E / 154C / 156T / 186D / 226I / 331R, 15Y / 14 9T / 154C / 186R / 195G / 224Y / 226I, 15P / 149R / 154C / 156T / 195G / 224Y / 331W,15E / 33L / 149R / 154C / 156T / 195G / 317A / 331L, 15E / 33L / 154C / 156T / 186R / 195G / 224Y / 317A / 331L, 15P / 149R / 186D / 195G / 224Y / 331L, 15P / 33L / 149R / 154C / 156T / 195G / 224Y / 331W, 15P / 149T / 154C / 156T / 224Y / 226I / 317A / 331W, 15P / 33L / 149T / 156T / 195G / 331W, 15P / 149T / 154C / 156T / 186D / 224Y / 226I / 331L, 154C / 156T / 195G / 224Y / 226I, 15Y / 33 L / 86G / 149R / 154C / 156T / 186D / 195G / 224Y / 317A / 331W, 15Y / 149R / 154C / 156T / 186R / 195G / 224Y / 226I, 15P / 33L / 14 9R / 195G / 317A / 331L, 15E / 149T / 154C / 224Y / 331R, 149T / 154C / 156T / 195G / 224Y / 226I / 331R, 15M / 149R / 154C / 195G / 331L, 15Y / 149R / 154C / 156T / 186R / 195G, 15E / 149T / 154C / 156T / 186R / 195G / 331R, 15M / 33L / 149T / 154C / 156T / 186 33. The modified RNA ligase of any one of claims 28, 29, and 32, comprising the amino acid positions 149R / 195G, 15P / 33L / 86G / 149R / 154C / 195G / 224Y / 317A / 331R, or 149R / 186D / 195G / 224Y / 226I / 331R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42, or relative to the reference sequence corresponding to SEQ ID NO:

42.

34. 20. The modified RNA ligase of claim 19, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of SEQ ID NO:204, or to the reference sequence corresponding to SEQ ID NO:204, wherein the amino acid sequence comprises one or more substitutions to the reference sequence corresponding to residues 12-346 of SEQ ID NO:204, or to the reference sequence corresponding to SEQ ID NO:

204.

35. 21. The modified RNA ligase of claim 20, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12-346 of an even-numbered SEQ ID NO:480-582, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:204, or relative to the reference sequence corresponding to SEQ ID NO:

204.

36. The amino acid sequence of the modified RNA ligase may comprise at least one substitution or set of substitutions at amino acid position(s): 2 / 39 / 69 / 144 / 156 / 316 / 330, 2 / 39 / 69 / 119 / 185 / 316, 69 / 212 / 256 / 330, 39 / 119 / 256 / 316 / 326 / 330, 69 / 116 / 119 / 185 / 330, 2 / 39 / 119 / 144 / 156 / 159 / 202 / 256 / 316 / 326 / 330, 81 / 183 / 185 / 230, 2 / 39 / 69 / 116 / 144 / 156 / 185, 2 / 14 / 39 / 69 / 116 / 1 19 / 159 / 185 / 256 / 326, 2 / 116 / 119 / 316, 116 / 119 / 144 / 185 / 314 / 316, 39 / 69 / 116 / 144 / 181 / 185 / 256 / 330, 2 / 39 / 69 / 119 / 144 / 156 / 185 / 314, 14 / 39 / 69 / 1 44 / 156 / 212 / 256 / 314 / 316 / 326 / 330, 39 / 69 / 144 / 156 / 185 / 316 / 330, 156 / 185 / 334 / 335, 39 / 63 / 156 / 230 / 257 / 275 / 330 / 335, 156 / 266 / 316 / 330 / 334 / 33 5, 39 / 63 / 156 / 183 / 185 / 230 / 330 / 334, 69 / 144 / 316, 116 / 119 / 156 / 202 / 212 / 326 / 330, 39 / 69 / 119 / 138 / 181 / 185 / 316 / 326, 39 / 81 / 156 / 230, 2 / 14 / 69 / 11 6 / 119 / 144 / 159 / 326 / 330, 39 / 156 / 334 / 345, 230 / 266 / 334, 2 / 116 / 144 / 156 / 159, 156 / 183 / 185 / 230, 2 / 14 / 116 / 316 / 326 / 330, 39 / 63 / 156 / 230 / 316 / 345 , 69 / 138 / 144 / 159 / 185 / 202 / 316, 39 / 156 / 257 / 266 / 316 / 334, 156 / 257 / 275 / 334, 183, 89 / 156 / 185 / 230 / 316 / 345, 2 / 14 / 119, 144 / 156 / 316 / 330, 39 / 81 / 185 / 316 / 334, 89 / 230 / 266 / 345, 39 / 156 / 230 / 275 / 316, 39 / 156 / 230 / 266 / 334 / 335, 156 / 185 / 275 / 316 / 330, 119 / 156 / 202, 230 / 257 / 275 / 316 / 330 / 345,230, 81 / 156 / 330 / 335 / 345, 156 / 183 / 230 / 266, 39 / 185, 39 / 81 / 183 / 275 / 316 / 334 / 345, 156 / 230 / 257 / 316, 39 / 116 / 156, or 183 / 185 / 257 / 275 / 316 / 330 / 334, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO:204 or relative to the reference sequence corresponding to SEQ ID NO:

204.

37. The amino acid sequence of the modified RNA ligase may contain at least one substitution or set of substitutions: 2E / 39S / 69D / 144W / 156L / 316L / 330M, 2V / 39S / 69D / 119T / 185K / 316L, 69D / 212I / 256A / 330M, 39S / 119T / 256A / 316L / 326R / 330M, 69D / 116N / 119T / 185K / 330M, 2E / 39S / 119T / 144W / 156N / 159F / 202Y / 256A / 316L / 326R / 330M, 81Y / 183V / 185G / 230D ...30M, 2V / 39S / 69D / 119T / 185K / 330M, 2V / 39S / 69D / 119T / 185K / 330M, 2V / 39S / 69D / 119T / 185K / 330 / 69D / 116N / 144W / 156L / 185K, 2E / 14K / 39S / 69D / 116N / 119T / 159F / 185K / 25 6A / 326R, 2E / 116N / 119T / 316L, 116N / 119T / 144W / 185K / 314W / 316L, 39S / 69 D / 116N / 144W / 181D / 185K / 256A / 330M, 2E / 39S / 69D / 119T / 144W / 156L / 185K / 314W, 14K / 39S / 69D / 144W / 156N / 212I / 256A / 314W / 316L / 326R / 330M, 39S / 69D / 144W / 156L / 185K / 316L / 330M, 156Q / 185G / 334R / 335H, 39Q / 63V / 156Q / 230D / 257L / 275N / 330R / 335H, 156Q / 266L / 316L / 330R / 334R / 335H, 39Q / 63V / 156Q / 183V / 185G / 230D / 330R / 334R, 69D / 144W / 316L, 116N / 119T / 156L / 20 2Y / 212I / 326R / 330M, 39S / 69D / 119T / 138R / 181D / 185K / 316L / 326R, 39Q / 81 Y / 156Q / 230D, 2V / 14K / 69D / 116N / 119T / 144W / 159F / 326R / 330M, 39Q / 156A / 334R / 345V, 230D / 266L / 334R, 2V / 116N / 144W / 156L / 159F, 156A / 183V / 185G / 230D, 2E / 14K / 116N / 316L / 326R / 330M, 39Q / 63V / 156A / 230D / 316L / 345V, 6 9D / 138R / 144W / 159F / 185K / 202Y / 316L, 39Q / 156Q / 257L / 266L / 316L / 334R,156Q / 257L / 275N / 334R, 183V, 89W / 156A / 185G / 230D / 316L / 345V, 2E / 14K / 119T , 144W / 156L / 316L / 330M, 39Q / 81Y / 185G / 316L / 334R, 89H / 230D / 266L / 345V, 39 Q / 156Q / 230D / 275N / 316L, 39Q / 156Q / 230D / 266L / 334R / 335H, 156Q / 185G / 275N / 316L / 330R, 119T / 156N / 202Y, 230D / 257L / 275N / 316L / 330R / 345V, 230D, 81Y / 37. The modified RNA ligase of any one of claims 34 to 36, comprising 156Q / 330R / 335H / 345V, 156A / 183V / 230D / 266L, 39S / 185K, 39Q / 81Y / 183V / 275N / 316L / 334R / 345V, 156A / 230D / 257L / 316L, 39S / 116N / 156N, or 183V / 185G / 257L / 275N / 316L / 330R / 334R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 346 of SEQ ID NO:204, or relative to the reference sequence corresponding to SEQ ID NO:

204.

