RNA ligase mediated oligonucleotide synthesis

Abstract

The present disclosure relates to methods of using single-stranded RNA ligase for synthesis of oligonucleotides, including oligonucleotides containing a conjugate moiety. In some embodiments, the present disclosure further provides recombinant single-stranded RNA ligases, polynucleotides encoding the recombinant single-stranded RNA ligases, and compositions of the single-stranded RNA ligases.

Description

Docket Number CX10-269WO4 RNA LIGASE MEDIATED OLIGONUCLEOTIDE SYNTHESIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 634,859, filed April 16, 2024; U.S. Provisional Application No.63 / 646,841, filed May 13, 2024; and U.S. Provisional Application No. 63 / 718,884, filed November 11, 2024; the contents of all of which are incorporated by reference herein. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX10-269WO4_ST26.xml, created on April 16, 2025, with a file size of 3,694,688 bytes, is part of the specification and is incorporated by reference herein. BACKGROUND

[0003] Oligonucleotide therapeutics, either as synthetic single-stranded or double stranded polynucleotides, represent a class of drugs that exert their biological effects by modulating gene expression. The oligonucleotides are designed primarily to target pre-mRNA, mRNA, or non-coding RNA (e.g., miRNA) to promote RNA degradation, modulate splicing, interrupt translation, or in some instances activate expression. Small interfering RNAs (siRNA) are a class of oligonucleotide therapeutics that are typically double stranded oligonucleotides that act via RISC (RNA-induced silencing complex) pathway, where the strand complementary to an RNA target, also referred to as the guide strand, targets the RNA for degradation or translation inhibition. Antisense oligonucleotides (ASOs) are single-stranded and designed to bind sequence specifically to a target RNA and modulate protein expression via several different mechanisms. Targets of ASOs include pre-mRNA, mRNA, and non-coding RNA.

[0004] siRNA and ASO oligonucleotides are designed to include various modifications to improve in vivo stability, cellular delivery, specificity, and efficacy. Sugar modifications include 2’-O-methyl, 2’-O-ethyl, 2’- O-methoxyethyl, 2’-fluoro, and locked nucleic acid (LNA); modification of internucleoside linkages include phosphorothioate and phosphoramidate morpholino linkages; and nucleobase modifications include 5’- methylcytosine and G-clamp. In addition, conjugating targeting moieties to the oligonucleotide, such as GalNac and lipid groups, can enhance delivery to cells and tissues.

[0005] Modified oligonucleotides are generally synthesized chemically by solid-phase synthesis using phosphoramidite chemistry. Disadvantages of chemical synthesis include low efficiency in synthesis of long oligonucleotides, difficulty in scale-up of manufacturing, and toxic chemical waste and solvent consumption, e.g., toluene and acetonitrile. Accordingly, desirable are alternative approaches to chemical synthesis. SUMMARY

[0006] The present disclosure provides single-stranded RNA ligase polypeptides and compositions thereof, and methods of using the single-stranded RNA ligases for synthesis of oligonucleotides, including modified oligonucleotides with conjugate moieties.Docket Number CX10-269WO4

[0007] In some embodiments, a method of extending an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor.

[0008] In some embodiments, the nucleotide donor, the oligonucleotide(A), or both the nucleotide donor and the oligonucleotide(A) comprise a modified nucleotide.

[0009] In some embodiments, the oligonucleotide(A)comprises at least one modified nucleoside, wherein the modified nucleoside comprises a conjugate moiety, reactive group, or linker.

[0010] In some embodiments, the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

[0011] In some embodiments, the reactive group comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

[0012] In some embodiments, the modified nucleoside on the oligonucleotide(A)is at the 5’-terminal nucleotide, an internal nucleotide, or the 3’-terminal nucleotide.

[0013] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A).

[0014] In some embodiments, the conjugate moiety or reactive group is attached to the nucleoside via a linker. In some embodiments, the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A). In some embodiments, the linker comprises a cleavable linker.

[0015] In some embodiments, the oligonucleotide(A) comprises one or more terminal groups. In some embodiments, the terminal group is at the 5’-terminal nucleotide of the oligonucleotide(A). In some embodiments, the terminal group is a 5’-phosphonate (E- or Z-vinylphosphonate), 4’-amino, 4’-aminoalkyl, abasic nucleotide, or inverted abasic nucleotide. In some embodiments, the oligonucleotide(A) comprises a 5’- OH or a 5’-blocking group that inhibits ligation by the single-stranded RNA ligase.

[0016] In some embodiments, the oligonucleotide(A)comprises the formula (I): A1[•A2]m•A3-OH(I)wherein each of A1, A2and A3is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage.

[0017] In some embodiments, each of A2 is the same or different nucleoside. In some embodiments, the oligonucleotide(A) comprises a modified nucleoside of at least one or more of A1, A2, or A3. In some embodiments, at least one of A1, A2, or A3 is modified with a conjugate moiety or conjugate reactive group.Docket Number CX10-269WO4

[0018] In some embodiments, one or more of the internucleoside linkage is a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphorothioate or a phosphorodithioate.

[0019] In some embodiments, the modified nucleoside on the oligonucleotide(A) comprises the formula (II): A-[L]g-[M]h(II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0.

[0020] In some embodiments, the oligonucleotide(A)is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

[0021] In some embodiments, wherein when g is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside. In some embodiments, wherein when g is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside.

[0022] In some embodiments, the nucleotide donor comprises a nucleotide(D) or an oligonucleotide(D). In some embodiments the nucleotide(D) or oligonucleotide(D) comprises a modified nucleoside. In some embodiments, the modified nucleoside of nucleotide(D) or oligonucleotide(D) comprises a conjugate moiety, a reactive group, or linker.

[0023] In some embodiments, the conjugate moiety on the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

[0024] In some embodiments, reactive group on the nucleotide donor comprises an amino, -CN (cyano), N3(azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

[0025] In some embodiments, the modified nucleoside on the oligonucleotide(D) is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.

[0026] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the modified nucleoside.

[0027] In some embodiments, the conjugate moiety or the conjugate reactive group is attached to the nucleoside via a linker L. In some embodiments, the linker comprises a cleavable linker.

[0028] In some embodiments, the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb)Docket Number CX10-269WO4 wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120.

[0029] In some embodiments, the nucleotide donor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

[0030] In some embodiments, each of D2is the same or different nucleoside. In some embodiments, wherein the nucleotide donor comprises a modified nucleoside, at least one or more of D1, D2, or D3is modified. In some embodiments, at least one of D1, D2, or D3is modified with a conjugate moiety, reactive group, or linker.

[0031] In some embodiments, D comprises a modified nucleoside. In some embodiments, D is modified with a conjugate moiety, reactive group, or linker.

[0032] In some embodiments, the modified nucleoside on nucleotide(D) or oligonucleotide(D) has the formula (IV): D-[L]q-[M]r(IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0.

[0033] In some embodiments, wherein when q is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside. In some embodiments, wherein when q is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside.

[0034] In some embodiments, the nucleotide(D)or pD includes a 3’-phosphate (e.g., pDp).

[0035] In some embodiments, the nucleotide donor (i.e., nucleotide(D) or oligonucleotide(D)) comprises a 3’- blocking group that inhibits ligation by the single-stranded RNA ligase to a 3’-OH group or inhibits incorporation of a nucleotide onto an oligonucleotide by a terminal nucleotidyl transferase. In some embodiments, the reaction with the single-stranded RNA ligase and a 3’-blocked nucleotide(D) or 3’-blocked oligonucleotide(D) results in a 3’-blocked extended oligonucleotide product. In some embodiments, the 3’- blocking group comprises a reversible blocking group.

[0036] In some embodiments, wherein the product is a 3’-blocked extended oligonucleotide, the 3’-blocked extended oligonucleotide is separated or removed from the single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligase is inactivated.Docket Number CX10-269WO4

[0037] In some embodiments, where the 3’-blocking group is a reversible blocking group, the method further comprises removing or cleaving the 3’-blocking group on 3’-blocked extended oligonucleotide with a deblocking agent to form an unblocked extended oligonucleotide.

[0038] In some embodiments, the method further comprises inactivating the deblocking agent or removing or separating the unblocked extended oligonucleotide from the deblocking agent.

[0039] In some embodiments, the method further comprises reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single-stranded RNA ligase.

[0040] In some embodiments, the method further comprises one or more cycles of extension with a nucleotide donor; separation of 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivation of the single-stranded RNA ligase; removing or cleaving the reversible 3’-blocking group with a deblocking agent; and separating the unblocked extended oligonucleotide, wherein each cycle uses a new nucleotide donor.

[0041] In some embodiments, the nucleotide donor for at least one cycle comprises a mixture of different nucleotide donors, such as a mixture of different pD or pDp.

[0042] In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor (e.g., nucleotide(D) or oligonucleotide(D)) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence. In particular, the nucleotide(D) comprises a selected or predetermined nucleotide(D) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence.

[0043] In some embodiments of the method, the single-stranded RNA ligase comprises RNA ligase 1. In some embodiments, the single-stranded RNA ligase comprises a recombinant single-stranded RNA ligase described herein.

[0044] In some embodiments of the method, the single-stranded RNA ligase is immobilized on a support medium. In some embodiments, the oligonucleotide acceptor (oligonucleotide(A)) and nucleotide donor are provided in solution or aqueous phase.

[0045] In some embodiments of the method, the oligonucleotide acceptor (oligonucleotide(A)) is immobilized is attached to a support medium. In some embodiments, the single-stranded RNA ligase and the nucleotide donor are provided in solution or aqueous phase.

[0046] In some embodiments of the method, the reaction further comprises a pyrophosphatase for cleaving of pyrophosphate product.

[0047] In some embodiments of the method, the reaction further comprises an ATP recycling system.

[0048] In some embodiments of the method, the suitable reaction conditions comprises one or more of an NTP, a divalent metal ion, and buffer.

[0049] In some embodiments of the method, the suitable reaction conditions comprises a reaction temperature of 5-60 ℃.Docket Number CX10-269WO4

[0050] In some embodiments of the method, the suitable reaction conditions comprise a reaction pH of about 5-8.

[0051] In another aspect, the present disclosure provides a recombinant single-stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

[0052] In some embodiments, the amino acid sequence of the recombinant single-stranded RNA ligase comprises the sequence comprising amino acid residue 12 to the carboxy terminus of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

[0053] In some embodiments, the single-stranded RNA ligase is the single-stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1.

[0054] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0055] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.Docket Number CX10-269WO4

[0056] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0057] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0058] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0059] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0060] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.Docket Number CX10-269WO4

[0061] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0062] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0063] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0064] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.Docket Number CX10-269WO4

[0065] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0066] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0067] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34 / 45 / 269, 34 / 45 / 173 / 297, 34 / 173 / 269 / 380, 173 / 269, 156 / 269 / 380, 173 / 269 / 380, 34 / 173, 269, 34 / 380, 34 / 269 / 380, or 173 / 380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0068] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94 / 263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0069] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283 / 337, 347, 323, 354, 343, 118, 345, 314, 268 / 269, 363, 356, 358, 348, 162, 324 / 330, 346, 160, 362, 361, 341 / 349, 353, 369, 248, 146 / 346, 332, 170, or 269 / 275, wherein the amino acid positions are relative to the reference sequence corresponding toDocket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.

[0070] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162 / 337 / 358 / 362, 162 / 236 / 237 / 320 / 337 / 358 / 362, 151 / 231 / 237 / 337, 199 / 231 / 237 / 337, 231 / 237 / 314 / 337, 310 / 314 / 337, 115 / 162 / 310 / 314, 199 / 237 / 337, 151 / 199 / 205 / 310 / 314, 199 / 204 / 205 / 231 / 236 / 310 / 314, 320 / 337 / 358 / 362, 135 / 320 / 337 / 358 / 362, 151 / 212 / 214 / 345 / 347 / 358, 135 / 337 / 358 / 362, 162 / 358 / 362, 337 / 358 / 362, 337 / 358, 92 / 337, 151 / 196 / 199 / 205 / 231 / 237 / 323, 151 / 214 / 347 / 358, 337, 151 / 345 / 347 / 358, 199 / 314, 199 / 205 / 231 / 237 / 323, 151 / 205 / 314, 151 / 212 / 345 / 347, 92 / 214 / 347 / 358, 162 / 204 / 205 / 310 / 314 / 358, 236 / 314, 151 / 345 / 347, 214 / 347 / 358, 151 / 212 / 358, 204 / 283 / 314 / 358, 236 / 237 / 358 / 362, 151 / 199 / 204 / 231 / 236 / 323, 231 / 236 / 237 / 358 / 362, 162 / 314 / 358, 92 / 151 / 347 / 358, 151 / 236, 212 / 345 / 347, 115 / 314, 345 / 347 / 358, 314, 358 / 362, 115 / 358, 310 / 314, 214 / 347 / 358, 237 / 314, 345 / 347, 151 / 310 / 323 / 343 / 347, 151 / 358, 347 / 358, 93 / 358 / 362, 231 / 236 / 237 / 320 / 358 / 362, 151 / 230 / 345 / 347 / 358, 135 / 231 / 236 / 237 / 358, 310 / 314 / 358 / 362, 151 / 230 / 347 / 358, 231 / 237 / 323, 135 / 358 / 362, 283 / 314 / 358 / 362, 199 / 205, 92 / 151 / 230 / 345 / 347 / 358, 314 / 358 / 362, 199 / 237, or 199 / 204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.

[0071] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161 / 162, 311 / 320, 123, 320 / 326, 174, 162 / 167, 32, 125, 162 / 166, 320, 283, 330, 278, 303, 281, 333 / 337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.

[0072] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123 / 256 / 320, 260, 256 / 260, 256, 260 / 281, 320, 123 / 260, 123 / 320, 256 / 281, or 260 / 273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0073] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118 / 123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.

[0074] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 254, 240, 118, 347, 167, 281, 303, 205, or 50, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.Docket Number CX10-269WO4

[0075] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118 / 220, 118 / 220 / 303 / 347, 347, 118 / 220 / 254 / 347, 220, 38 / 220, 220 / 254 / 303 / 347, 220 / 254, 220 / 303 / 347, 48 / 220 / 347, 49 / 220, 217 / 220 / 347, 38 / 220 / 254, 49 / 118 / 220 / 254 / 347, 49 / 220 / 254, 38 / 48 / 49 / 118 / 220, 49 / 217 / 220 / 254, 49 / 220 / 347, 49 / 347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248 / 357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.

[0076] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 54 / 127 / 205 / 254, 165 / 255 / 258 / 259, 205 / 254 / 258, 33 / 38 / 127 / 165 / 254, 38 / 54 / 205 / 258, 127, 127 / 165 / 258, 127 / 205 / 254 / 255 / 258 / 259, 205 / 258, 33 / 38 / 254 / 255, 127 / 254 / 258 / 259, 127 / 165, 38 / 127 / 165 / 259, 38 / 127 / 258 / 259, 38 / 254 / 255, 127 / 205 / 254 / 258 / 259, 127 / 165 / 258 / 259, or 165 / 205 / 258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.

[0077] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127 / 222 / 223 / 225 / 255 / 272 / 276, 127 / 255 / 259, 276, 255 / 259, 127 / 255, 127 / 162 / 223 / 255, 127 / 162 / 255 / 259 / 272 / 276, or 259 / 272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0078] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 165 / 259 / 281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.

[0079] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279 / 281, 303, 165 / 166, 281 / 282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.

[0080] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44 / 118, 44 / 118 / 246 / 272 / 276, 44 / 118 / 246 / 280, 44 / 118 / 246 / 280 / 302, 44 / 118 / 272 / 276, 44 / 246, 44 / 246 / 272 / 276 / 302, 44 / 246 / 276 / 279 / 280, 44 / 246 / 276 / 280, 44 / 272 / 276 / 280, 44 / 272 / 279, 44 / 272 / 280, 44 / 276, 44 / 276 / 279, 44 / 276 / 280 / 295 / 302, 44 / 280, 44 / 302, 118 / 246 / 276 / 280 / 302, 118 / 246 / 280 / 295, 118 / 272 / 276, 118 / 280 / 302, 246 / 272 / 276 / 279 / 280 / 302, 246 / 272 / 279 / 280, 246 / 276, 246 / 276 / 302, 246 / 279 / 280, 246 / 279 / 280 / 302, 246 / 280, 272 / 276 / 279 / 280 / 302, 272 / 280, 272 / 280 / 302, 276 / 279 / 280 / 302, 276 / 280, 279 / 280 / 302, or 280, wherein the amino acid positions areDocket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768.

[0081] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.

[0082] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 127 / 238 / 284, 38 / 127 / 255, 38 / 127 / 255 / 359, 38 / 127 / 284, 38 / 238 / 255, 38 / 238 / 255 / 359, 38 / 238 / 255 / 359 / 381, 38 / 238 / 284 / 359, 127, 127 / 212 / 238 / 284 / 359, 127 / 238 / 255, 127 / 238 / 255 / 359 / 381, 127 / 238 / 284 / 359, 127 / 238 / 284 / 359 / 381, 127 / 238 / 359, 127 / 255 / 359 / 381, 127 / 255 / 381, 238, 238 / 255, 238 / 255 / 359, 238 / 255 / 381, 238 / 284, 238 / 359, 255, 255 / 284, 255 / 359, 255 / 359 / 362 / 381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882.

[0083] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34 / 35 / 38, 34 / 38 / 170, 34 / 135 / 170 / 271 / 357, 34 / 170 / 271, 34 / 271, 35 / 38 / 170 / 357, 35 / 38 / 271 / 357, 35 / 170 / 271 / 357, 38 / 170, 38 / 170 / 271, 38 / 170 / 357, 56 / 135 / 170 / 357, 56 / 135 / 271 / 357, 135 / 170 / 271, 170, 170 / 271 / 272, 170 / 271 / 272 / 357, 170 / 271 / 357, 170 / 357, 254 / 260, 254 / 281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.

[0084] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9 / 369, 34 / 38 / 357, 34 / 135 / 170 / 271 / 357, 34 / 271, 56 / 135 / 170 / 357, 56 / 135 / 271 / 357, 113, 115, 141, 144, 145, 177, 214, 254 / 260, 254 / 281, 260, 274, 340, 341, 350, 354 / 359, 359 / 360, 359 / 362, 368, 369, 371, 377 / 381, or 381 / 384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.

[0085] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113 / 115, 113 / 115 / 177 / 254 / 281, 113 / 115 / 177 / 254 / 281 / 350 / 359, 113 / 115 / 177 / 254 / 350, 113 / 115 / 177 / 254 / 359, 113 / 115 / 177 / 281 / 359, 113 / 115 / 254, 113 / 115 / 254 / 281, 113 / 115 / 254 / 350, 113 / 115 / 254 / 359, 113 / 115 / 350, 113 / 115 / 359, 113 / 177 / 254 / 350, 113 / 177 / 281 / 359, 113 / 177 / 350 / 354 / 359, 113 / 254, 113 / 254 / 281 / 350 / 359, 113 / 254 / 281 / 359, 113 / 254 / 350 / 354 / 359, 113 / 254 / 354 / 359, 113 / 254 / 359, 113 / 359, 115 / 177, 115 / 177 / 254, 115 / 177 / 254 / 359, 115 / 177 / 359, 115 / 254, 115 / 254 / 350 / 354 / 359, 115 / 254 / 359, 115 / 350, 144 / 145 / 260, 144 / 145 / 260 / 341, 144 / 145 / 260 / 341 / 366 / 371, 144 / 260 / 341 / 366, 144 / 260 / 366 / 369, 145 / 260 / 263, 145 / 260 / 341, 145 / 260 / 341 / 366 / 369, 145 / 341, 145 / 341 / 371, 145 / 371, 177, 177 / 254, 177 / 254 / 281 / 359, 177 / 254 / 354 / 359, 177 / 254 / 359, 177 / 359, 254, 254 / 281 / 354 / 359, 254 / 350, 254 / 354 / 359, 254 / 359, 260 / 366, 341, or 359, whereinDocket Number CX10-269WO4 the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992.