38. 2. The modified RNA ligase of claim 1, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution as provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, and the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or relative to the reference sequence corresponding to SEQ ID NO:42 or 204.

39. 2. The modified RNA ligase of claim 1, wherein the amino acid sequence of the modified RNA ligase comprises at least one substitution or set of substitutions of an RNA ligase variant provided in Tables 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, and the amino acid positions are relative to the reference sequence corresponding to residues 12-346 of SEQ ID NO:42 or 204, or relative to the reference sequence corresponding to SEQ ID NO:42 or 204.

40. 2. The modified RNA ligase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, and 12.1, wherein the amino acid positions correspond to residues 12-346 of SEQ ID NO: 14, 42, or 204, or to the reference sequence corresponding to SEQ ID NO: 14, 42, or 204.

41. 2. The modified RNA ligase of claim 1, wherein the RNA ligase comprises an amino acid sequence comprising residues 12-346 of an even-numbered SEQ ID NO:24-582, or an amino acid sequence comprising an even-numbered SEQ ID NO:24-582, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

42. 42. The modified RNA ligase of any one of claims 1 to 41, comprising a fusion polypeptide.

43. A modified RNA ligase according to any one of claims 1 to 42, having RNA ligase 2 activity.

44. 44. A modified RNA ligase according to any one of claims 1 to 43, having at least one improved property compared to a reference RNA ligase.

45. 45. The modified RNA ligase of claim 44, wherein the improved property is selected from: i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased activity towards polynucleotide ligase substrates comprising phosphorothioate internucleotide linkages, vi) increased activity towards oligonucleotides comprising 2'-modifications, vii) increased substrate tolerance, or any combination of i), ii), iii), iv), v), vi), and vii), relative to a reference RNA ligase.

46. 46. ​​The modified RNA ligase of Claim 44 or 45, wherein the reference RNA ligase has the sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, 42, or 204, or the sequence corresponding to SEQ ID NO: 14, 42, or 204.

47. 46. ​​The modified RNA ligase of claim 44 or 45, wherein the reference RNA ligase has the sequence corresponding to residues 12 to 346 of SEQ ID NO: 14, or the sequence corresponding to SEQ ID NO:

14.

48. 48. The modified RNA ligase of any one of claims 1 to 47, which is purified.

49. A recombinant polynucleotide comprising a polynucleotide sequence encoding the modified RNA ligase of any one of claims 1 to 47.

50. 50. The recombinant polynucleotide of Claim 49, wherein the polynucleotide sequence has at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1038 of SEQ ID NO: 13, 41, or 203, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 13, 41, or 203, and wherein the recombinant polynucleotide encodes an RNA ligase.

51. 50. The recombinant polynucleotide of Claim 49, wherein the polynucleotide sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1038 of an odd-numbered SEQ ID NO:23-581, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO:23-581, and wherein the recombinant polynucleotide encodes an RNA ligase.

52. 52. The recombinant polynucleotide of any one of claims 49 to 51, wherein said polynucleotide sequence is codon-optimized for expression of said encoded modified RNA ligase.

53. (a) a polynucleotide sequence comprising nucleotide residues 34 to 1038 of SEQ ID NO: 13, 41, or 203, or a polynucleotide sequence comprising SEQ ID NO: 13, 41, or 203; or (b) a polynucleotide sequence comprising nucleotide residues 34 to 1038 of an odd-numbered SEQ ID NO: 23-581, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 23-581.

54. An expression vector comprising the recombinant polynucleotide of any one of claims 49 to 53.

55. 55. The expression vector of claim 54, wherein the polynucleotide is operably linked to a regulatory sequence.

56. 56. The expression vector of claim 55, wherein the control sequence comprises at least a promoter.

57. A host cell comprising the expression vector of any one of claims 54 to 56.

58. 58. The host cell of claim 57, comprising a prokaryotic or eukaryotic cell.

59. 59. The host cell of claim 58, comprising a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

60. 60. A method of producing a modified RNA ligase polypeptide in a host cell, comprising culturing the host cell of any one of claims 57 to 59 under suitable culture conditions so that the modified RNA ligase is produced.

61. 61. The method of claim 60, further comprising recovering the modified RNA ligase from the culture and / or host cell.

62. 62. The method of claim 60 or 61, further comprising purifying the modified RNA ligase.