[0086] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.

[0087] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27 / 127 / 374, 27 / 127 / 374 / 376, 27 / 275 / 356, 27 / 351, 34 / 35 / 118 / 127 / 275 / 351 / 374 / 376, 122, 127 / 275 / 374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

[0088] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

[0089] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

[0090] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

[0091] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19 / 21 / 65, 19 / 21 / 65 / 66, 19 / 21 / 65 / 66 / 90 / 93, 19 / 21 / 65 / 66 / 93, 19 / 21 / 65 / 93, 19 / 21 / 65 / 190, 19 / 21 / 65 / 197, 19 / 21 / 66, 19 / 21 / 190, 19 / 65, 19 / 65 / 66, 19 / 65 / 66 / 90 / 93 / 190, 19 / 65 / 66 / 93, 19 / 65 / 66 / 190, 19 / 65 / 66 / 197, 19 / 66, 19 / 66 / 90 / 93 / 197, 25, 25 / 54, 25 / 122, 65, 65 / 66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.

[0092] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351 / 352, 351 / 353, or 351 / 358, wherein the amino acid positions are relative to the referenceDocket Number CX10-269WO4 sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.

[0093] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351 / 352, 351 / 353, 351 / 355, or 372 / 374 / 376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.

[0094] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21 / 54, 21 / 65 / 66 / 151, 21 / 90 / 93 / 122, 21 / 93, 54 / 58, 65 / 93, 65 / 151, 66 / 90 / 151 / 190 / 197, 90 / 93 / 122, 90 / 190 / 197, 90 / 197, 93, or 93 / 151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.

[0095] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.

[0096] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.

[0097] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0098] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to theDocket Number CX10-269WO4 reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0099] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0100] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526.

[0101] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0102] In some embodiments, the recombinant single stranded RNA ligase comprising an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244- 912, and 934-1526.

[0103] In some embodiments, the recombinant single stranded RNA ligase exhibits single stranded RNA ligase activity and at least an improved property as compared to a reference single stranded RNA ligase.

[0104] In some embodiments, the recombinant single stranded RNA ligase exhibits an improved property selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), as compared to a reference single stranded RNA ligase.Docket Number CX10-269WO4

[0105] In some embodiments, the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, or 768, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, or 768. In some embodiments, the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or an amino acid sequence corresponding to SEQ ID NO: 14.

[0106] In some embodiments, the recombinant single-stranded RNA ligase further comprises a fusion protein.

[0107] In some embodiments, the recombinant single stranded RNA ligase is provided as a purified preparation.

[0108] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding any of the recombinant single stranded RNA ligase disclosed herein.

[0109] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 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 the sequence from nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, or to a reference nucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

[0110] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 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 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, wherein the recombinant polynucleotide encodes a single stranded RNA ligase.

[0111] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 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 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, wherein the recombinant polynucleotide encodes a single stranded RNA ligase.

[0112] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon- optimized for expression of the encoded recombinant single stranded RNA ligase.

[0113] In some embodiments, polynucleotide sequence encoding the single stranded RNA ligase comprises nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-Docket Number CX10-269WO4 1523, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243- 911, and 933-1523.

[0114] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide encoding the single stranded RNA ligase comprises nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or a polynucleotide sequence comprising SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499.

[0115] In a further aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding any of the single stranded RNA ligases described herein. In some embodiments, the expression vector comprises a control sequence operably linked to the recombinant polynucleotide. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter.

[0116] In a further aspect, the present disclosure provides a host cell comprising an expression vector for expression or production of the recombinant single stranded RNA ligase. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0117] In a further aspect, the present disclosure provides a method of producing a recombinant single stranded RNA ligase, the method comprising culturing a host cell described herein under suitable culture conditions such that the encoded recombinant single stranded RNA ligase is produced. In some embodiments, the method further comprises recovering the recombinant single stranded RNA ligase polypeptide from the culture and / or host cell. In some embodiments, the method further comprises purifying the recombinant single stranded RNA ligase polypeptide.

[0118] In another aspect, the present disclosure provides a composition comprising a recombinant single stranded RNA ligase. In some embodiments, the composition further comprises one or more of a buffer, nucleotide cofactor, divalent metal, ligation enhancer, and / or one or more polynucleotide substrates for the single stranded RNA ligase.

[0119] In some embodiments, the composition further comprises at least an oligonucleotide acceptor and / or a nucleotide donor (e.g., nucleotide(D)or oligonucleotide(D)). In some embodiments, the oligonucleotide donor comprises a modified oligonucleotide donor. In some embodiments, the nucleotide donor nucleotide(D)comprises a modified nucleotide(D). In some embodiments, the nucleotide donor oligonucleotide(D)comprises a modified oligonucleotide(D).

[0120] In another aspect, the present disclosure further provides a kit comprising a recombinant single stranded RNA ligase described herein.

[0121] In some embodiments, the kit further comprises one or more of a buffer, nucleotide cofactor, divalent metal, ligation enhancer, and / or one or more polynucleotide substrates for the single stranded RNA ligase. DETAILED DESCRIPTIONDocket Number CX10-269WO4

[0122] The present disclosure provides recombinant single-stranded RNA ligases and methods of synthesizing oligonucleotide using the recombinant single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligases are used to produce modified oligonucleotide, particularly modified oligonucleotides having a conjugate moiety or a reactive group. Abbreviations and Definitions

[0123] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0124] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide.

[0125] Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0126] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0127] “About” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.

[0128] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0129] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0130] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0131] “Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0132] “Amino acids” and “amino acid” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (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),Docket Number CX10-269WO4 threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about α-carbon (Cα). For example, whereas “Ala” designates alanine without specifying the configuration about the α-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the α-carbon and lower case letters designate amino acids in the D-configuration about the α-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0133] “Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).

[0134] “RNA ligase” refers to enzymes that covalently joins the 5’-phosphoryl termini of RNA or DNA to the 3’-hydroxyl termini of RNA or DNA. Families of known RNA ligases include RNA ligase 1, also referred to as single-stranded RNA ligase or ssRNA ligase, which catalyzes the covalent joining of single-stranded 5’- phosphoryl termini of RNA or DNA to single-stranded 3--hydroxyl termini of RNA or DNA. RNA ligase 2, also referred to as double stranded RNA ligase or dsRNA ligase, also catalyzes the covalent joining of a 3’- hydroxyl terminus of RNA to a 5’-phosphorylated RNA or DNA but shows preference for double stranded substrates. In some embodiments, RNA ligases include those enzymes classified in EC 6.5.1.3. It is to be understood that the ligation reaction is not limited to naturally occurring RNA and DNA substrates also includes nucleotide substrates that contain modified nucleotides and / or nucleotide analogs.

[0135] “Polynucleotide,” “nucleic acid,” or “oligonucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2’-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’-deoxyribonucleosides on an individual basis or on an aggregate basis. When a polynucleotide, nucleic acid, or oligonucleotide sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. The polynucleotide or nucleic acid may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions.

[0136] In some embodiments, the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide or nucleic acid or oligonucleotide, which include, among others, nucleosides linked together via other than standard phosphodiester linkages, such as non-standard linkages of phosphoramidates, phosphorothioates, amideDocket Number CX10-269WO4 linkages, etc.; nucleosides with modified and / or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’-O-alkyl, 2’-halo, 2,3-dideoxy, 2’- halo-2’-deoxy, β-D-ribo LNA, α-L-ribo-LNA (e.g., locked nucleic acids), etc.; and / or 5’-phosphate analogs, including, among others, phosphorothioate, phosphoacetate, phosphoramidate, monomethylphosphate, methylphosphonate, or phosphonocarboxylate.

[0137] “Nucleobase” refers to means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T). cytosine (C). uracil (U). or guanine (G). A “modified nucleobase” refers to a group of atoms other than unmodified A, T, C, U. or G capable of pairing with at least one unmodified nucleobase.

[0138] “Nucleoside” refers to a compound comprising a nucleobase and a sugar moiety. The nucleobases and sugar moiety are each, independently, unmodified or modified.

[0139] “Internucleoside linkage” refers to as a linkage that covalently couples two nucleosides together. In the oligonucleotides herein, internucleoside linkages covalently couple adjacent nucleosides together, typically forming a bond between the sugar moieties of the adjacent nucleosides. Non-limiting examples of internucleoside linkages include phosphodiester -O-P(O)2-O- linkages and modified internucleoside linkages, such as phosphorothioate -O-P-(O, S)-O- and phosphorodithioate -O-P(S)2-O-.

[0140] “Modified oligonucleotide” refers to an oligonucleotide which contains at least one modified internucleoside linkage and / or a modified nucleoside, or a modified terminal group.

[0141] “Modified nucleotide” refers to a nucleotide (e.g., NMP, NDP, NTP) in which at least one of the phosphate is a modified phosphate group and / or a modified nucleoside.

[0142] “Modified nucleoside” or “nucleoside modification” refers to a nucleoside modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase. The modified nucleoside comprises a modified nucleobase and / or a modified sugar residue. The term “modified nucleoside” may also be used herein interchangeably with the term “nucleoside analogue.” Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the nucleobase of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson-Crick base pairing.

[0143] “Modified internucleoside linkage” refers to as a linkage other than a phosphodiester (PO) linkage that covalently connects two nucleosides together. In some embodiments, exemplary modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. Other modified phosphorus-containing internucleoside linkages include phosphotriesters, methylphosphonates, and phosphoramidates (P-NH2). See, e.g., Clave et al., RSC Chem Biol., 20212(1): 94–150). In some embodiments, the modified internucleoside linkage is a non-phosphorus containing internucleoside linkage, including but not limited tomethylenemethylimino (-CH2-N(CH3)-O-CH2), thiodiestcr, thionocarbamate (-O- C(=O)(NH)-S-); siloxane (-O-SiH2-O-); N,N’-dimethylhydrazine (-CH2-N((CH3)-N((CH3)-); MMI (3'-CH2- N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-Docket Number CX10-269WO4 5'), methoxypropyl, and thioformacetal (3’-S-CH2-O-5'). In some embodiments, internucleoside linkages having a chiral atom can be prepared as a mixture of the stereoisomers, or as separate stereoisomers.

[0144] “Phosphorothioate internucleoside linkage” refers to an internucleoside linkage in which one of the oxygen atom in a phosphodiester linkage is replaced with a sulfur atom. In some embodiments, a phosphorothioate linkage may be represented as -O-P(O,S)-O-, wherein one of the non-bridging oxygen atoms is replaced with a sulfur atom. Phosphorothioate internucleoside linkages are chiral (see, for example, Jahns et al.2022, Nucleic Acids Research Vol.50, No 3, 1221-1240), with right-handed (Rp) and left-handed (Sp) isomers. In some embodiments, the Rp diastereomer may be referred to as an R-PS internucleoside linkage or an srP internucleoside linkage. The Sp diastereomer may be referred to as an S-PS internucleoside linkage or ssP internucleoside linkage. In some embodiments, the oligonucleotide comprises one or more srP internucleoside linkages. In some embodiments, the oligonucleotide comprises one or more ssP internucleoside linkages. Where the chirality of a phosphorothioate internucleoside linkage is not specified, that phosphorothioate internucleoside linkage may be either an srP linkage or an ssP linkage.

[0145] “Non-bridging phosphorothioate internucleoside linkage” refers to a phosphorothioate internucleoside linkage in which the sulfur atom attached to the phosphorous atom is in place of a non-bridging oxygen atom.

[0146] “Non-bridging phosphorodithioate internucleoside linkage” refers to a modified internucleoside linkage which is a non-bridging phosphorodithioate internucleoside linkage. A non-bridging phosphorodithioate internucleoside linkage has two identical sulfur atoms attached to the phosphorous atom, achieved by replacing the non-bridging oxygen atom in the phosphorothioate linkage with a sulfur atom.

[0147] “Abasic sugar moiety” refers to a sugar moiety of a nucleoside that is not attached to a nucleobase. In some embodiments, such abasic sugar moieties are referred to as “abasic nucleoside.”

[0148] “Inverted nucleoside” refers to a nucleotide having a 3’ to 3’ and / or 5’ to 5’ internucleoside linkage. Similarly, and “inverted sugar moiety” refers to the sugar moiety of an inverted nucleoside or an abasic sugar moiety having a 3’ to 3’ and / or 5’ to 5’ internucleoside linkage.

[0149] “LNA nucleoside” or “locked nucleoside” refers to 2'-modified nucleoside which comprises a biradical linking the C2' and C4' of the ribose sugar ring of said nucleoside (also referred to as a "2'- 4' bridge"), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex.

[0150] Non-limiting, exemplary LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett.2002, 12, 73-76, Seth et al. J. Org. Chem.2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.Docket Number CX10-269WO4

[0151] “Terminal group” as used herein refers to a group located at the first or last nucleoside in a polynucleotide or oligonucleotide. A 5’-terminal group refers to the terminal group bonded to 5′-or 4’-carbon atom of the first nucleoside within a polynucleotide. A 3’-terminal group is a terminal group bonded to 3′- carbon atom of the last nucleoside within a polynucleotide or oligonucleotide.

[0152] “5’-blocking group” as used herein refers to a moiety or chemical group that prevents or inhibits attachment of another nucleoside, nucleotide or oligonucleotide to the 5’-terminal nucleoside. In context enzymes active on the 5’-terminal nucleoside, a 5’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide or oligonucleotide to the to the 5’-terminal nucleoside, particularly the 5’-OH of the 5’-terminal nucleoside.

[0153] “3’-blocking group” refers to moiety or chemical group that prevents or inhibits attachment off another nucleoside, nucleotide, or oligonucleotide to the 3’-terminal nucleoside. In context of single-stranded RNA ligase or other enzymes active on the 3’-terminal nucleoside, a 3’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide, or oligonucleotide to the 3’-terminal nucleoside, particularly the 3’-OH of the 3’-terminal nucleoside.

[0154] “Reversible blocking group” refers to a blocking group that can be removed or cleaved off to provide a free 3’-OH. In some embodiments, the blocking group is removable with a deblocking agent, which can be a chemical or enzymatic deblocking agent.

[0155] “Enzymatically reversible blocking group” refers to a blocking group that is susceptible to removal or cleaving by an enzyme.

[0156] “Duplex” and “ds” refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule comprised of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs to T or U, C pairs to G), arranged in an antiparallel 5’ to 3’ orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).

[0157] “Complementary” is used herein to describe the structural relationship between nucleotide bases that are capable of forming base pairs with one another. For example, a purine nucleotide base present on a polynucleotide that is complementary to a pyrimidine nucleotide base on a polynucleotide may base pair by forming hydrogen bonds with one another. Complementary nucleotide bases can base pair via Watson / Crick base pairing or in any other manner than forms stable duplexes or other nucleic acid structures.

[0158] “Watson / Crick Base-Pairing” refers to a pattern of specific pairs of nucleobases and analogs that bind together through sequence-specific hydrogen-bonds, e.g., A pairs with T or U, and G pairs with C.

[0159] “Annealing” or “Hybridization” refers to the base-pairing interactions of one nucleobase polymer (e.g., poly- and oligonucleotides) with another that results in the formation of a double-stranded structure, a triplex structure or a quaternary structure. Annealing or hybridization can occur via Watson-Crick base- pairing interactions, but may be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing. In some embodiments, the nucleobase polymer that anneals or hybridizes to another is a single nucleobase polymer while in other embodiments, the nucleobase polymers are separate nucleobase polymers.Docket Number CX10-269WO4

[0160] “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and / or by manipulation using recombinant techniques.

[0161] “Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0162] “Coding sequence” and synonymously “encoding” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0163] “Percent (%) sequence identity” refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far asDocket Number CX10-269WO4 the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0164] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0165] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0166] “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a recombinant primase,Docket Number CX10-269WO4 can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0167] “Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0168] “Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The amino acid positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. In some instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances, the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in the reference sequence. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a “-“ where appropriate. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino acid residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.

[0169] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “ / ”. The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either / or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0170] “Amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set ofDocket Number CX10-269WO4 amino acid substitutions that is present in any of the variant primase polypeptides listed in any of the Tables in the Examples. In some embodiments, the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set. In the substitution sets, the individual substitutions are separated by a semicolon “;” or slash “ / ”.

[0171] “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0172] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0173] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity and / or retaining the improved properties of an recombinant primase. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated by “-“, and may be present in substitution sets.

[0174] “Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0175] “Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to whichDocket Number CX10-269WO4 it is being compared (e.g., a full length recombinant primase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0176] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant primase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant primase polypeptides provided herein are isolated polypeptides.

[0177] “Substantially pure polypeptide” or “purified” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure primase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant primase polypeptides are substantially pure polypeptide compositions.

[0178] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the primase are codon optimized for optimal production from the host organism selected for expression.

[0179] “Control sequence” refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present disclosure. Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0180] “Operably linked” or “operatively linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest suchDocket Number CX10-269WO4 that the control sequence directs or regulates the expression of the polynucleotide of interest, and where appropriate, expression of the encoded polypeptide of interest.

[0181] “Promoter” or “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0182] “Suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co- solvents, co-factors, etc.) under which an RNA ligase is capable of attaching a nucleotide donor to a nucleotide acceptor. Exemplary “suitable reaction conditions” are provided herein (see, the Examples).

[0183] “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the primase polypeptide on the substrate.

[0184] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0185] “Vector” is a recombinant construct for introducing a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide or a polypeptide encoded in the polynucleotide. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0186] “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0187] “Produces” refers to the production of proteins and / or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0188] “Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

[0189] “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g., a polynucleotide sequences encoding a recombinant primase). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques, and progeny thereof, as known in the art.Docket Number CX10-269WO4

[0190] “Alkyl” refers to straight or branched chain hydrocarbon groups having the number of carbon atoms designated, for example 1 to 20 carbon atoms (C1-C20), particularly 1 to 12 carbon atoms (C1-C12 or C1-12), and more particularly (C1-C8 or C1-8) carbon atoms. Exemplary “alkyl” includes, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, and s-pentyl.

[0191] “Alkenyl” refers to straight or branched chain hydrocarbon having the number of carbon atoms designated, for example 2 to 20 carbon atoms (C2-C20), particularly 2 to 12 carbon atoms (C2-C12 or C2-12), and most particularly 2 to 8 (C2-C8 or C2-8)carbon atoms, having at least one double bond. Exemplary “alkenyl” includes, but are not limited to, vinyl ethenyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl.

[0192] “Alkynyl” refers to a straight or branched chain hydrocarbon having the number of carbon atoms designated, for example 2 to 12 carbon atoms (C2-C12or C2-12), particularly 2 to 8 carbon atoms (C2-C8or C2-8), containing at least one triple bond. Exemplary “alkynyl” includes ethynyl, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3- hexynyl, 4-hexynyl and 5-hexynyl.

[0193] “Alkylene”, “alkenylene” and “alkynylene” refers to a straight or branched chain divalent hydrocarbon radical of the corresponding alkyl, alkenyl, and alkynyl, respectively. The “alkylene”, “alkenylene” and “alkynylene” may be optionally substituted, for example with alkyl, alkyloxy, hydroxyl, carbonyl, carboxyl, halo, nitro, and the like.

[0194] “Lower” in reference to substituents refers to a group having between one and six carbon atoms.

[0195] “Heteroalkyl,” heteroalkenyl,” and “heteroalkynyl” refers to the corresponding alkyl, alkenyl, and akynyl in which one or more of the carbon atoms is replaced with a heteroatom, such as O, S and N.

[0196] “Cycloalkyl” refers to any stable monocyclic or polycyclic system which consists of carbon atoms, any ring of which being saturated. “Cycloalkenyl” refers to any stable monocyclic or polycyclic system which consists of carbon atoms, with at least one ring thereof being partially unsaturated. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicycloalkyls and tricycloalkyls (e.g., adamantyl).