63. A composition comprising the RNA ligase of any one of claims 1 to 48.

64. 64. The composition of claim 63, further comprising a buffer, ATP or dATP, and one or more polynucleotide substrates for the RNA ligase.

65. 100. A method of ligating at least a first polynucleotide strand and a second polynucleotide strand, comprising contacting the first polynucleotide strand and the second polynucleotide strand with the modified RNA ligase of any one of claims 1 to 48 in the presence of a nucleotide substrate under conditions suitable for ligation of the first polynucleotide strand to the second polynucleotide strand, wherein the first polynucleotide strand comprises a ligatable 5' end and the second polynucleotide strand comprises a 3' end that is ligatable to the 5' end of the first polynucleotide.

66. 66. The method of claim 65, further comprising a third polynucleotide strand, wherein the first polynucleotide strand and the second polynucleotide strand hybridize adjacent to each other on the third polynucleotide strand, and the 5' end of the first polynucleotide strand is positioned adjacent to the 3' end of the second polynucleotide strand.

67. 67. The method of claim 66, wherein the third polynucleotide strand is contiguous with the first polynucleotide strand or the second polynucleotide strand.

68. 68. The method of claim 67, wherein the third polynucleotide strand is contiguous with the first polynucleotide strand and the second polynucleotide strand to form a single continuous polynucleotide substrate.

69. 67. The method of claim 66, wherein the third polynucleotide strand comprises a splint or bridge polynucleotide, and the 5' terminal sequence of the first polynucleotide strand and the 3' terminal sequence of the second polynucleotide strand hybridize adjacent to each other on the splint or bridge polynucleotide, positioning the 5' end of the first polynucleotide strand adjacent to the 3' end of the second polynucleotide strand.

70. 67. The method of Claim 66, wherein the first and third polynucleotide strands hybridize to each other to form a first double-stranded polynucleotide fragment, and the second polynucleotide strand hybridizes to a fourth polynucleotide strand to form a second double-stranded fragment, and the first and second double-stranded fragments have complementary ends that can base-pair to form a substrate for the modified RNA ligase.

71. 71. The method of any one of claims 65 to 70, wherein the first polynucleotide strand and / or the second polynucleotide strand comprises RNA or a mixture of RNA and DNA.

72. 72. The method of any one of claims 65 to 71, wherein the 3' end of the second polynucleotide strand is 3'-OH.

73. 73. The method of any one of claims 65 to 72, wherein the 5' end of the first polynucleotide strand is a 5'-phosphate.

74. 74. The method of any one of claims 65 to 73, wherein the internucleotide linkage comprises a phosphate linkage.

75. 75. The method of any one of claims 65 to 74, wherein the first polynucleotide strand and / or the second polynucleotide strand comprises one or more nucleotide analogues, wherein the nucleotide analogues comprise a modified nucleobase, a modified nucleoside sugar residue, a modified nucleobase or nucleobase analogue, a modified internucleotide linkage, and / or a modified 5'-terminal phosphate group.

76. 76. The method of claim 75, wherein the modified 5'-terminal phosphate group is a phosphate analog.

77. 77. The method of claim 76, wherein the phosphate analog is phosphorothioate or monomethyl phosphate.

78. 76. The method of claim 75, wherein the modified nucleoside sugar moiety is 2'-O-alkyl, 2'-fluoro, β-D-ribo-LNA, or α-L-ribo-LNA (locked nucleic acid).

79. 76. The method of claim 75, wherein said modified nucleobase or nucleobase analog is xanthine, hypoxanthine, inosine, 7-methylguanine, 2,6-diaminopurine, 5-methylcytosine, 5-hydroxycytosine, 5-bromocytosine, 5-iodocytosine, 2-thiothymine, 5-fluorouracil, 5-bromouracil, 8-bromoguanine, 8-aminoguanine, or 8-aza-7-deazaguanine.

80. 76. The method of claim 75, wherein the internucleotide linkage is phosphorothioate, phosphoacetate, phosphoramidate, methylphosphonate, or phosphonocarboxylate.

81. 81. The method of any one of claims 65 to 80, wherein the nucleotide substrate is ATP or dATP.

82. A kit comprising at least one RNA ligase according to any one of claims 1 to 48.

83. 83. The kit of claim 82, further comprising one or more of a buffer, a nucleotide substrate, a polynucleotide substrate for a ligase, and / or a ligation enhancer.

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