[0197] “Heterocycloalkyl” or “heterocyclyl” refers to a substituted or unsubstituted 3 to 14 membered, mono- or bicyclic, non-aromatic hydrocarbon, wherein 1 to 3 carbon atoms a (e replaced by a heteroatom. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR’-, -PH-, -S(O)-, -S(O)2-, -S(O) NR’-, -S(O)2NR’-, and the like, including combinations thereof, where each R’ is independently hydrogen or lower alkyl. Examples include oxiranyl, oxetanyl, azetidynyl, oxazolyl, thiazolidinyl, thiazolyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyranyl, tetrahydrofuranyl, tetrahydropyranyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, azapanyl, and the like.Docket Number CX10-269WO4

[0198] “Aryl” refers to a six- to fourteen-membered, mono- or bi-carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings in the bicyclic ring is aromatic. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. Examples of “aryl” groups include phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, and the like.

[0199] “Heteroaryl” refers to an aromatic heterocyclic ring, including both monocyclic and bicyclic ring systems, where at least one carbon atom of one or both of the rings is replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur, or at least two carbon atoms of one or both of the rings are replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl can be a 5 to 6 membered monocyclic, or 7 to 11 membered bicyclic ring systems. Examples of “heteroaryl” groups include pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl, isoquinolyl, quinoxalinyl, quinolyl, and the like.

[0200] “Bridged bicyclic” refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 5 to 12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Such bridged bicyclic groups include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include: ,

[0201] In some embodiments, a locked nucleoside is a bridged bicyclic compound.

[0202] “Fused ring” refers a ring system with two or more rings having at least one bond and two atoms in common. A “fused aryl” and a “fused heteroaryl” refer to ring systems having at least one aryl and heteroaryl, respectively, that share at least one bond and two atoms in common with another ring.Docket Number CX10-269WO4

[0203] “Carbonyl” refers to -C(O)-. The carbonyl group may be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones. For example, an -C(O)R’, wherein R’ is an alkyl is referred to as an alkylcarbonyl. In some embodiments, R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0204] “Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

[0205] “Haloalkyl” refers to an alkyl substituted with 1 or more halogen atoms. Preferably, the alkyl is substituted with 1 to 3 halogen atoms.

[0206] “Hydroxy” refers to –OH.

[0207] “Oxy” refers to group -O-, which may have various substituents to form different oxy groups, including ethers and esters. In some embodiments, the oxy group is an –OR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0208] “Acyl” refers to -C(O)R’, where R is hydrogen, or an optionally substituted alkyl, heteroalkyl, cylcoalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl as defined herein. Exemplary acyl groups include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

[0209] “Alkyloxy” or “alkoxy” refers to –OR’, wherein R’ is an optionally substituted alkyl.

[0210] “Aryloxy” refers to –OR’, wherein R’ is an optionally substituted aryl.

[0211] “Carboxy” refers to –COO- or COOM, wherein H or a M+counterion.

[0212] “Carbamoyl” refers to -C(O)NR’R’, wherein each R’ is independently selected from H or an optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocylcoalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.

[0213] “Cyano” refers to –CN.

[0214] “Ester” refers to a group such as -C(=O)OR’, alternatively illustrated as –C(O)OR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocyclolalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0215] “Silyl” refers to Si, which may have various substituents, for example –SiR’R’R’, where R’ is as defined in the specification. For example, each R’ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. As defined herein, any heterocyloalkyl or heteroaryl group present in a silyl group has from 1 to 3 heteroatoms selected independently from O, N, and S.

[0216] “Thiol” or “sulfhydryl” refers to –SH.

[0217] “Disulfied” refers to -S-S- groups.Docket Number CX10-269WO4

[0218] “Sulfanyl” refers to –SR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. For example, -SR, wherein R is an alkyl is an alkylsulfanyl.

[0219] “Sulfonyl” refers to -S(O)2-, which may have various substituents to form different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones. For example, -S(O)2R’, wherein R’ is an alkyl refers to an alkylsulfonyl. In some embodiments of -S(O)2R’, R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0220] “Amino” or “amine” refers to the group –NR’R’ or –NR’R’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, heterocycloalkyl, alkyloxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkyloxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

[0221] “Amide” refers to a group such as, -C(=O)NR’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0222] “Optional” or “optionally” refers to a described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not. For example, “optionally substituted alkyl” refers to an alkyl group that may or may not be substituted and that the description encompasses both substituted alkyl group and unsubstituted alkyl group.

[0223] “Substituted” as used herein means one or more hydrogen atoms of the group is replaced with a substituent atom or group commonly used in pharmaceutical chemistry. Each substituent can be the same or different. Examples of suitable substituents include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, OR’(e.g., hydroxyl, alkyloxy (e.g., methoxy, ethoxy, and propoxy), aryloxy, heteroaryloxy, arylalkyloxy, ether, ester, carbamate, etc.), hydroxyalkyl, alkyloxycarbonyl, alkyloxyalkyloxy, perhaloalkyl, alkyloxyalkyl, SR’(e.g., thiol, alkylthio, arylthio, heteroarylthio, arylalkylthio, etc.), S+R’2, S(O)R’, SO2R’, NR’R”(e.g., primary amine (i.e., NH2), secondary amine, tertiary amine, amide, carbamate, urea, etc.), hydrazide, halo, nitrile, nitro, sulfide, sulfoxide, sulfone, sulfonamide, thiol, carboxy, aldehyde, keto, carboxylic acid, ester, amide, imine, and imide, including seleno and thio derivatives thereof, wherein each of the substituents can be optionally further substituted. In embodiments in which a functional group with an aromatic carbon ring is substituted, such substitutions will typically number less than about 10 substitutions, more preferably about 1 to 5, with about 1 or 2 substitutions being preferred.

[0224] “Stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. Thus, “stereoisomer thereof” with respect to a compound includes any stereoisomer of the compound and mixtures of stereoisomers, and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposable mirrorDocket Number CX10-269WO4 images of one another. A compound may have more than one chiral center such that the compound may exist as either an individual diastereomer or as a mixture of diastereomers. Ligation of Oligonucleotides with single-stranded RNA Ligases

[0225] In one aspect, the present disclosure provides a method of ligating an acceptor to a donor substrate of single-stranded RNA ligase to form a ligated oligonucleotide product. In some embodiments, the acceptor or donor, or both the acceptor and donor of the single-stranded RNA ligase substrates are modified to generate a ligated modified oligonucleotide product.

[0226] In some embodiments, a method of synthesizing an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor to form an extended oligonucleotide.

[0227] In some embodiments, the oligonucleotide(A), the nucleotide donor, or both the oligonucleotide(A) and the nucleotide donor comprise a modified nucleotide.

[0228] In some embodiments, the single stranded RNA ligase can be used to synthesize, among others, antisense oligonucleotides (ASOs), polynucleotide strands for use in synthesis of double stranded polynucleotides (e.g., siRNA compounds), and guide RNA used in CRISPR or related genome editing technology, and the like. In some embodiments, the single stranded RNA ligase is used to add a nucleotide comprising a conjugate moiety, such as cell targeting moieties (e.g., GalNAc, lipids, steroids, etc.), to an oligonucleotide. Oligonucleotide Acceptor

[0229] In some embodiments, the oligonucleotide acceptor (oligonucleotide(A)) comprises a free 3’-OH group, or a functional form thereof, at the 3’-terminal nucleotide suitable as an acceptor for or reaction with the nucleotide donor in the single-stranded RNA ligase reaction.

[0230] In some embodiments, the oligonucleotide acceptor is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotide acceptor is up to 122 nucleotides in length. In some embodiments, the oligonucleotides is up to 150 or more nucleotides in length. In some embodiments, the oligonucleotides is up to 200 or more nucleotides in length.

[0231] In some embodiments, the oligonucleotide acceptor is 2 nucleotides in length. In some embodiments, the oligonucleotide acceptor is 3 nucleotides in length. In some embodiments, the oligonucleotide acceptors is 4 nucleotides in length. In some embodiments, the oligonucleotide acceptor is 5 nucleotides in length. In some embodiments, the oligonucleotide is 6 nucleotides in length. In some embodiments, the oligonucleotide is 7 nucleotides in length. In some embodiments, the oligonucleotide is 8 nucleotides in length. In some embodiments, the oligonucleotide is 9 nucleotides in length. In some embodiments, the oligonucleotide is 10 nucleotides in length.Docket Number CX10-269WO4

[0232] In some embodiments, the oligonucleotide acceptor comprises a 5’-OH group. In some embodiments, the presence of a 5’-OH group prevents the oligonucleotide acceptor from acting as a nucleotide donor for another RNA ligase reaction.

[0233] In some embodiments, the oligonucleotide acceptor comprises a 5’-blocking group that prevents or inhibits the oligonucleotide acceptor to act as a nucleotide donor in another RNA ligase reaction, as further described herein. In some embodiments, use of an oligonucleotide acceptor with a 5’-OH or a 5’-blocking group in combination with a nucleotide donor having a 3’-blocking group can be used to direct or guide the ligation reaction to generate specific ligated products.

[0234] In some embodiments, the oligonucleotide acceptor comprises at least a modified nucleotide. In some embodiments, the modified nucleotide on the oligonucleotide acceptor comprises a modified internucleoside linkage, a modified nucleoside, a 5’-terminal group, or any combinations thereof.

[0235] In some embodiments, a modified nucleoside on the oligonucleotide acceptor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. In some embodiments, the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide acceptor, as limited by the length of the oligonucleotide acceptor. In some embodiments, all nucleosides of the oligonucleotide acceptor comprises a modified nucleoside.

[0236] In some embodiments, the modified 3’-terminal nucleoside of the oligonucleotide acceptor comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, the modified 3’-terminal nucleoside is a 2’-fluoro adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro thymidine, 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine, 2’-O-methyl uridine, or 2’-O-methyl thymidine.

[0237] In some embodiments, the oligonucleotide acceptor comprises at least one modified nucleoside or modified terminal group, wherein the modified nucleoside or terminal group comprises a conjugate moiety, a reactive group, or a linker.

[0238] In some embodiments, the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

[0239] In some embodiment, the reactive group comprises an amino, -CN (cyano), N3(azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

[0240] In some embodiments, the reactive group includes those used in click chemistry, such as copper-free click chemistry, and include, by way example and not limitation, azido, alkynyl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine groups.

[0241] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the nucleotide acceptor. In some embodiments, the conjugate moiety or reactive group is attached to the nucleoside via a linker. In some embodiments, the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A).Docket Number CX10-269WO4

[0242] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the 2’-position of the sugar moiety.

[0243] In some embodiments, wherein the nucleoside is an internal nucleoside or a 3’-terminal nucleoside, the conjugate moiety, reactive group, or linker is attached to the nucleobase and / or 2’-position of the sugar moiety.

[0244] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the 5’-OH group, a 5’-phosphate group, 4’-carbon of the sugar moiety, or through an abasic nucleoside, or an inverted abasic nucleoside on the 5’-terminal nucleotide.

[0245] In some embodiments, the oligonucleotide acceptor comprises one or more terminal groups. In some embodiments, the terminal group is present at the 5’-terminal nucleotide of the nucleotide acceptor. In some embodiments, the terminal group present on the oligonucleotide acceptor is a 5’-phosphonate, (e.g., E or Z vinylphosphonate), 5’-phosphoalkyl (e.g., 5’-phosphomethyl or 5’-phosphoethyl, etc.), 4’-amino, 4’- aminoalkyl, abasic nucleoside, or inverted abasic nucleoside.

[0246] In some embodiments, the oligonucleotide acceptor comprises one or more modified internucleoside linkages. In some embodiments, the internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or a mixture of Sp and Rp stereoisomer.

[0247] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 5’ terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide acceptor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 5’-terminal region with modified internucleoside linkages.

[0248] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 3’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide acceptor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 3’-terminal region with modified internucleoside linkages.

[0249] In some embodiments, the oligonucleotide acceptor (oligonucleotide(A)) comprises the formula (I): A1[•A2]m•A3-OH (I) wherein each of A1, A2 and A3 is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage.

[0250] In some embodiments, m is 0, 1, 2, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, when m is 2 or greater, each of A2 is same or different.Docket Number CX10-269WO4

[0251] In some embodiments, at least one or more of A1, A2, or A3 comprises a modified nucleoside. In some embodiments, A1 is modified. In some embodiments, at least one of A2 is modified. In some embodiments, at least 2, 3, 4, 5, 6, or all of A2 is modified. In some embodiments, A3 is modified. In some embodiments, at least one of A1, A2, or A3 is modified with a conjugate moiety, reactive group, or linker.

[0252] In some embodiments, A3 comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, A3 comprises a 2’-fluoro-adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine.

[0253] In some embodiments, at least one internucleoside linkage “•“ comprises a modified internucleoside linkage. In some embodiments, the internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp or Rp stereoisomer

[0254] In some embodiments, the linker on the modified oligonucleotide acceptor comprises any suitable linker, for example, linker comprised of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like. In some embodiments the linker comprises an C2-C20alkylene or polyethylene linker. Various linkers suitable for the purpose herein are described herein and also known in the art.

[0255] In some embodiments, the modified nucleoside on the oligonucleotide acceptor comprises the formula (II): A-[L]g-[M]h (II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0.

[0256] In some embodiments, the oligonucleotide acceptor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

[0257] In some embodiments, wherein for formula (II) when g is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside, or wherein when g is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside. Nucleotide Donor or Donor SubstrateDocket Number CX10-269WO4

[0258] In some embodiments, the nucleotide donor or donor substrate for the RNA ligase comprises a 5’- phosphate group, or functional form thereof, at the 5’-terminal nucleotide suitable for attachment or ligation to any of the oligonucleotide acceptors described herein.

[0259] In some embodiments, the nucleotide donor comprises a single nucleotide donor, also referred to herein as nucleotide(D) or D, or an oligonucleotide donor, also referred to as oligonucleotide(D).

[0260] In some embodiments, the oligonucleotide donor is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotide donor is up to 122 nucleotides in length. In some embodiments, the oligonucleotides donor is up to 150 or more nucleotides in length. In some embodiments, the oligonucleotide donor is up to 200 or more nucleotides in length.

[0261] In some embodiments, the oligonucleotide donor is 2 nucleotides in length. In some embodiments, the oligonucleotide donor is 3 nucleotides in length. In some embodiments, the oligonucleotide donor is 4 nucleotides in length. In some embodiments, the oligonucleotide donor is 5 nucleotides in length. In some embodiments, the oligonucleotide donor is 6 nucleotides in length. In some embodiments, the oligonucleotide donor is 7 nucleotides in length. In some embodiments, the oligonucleotide donor is 8 nucleotides in length. In some embodiments, the oligonucleotide donor is 9 nucleotides in length. In some embodiments, the oligonucleotide donor is 10 nucleotides in length.

[0262] In some embodiments, the nucleotide donor, either as a nucleotide(D) or oligonucleotide(D), comprises a modified nucleotide. In some embodiments, the oligonucleotide(D) comprises at least one modified nucleotide.

[0263] In some embodiments, the modified nucleotide on nucleotide(D) comprises a modified nucleoside, a 3’-terminal group, or any combinations thereof.

[0264] In some embodiments, the modified nucleotide on an oligonucleotide(D)comprises a modified internucleoside linkage, a modified nucleoside, a 3’-terminal group, or any combinations thereof.

[0265] In some embodiments, a modified nucleoside on the oligonucleotide donor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. In some embodiments, the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide donor, as limited by the length of the oligonucleotide donor. In some embodiments, all nucleosides of an oligonucleotide donor comprises a modified nucleoside.

[0266] In some embodiments, a modified 5’-terminal nucleoside of the oligonucleotide donor comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, the modified 5’-terminal nucleoside is a 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O- methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine.

[0267] In some embodiments, the modified nucleoside of nucleotide donor nucleotide(D)or oligonucleotide(D)comprises a conjugate moiety, a reactive group, or a linker.Docket Number CX10-269WO4

[0268] In some embodiments, the conjugate moiety on the modified nucleoside of the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

[0269] In some embodiment, the reactive group on the modified nucleoside comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. In some embodiments, the reactive group used in click chemistry, including copper-free click chemistry, such as azido, alkynyl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine groups.

[0270] In some embodiments, the linker on the modified nucleotide donor comprises any suitable linker, for example, linker comprised of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like. In some embodiments the linker comprises a C2-C20alkylene or polyethylene linker. As noted above, various linkers suitable for the modified nucleoside of the nucleotide donor are described herein and also known in the art.

[0271] In some embodiments, when the nucleotide donor comprises oligonucleotide(D), the nucleoside modified with a conjugate moiety, reactive group, or linker on the oligonucleotide donor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. In some embodiments, the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide(D), as limited by the length of the oligonucleotide donor.

[0272] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase, the sugar moiety of a nucleoside of a nucleotide donor, or a 3’-terminal group.

[0273] In some embodiments, where the nucleotide donor comprises nucleotide(D), the conjugate moiety, reactive group, or linker can be attached to the nucleobase, the 2’- or 3’-position of the sugar moiety, a 3’- terminal group, or any combinations thereof.

[0274] In some embodiments, where the nucleotide donor comprises an oligonucleotide(D), the conjugate moiety, reactive group, or linker can be attached to the nucleobase or the 2’-position of the sugar moiety of the 5’-terminal nucleoside and / or the internal nucleoside of oligonucleotide(D). In some embodiments, the conjugate moiety, reactive group, or linker can be attached to the nucleobase, the 2’- or the 3’-position of the sugar moiety, or a 3’-terminal group, or any combinations thereof, of the 3’-terminal nucleotide.

[0275] In some embodiments, the nucleotide donor comprises one or more terminal groups. In some embodiments, the terminal group is present on nucleotide(D), or where the nucleotide donor comprises oligonucleotide(D), at the 3’-terminal nucleotide of nucleotide(D)or oligonucleotide(D). In some embodiments, the terminal group present on the nucleotide donor is a 3’-phosphate, 3’-phosphorothioate, 3’- phosphorodithioate, 3’-phosphonate (e.g., E or Z vinylphosphonate), 3’-phosphoalkyl (e.g., 3-phosphomethyl or 3’-phosphoethyl, etc.), an abasic nucleoside, or inverted abasic nucleoside.

[0276] In some embodiments, wherein the nucleotide donor comprises nucleotide(D), the 3’-terminal group comprises a 3’-phosphate group for enhancing the efficiency of ligation of the nucleotide donor to the oligonucleotide acceptor, e.g., pDp, where the prefix p is a 5’-phosphate group and suffix p is a 3’-phosphateDocket Number CX10-269WO4 group. In some embodiments, a 3’-phosphate group can also act as a 3’-blocking group, either on the single nucleotide donor or oligonucleotide donor.

[0277] In some embodiments, where the nucleotide donor comprises an oligonucleotide(D), the oligonucleotide(D) comprises one or more modified internucleoside linkages. In some embodiments, the modified internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or mixtures thereof.

[0278] In some embodiments, the oligonucleotide(D)has at least 1 nucleotide at the 5’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide donor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 5’-terminal region with modified internucleoside linkages.

[0279] In some embodiments, the oligonucleotide(D)has at least 1 nucleotide at the 3’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide donor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 3’-terminal region with modified internucleoside linkages.

[0280] In some embodiments, the nucleotide donor comprises a 3’-blocking group. In some embodiments, a 3’-blocking group prevents or inhibits reiterative extensions or attachments of the nucleotide donor to another nucleotide donor. In some embodiments, the 3’-blocking group is present on the 3’-OH group or the nucleobase of the 3’-terminal nucleoside of the nucleotide donor. In some embodiments, the 3’-blocking group is a reversible blocking group, which is removable or capable of being cleaved with a deblocking agent to produce a free 3’-OH. Various reversible 3’-blocking agents are described herein, and known in the art.

[0281] In some embodiments, the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb) wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120.

[0282] In some embodiments, for formula (IIIb), n is 0, 1, 2, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, when n is 2 or greater, each of D2 is same or different nucleoside.

[0283] In some embodiments, for formula (IIIa), D comprises a modified nucleoside. In some embodiments, D is modified with a conjugate moiety, reactive group, or a linker.

[0284] In some embodiments, for formula (IIIb), at least one or more of D1, D2, or D3comprises a modified nucleoside. In some embodiments, D1is modified. In some embodiments, at least one of D2is modified. In some embodiments, at least 2, 3, 4, 5, 6 or all of D2is modified. In some embodiments, D3is modified. InDocket Number CX10-269WO4 some embodiments, at least one of D1, D2, or D3 is modified with a conjugate moiety, reactive group, or linker.

[0285] In some embodiments, D1 comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, D1 comprises a 2’-fluoro-adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine.

[0286] In some embodiments, for formula (IIIb) at least one internucleoside linkage “•“ comprises a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp and Rp stereoisomers.

[0287] In some embodiments, the modified nucleoside on the nucleotide donor, e.g., of formula (IIIa) or formula (IIIb), comprises the formula (IV): D-[L]q-[M]r(IV)wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0.

[0288] In some embodiments, when q is 1, L is attached to the nucleobase or the sugar moiety, or when q is 0, M is attached to the nucleobase or the sugar moiety.

[0289] In some embodiments, when q is 1, L can be attached to the 2’-position or 3’-position of the sugar moiety, as appropriate for D, either for nucleotide(D) or for oligonucleotide(D). For example, for formula (IIIa), L can be attached to 2’-position or 3’-position of the sugar moiety. For formula (IIIb), where D is D1 or D2, L can be attached to the 2’-position of the sugar moiety. Where D is D3, the L can be attached to the 2’- or 3’- position of the sugar moiety.

[0290] In some embodiments, D or D3further comprises one or more 3’-terminal groups. In some embodiments, the terminal group is a 3’-phosphate, 3’-phosphorothioate, 3’-phosphorodithioate, 3’- phosphonate, (e.g., E or Z vinylphosphonate), 3’-phosphoalkyl (e.g., 5-phosphomethyl or 5’-phosphoethyl, etc.), an abasic nucleoside, or inverted abasic nucleoside.

[0291] In some embodiments, for formula (IIIa), D of the nucleotide donor comprises a 3’-phosphate group, where the nucleotide donor comprises pDp representing the 5’-phosphate and the 3’-phosphate groups on the nucleoside D. In some embodiments, the pDp is pUp, pTp, pCp, pAp, or pGp. In some embodiments, theDocket Number CX10-269WO4 pDp comprises a 2’-modification. In some embodiments, pDp is pmUp, pmTp, pmCp, pmAp, or pmGp, with “m” representing 2’-O-methyl. In some embodiments, pDp is pfUp, pfTp, pfCp, pfAp, or pfGp, with “f” representing 2’-F. In some embodiments, the nucleobase of nucleoside comprises a nucleobase selected from the list described below.

[0292] In some embodiments, for formula (IIIb), the nucleotide donor comprises the formula pD1[-D2]n-D3p, wherein the pD1 represents the 5’-phosphate on the 5-terminal nucleoside D1, and D3p represents the 3’- phosphate on the 3’-terminal nucleoside D3.

[0293] In some embodiments, for formula (IIIa), D further comprises a 3’-blocking group. In some embodiments, for formula (IIIb), D3further comprises a 3’-blocking group. In some embodiments, the 3’- blocking group prevents or inhibits attachment of the nucleotide donor to another nucleotide donor in the RNA ligase reaction, or reiterative extensions or attachments of a nucleotide substrate using a terminal nucleotidyl transferase. In some embodiments, the 3’-blocking group is present on the 3’-OH group or the nucleobase of D or D3. In some embodiments, the 3’-blocking group is a reversible blocking group. In some embodiments, the 3’-blocking is removable or capable of being cleaved with a deblocking agent, as further described herein. Reiterative Extensions or Additions with single-stranded RNA ligase and Optionally Terminal Nucleotidyl Transferase

[0294] In some embodiments, the method of synthesizing an oligonucleotide comprises reacting an oligonucleotide acceptor and a nucleotide donor in presence of a single-stranded RNA ligase under reaction conditions suitable for ligation of the nucleotide donor to the oligonucleotide acceptor, wherein the nucleotide donor comprises a 3’-blocking group to form a 3’-blocked extended oligonucleotide. In some embodiments, the presence of the 3’-blocking groups provides for attachment of a single nucleotide donor to the oligonucleotide acceptor. In some embodiments, attachment of 3’-blocked nucleotide donor allows for attaching one nucleotide(D) or one oligonucleotide(D) to the oligonucleotide acceptor. In some embodiments, where the nucleotide nucleotide(D) comprises a conjugate moiety, reactive moiety, and / or a linker, the presence of a 3’-blocking group provides for attachment of one nucleotide(D) comprising a conjugate moiety, reactive moiety, and / or a linker.

[0295] In some embodiments, the 3’-blocking group present on the nucleotide donor is a reversible 3’- blocking group, thereby resulting in formation of a reversible 3’-blocked extended oligonucleotide by the single-stranded RNA ligase reaction. In some embodiments, the reversible 3’-blocking agent on the blocked extended oligonucleotide is removable or cleavable with a deblocking agent appropriate for the particular 3’- blocking group. As further discussed below, an exemplary 3’-blocking groups comprises a 3’-phosphate, where the deblocking agent is a phosphatase.

[0296] In some embodiments, the method further comprises removing or separating the 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivating the single-stranded RNA ligase.

[0297] In some embodiments, optionally the method further comprises degrading or inactivating the by- products (e.g., unreacted nucleotide donor, pyrophosphate, etc.) of the single-stranded RNA ligase reaction.Docket Number CX10-269WO4

[0298] In some embodiments, wherein the 3’-blocking group is a reversible 3’-blocking group, the method further comprises removing or cleaving the 3’-blocking group from the 3’-blocked extended oligonucleotide to form an unblocked extended oligonucleotide, i.e., an extended oligonucleotide with a free 3’-OH group. In some embodiments, the removing or cleaving of the 3’-blocking group is carried out with a deblocking agent. In some embodiments, following the deblocking reaction, the deblocking agent is inactivated or the unblocked extended oligonucleotide is separated from the deblocking agent.

[0299] In some embodiments, the removing or cleaving the 3’-blocking group with the deblocking agent and the degrading or inactivating of the by-products of the single-stranded RNA ligase reaction are done concurrently.

[0300] In some embodiments, the method further comprises reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single-stranded RNA ligase under suitable reaction conditions for attachment or ligation of the second nucleotide donor to the unblocked extended oligonucleotide.

[0301] In some embodiments, the method further comprises one or more cycles of: extension with a nucleotide donor comprising a reversible 3’-blocking agent; separating the 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivating the single-stranded RNA ligase; removing or cleaving the 3’-blocking group with a deblocking agent; separating the unblocked extended oligonucleotide from the deblocking agent or inactivating the deblocking agent, wherein each cycle is with a new nucleotide donor. Repeating the cycle allows for step-wise extension of the extended oligonucleotide. In some embodiments, where the nucleotide donor is nucleotide(D), repeating each cycle with new nucleotide(D)allows for step-wise extensions of a single nucleotide with each cycle. In some embodiments, where the nucleotide donor is oligonucleotide(D), repeating each cycle with new oligonucleotide(D)allows for step-wise extensions with oligonucleotide(D).

[0302] In some embodiments, the 3’-blocked nucleotide donor is used in combination with a 5’-OH containing or 5’-blocked oligonucleotide acceptor. Use of an oligonucleotide acceptor with a 5’-OH or 5’- blocking group limits or inhibits ligation to the 3’-OH of another oligonucleotide acceptor, thereby limiting ligation to the 3’-blocked nucleotide donor.

[0303] In some embodiments, each nucleotide donor is a selected or predetermined nucleotide donor for producing an extended oligonucleotide with a defined nucleotide sequence.

[0304] In some embodiments, the use of a 3’-blocked nucleotide donor comprising a conjugate moiety, reactive moiety, or linker, where the 3’-blocking group is a reversible blocking group, allows for sequential or stepwise addition of nucleotides with the conjugate moiety, reactive group, or linker. In some embodiments, the method comprises sequential or stepwise attachment or addition of at least two nucleotide donors comprising a conjugate moiety, reactive moiety, or linker; at least three nucleotide donors comprising a conjugate moiety, reactive moiety, or linker; or at least four nucleotide donors comprising a conjugate moiety, reactive moiety, or linker. In some embodiments, the nucleotide donor in the sequential or stepwise addition of nucleotides is nucleotide(D) comprising a conjugate moiety, reactive moiety, or linker.Docket Number CX10-269WO4

[0305] In some embodiments, the unblocked extended oligonucleotide can serve as a substrate for a terminal nucleotidyl transferase. In some embodiments, the method further comprises reacting the unblocked extended oligonucleotide with a nucleotide substrate in presence of a terminal nucleotidyl transferase under reaction conditions suitable for the extension by or addition of at least one nucleotide to form at least a single nucleotide extended oligonucleotide. In some embodiments, more than one nucleotide is attached or added to the extended oligonucleotide by the terminal nucleotidyl transferase.

[0306] In some embodiments, the nucleotide substrate for the terminal nucleotidyl transferase comprises a 3’-blocked nucleotide substrate (i.e., a nucleotide substrate comprising a 3’-blocking group), providing for a single nucleotide addition or attachment to the oligonucleotide or the extended oligonucleotide to form a 3’- blocked single nucleotide extended oligonucleotide.

[0307] In some embodiments, the method further comprises removing or cleaving the 3’-blocking group to form an unblocked single nucleotide extended oligonucleotide. In some embodiments, the removing or cleaving of the 3’-blocking group is done with a deblocking agent appropriate for the specific 3’-blocking group.

[0308] In some embodiments, the method further comprises degrading the by-products (e.g., unreacted NTP, unreacted 3’-blocked NTP, NDP, NMP, 3’-blocked NMP, etc.) of the terminal nucleotidyl transferase reaction. For example, where the 3’-blocking group is a 3’-phosphate, a phosphatase can be used as the deblocking agent.

[0309] In some embodiments, following the deblocking reaction, the deblocking agent is inactivated or the deblocking agent separated from the unblocked single nucleotide extended oligonucleotide. In some embodiments, the inactivation and degrading of the by-products is done concurrently.

[0310] In some embodiments, the method further comprises repeating one or more cycles of reacting the unblocked extended oligonucleotide with a nucleotide substrate comprising a 3’-blocking group in presence of the terminal nucleotidyl transferase, removing or cleaving the 3’-blocking moiety (i.e., deblocking the 3’- blocked nucleoside) on the extended oligonucleotide with a deblocking agent, and inactivating the deblocking agent or separating the deblocking agent from the unblock extended oligonucleotide, where each cycle is with a new nucleotide substrate.

[0311] In some embodiments, each cycle of extension, deblocking, and inactivation / separation is with a new nucleotide substrate for step-wise, sequential attachment or addition of nucleotides to the extended oligonucleotide by the terminal nucleotidyl transferase.

[0312] In some embodiments, each nucleotide substrate for the terminal nucleotidyl transferase reaction is a selected or predetermined nucleotide substrate for producing an extended oligonucleotide with a defined nucleotide sequence. In some embodiments, the use of a modified nucleotide substrate (e.g., modified nucleobase and / or 2’-position of the sugar moiety) allows for stepwise, sequential addition of modified nucleotides to the extended oligonucleotide. Reaction Conditions and ProcessDocket Number CX10-269WO4

[0313] In the embodiments herein, the single-stranded RNA ligase reactions are carried out under suitable conditions for the ligase mediated attachment of the oligonucleotide acceptor to the nucleotide donor.

[0314] In some embodiments, the suitable reaction conditions include a nucleotide co-factor used by the single-stranded RNA ligase to catalyze the joining reaction. In some embodiments, the nucleotide cofactor is ATP. In some embodiments, the nucleotide cofactor is about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, or more as appropriate. In some embodiments, the nucleotide cofactor or substrate (e.g., ATP) is present at a concentration of about 0.05-25 mM, 1-20 mM, 2-18 mM, or 5-15 mM. In some embodiments, the nucleotide cofactor or substrate is present at a concentration of about 0.5 mM, 1 mM, 2 mM, 3 mM 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 20 mM, or 25 mM.

[0315] In some embodiments, the reaction conditions for the ligation include additional components, such as a divalent metal (e.g., Mg+2), buffer, and / or salts. Exemplary reaction components are provided in the Examples. In some embodiments, the salt in the reaction conditions, include, among others, NaCl, KCl, ammonium salts (e.g., NH4Cl), and acetate salts (e.g., sodium acetate). In some embodiments, the salt is present at 0.5 mM-300 mM, 1 mM-250 mM, 2 mM-200 mM, 5 mM-150 mM, 10 mM-100 mM, 20 mM-80 mM, or 40 mM-60 mM. In some embodiments, the salt is present at about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, or 300 mM. In some embodiments, the salt is present at sufficient concentration to reduce degradation or other undesired products.

[0316] In some embodiments, the reaction conditions also include a ligation enhancing reagent, including, among others, DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000).

[0317] In some embodiments, the single-stranded RNA ligase reaction is carried out at a suitable temperature and reaction time period for the ligation of the oligonucleotide acceptor and the nucleotide donor. In some embodiments, the RNA ligation reaction temperature is from about 2° C to about 60° C. In some embodiments, the RNA ligation reaction temperature is from 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 RNA 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, the reaction temperature is chosen based on the thermostability of the single-stranded RNA ligase and / or the efficiency of the ligation at defined temperatures and reaction conditions.

[0318] In some embodiments, the single-stranded RNA ligase reaction time can be a sufficient time for ligation of the oligonucleotide acceptor to the nucleotide donor. In some embodiments, the ligation reaction time is from 0.5-72 hr or longer. In some embodiments, the ligation reaction time is 1-72 hr, 2-48 hr, or 2-24 hr. In some embodiments, the ligation reaction time is 0.5, 1, 2, 4, 5, 12, 24, 48, or 72 hr or longer.

[0319] In the embodiments, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and / or during the course of the reaction. In some embodiments, the suitable reaction conditions comprise a solution pH from about 4 to aboutDocket Number CX10-269WO4 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0320] In some embodiments, the pH of the reaction mixture may change during the reaction. In some embodiments, the pH of the reaction solution is maintained at a desired pH or within a desired pH range, such as by the addition of an acid or a base, before and / or during the course of the reaction. In some embodiments, the pH is controlled by using an appropriate buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, phosphate, 2-(N- morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. In some embodiments, the buffer concentration is from 1 to 500 mM, 1 to 400 mM, 1 to 300 mM, 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM. In some embodiments, the buffer concentration is about 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM.

[0321] In some embodiments, the concentration of oligonucleotide acceptor and nucleotide donor can be varied based on, among others, the reaction conditions, the activity of the single-stranded ligase, factors affecting efficiency of the oligonucleotide acceptor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the acceptor, presence of secondary structure, 3’-terminal properties, etc.), and the efficiency of the nucleotide donor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the nucleotide donor, presence of secondary structure, 5’-terminal properties, etc.). If the process uses an oligonucleotide bound to a support medium, additional considerations include, among others, density of the oligonucleotide acceptor on the support medium, effect of support medium on the single-stranded RNA ligase reaction, and access of the single-stranded RNA ligase and other enzymes (e.g., pyrophosphatase, terminal nucleotidyl transferase, etc.) to the oligonucleotide acceptor bound to the support medium.

[0322] In some embodiments, the oligonucleotide acceptor is provided at a concentration of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, oligonucleotide acceptor concentration is at about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3 mM-0.6 mM. In some embodiments, the oligonucleotide acceptor concentration is at about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient ligation to the nucleotide donor.

[0323] In some embodiments, the nucleotide donor is provided at a concentration of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, nucleotide donor concentration is at about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3 mM-0.6 mM. In some embodiments, the nucleotide donor concentration is at about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM,Docket Number CX10-269WO4 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient ligation to the oligonucleotide acceptor.

[0324] In some embodiments, the ratio of nucleotide donor to oligonucleotide acceptor is 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or higher.

[0325] In some embodiments, the single stranded RNA ligase is provided at concentrations from about 0.01 g / L to about 50 g / L; about 0.01 to about 0.1 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L. Oligonucleotide Synthesis Using Oligonucleotide Bound to Support Medium

[0326] In some embodiments, the oligonucleotide acceptor is bound to a support medium (e.g., a solid support), and the ligation reaction carried out with the single-stranded RNA ligase and the nucleotide donor in solution to ligate the nucleotide donor to the support bound oligonucleotide acceptor. In some embodiments, the oligonucleotide is conjugated to the support medium via the 5’-terminal nucleotide to provide for a ligase accessible free 3’-OH.

[0327] In some embodiments, the single-stranded RNA ligase, any unreacted nucleotide donor, and by- products of the single-stranded RNA ligase reaction are removed or separated from the immobilized extended oligonucleotide. In some embodiments, the extended oligonucleotide is released or cleaved from the substrate medium to yield the extended oligonucleotide in solution. In some embodiments, the releasing agent is a chemical cleavage agent appropriate for the covalent attachment to the substrate medium. In some embodiments, a biological releasing or cleaving agent is used, for example a nuclease that can cleave at a specific site in the oligonucleotide bound to the substrate medium.

[0328] In some embodiments, for addition of one nucleotide donor, e.g., nucleotide(D), or for reiterative synthesis using the oligonucleotide acceptor bound to a support medium, the ligation reaction is carried out with the single-stranded RNA ligase and a 3’-blocked nucleotide donor in solution to form a 3’-blocked extended oligonucleotide bound to the support medium. The single-stranded RNA ligase, any unreacted nucleotide donor, and by-products of the single-stranded RNA ligase reaction are removed or separated from the immobilized 3’-blocked extended oligonucleotide. In some embodiments, where the 3’-blocking group is desired in the extended oligonucleotide, for example, a conjugate moiety, reactive group, or linker attached to the 3’-OH group of the sugar moiety, no further extensions are carried out and the extended oligonucleotide is released or cleaved from the support medium.

[0329] In some embodiments, for the addition of one nucleotide donor nucleotide(D), where removal or cleavage of the 3’-blocking groups is desired, a reversible 3’-blocking group is used and the support bound 3’- blocked extended oligonucleotide reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide. The unblocked extended oligonucleotide is then released or cleaved from the support medium. In some embodiments, the 3’-blocked extended oligonucleotide is released or cleaved from the support medium and the deblocking step carried out in solution.Docket Number CX10-269WO4

[0330] In some embodiments, for stepwise, sequential additions or extensions with two or more nucleotide donors, the support bound 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide bound to the support medium. In some embodiments, following the deblocking reaction, the deblocking agent is inactivated or the unblocked extended oligonucleotide separated from the deblocking agent. In some embodiments, the by-products of the single-stranded RNA ligase reaction are degraded with a degrading agent.

[0331] In some embodiments, for each new nucleotide donor in the sequential additions or extensions, the cycle of reaction with the extended oligonucleotide with RNA ligase and deblocking and associated inactivation / separation of deblocking agent and degrading of by-products is repeated for each nucleotide donor.

[0332] In some embodiments, following the addition of the last nucleotide donor in the stepwise, sequential additions, the support bound 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide. The unblocked extended oligonucleotide is then released or cleaved from the support medium.

[0333] In some embodiments, the desired 3’-blocked extended oligonucleotide is released or cleaved from the support medium and the deblocking step carried out in solution to yield the final extended oligonucleotide product.

[0334] In some embodiments, the final extended oligonucleotide product in solution can be purified, for example by chromatography (e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography), including HPLP and UPLC; ultrafiltration; and affinity techniques (e.g., capture tags, hybridization, etc.). Oligonucleotide Synthesis Using Immobilized Enzymes

[0335] In some embodiments, the ligation reaction is carried out with a single-stranded RNA ligase bound to a support medium, e.g., immobilized single-stranded RNA ligase. In some embodiments, the oligonucleotide acceptor and nucleotide donor in solution can be contacted with the support-bound single-stranded RNA ligase to ligate the oligonucleotide acceptor to the nucleotide donor. In some embodiments, the substrates in solution are passed through a bed (e.g., a column) of support-bound single-stranded RNA ligase until a desired level of extended product has formed. In some embodiments, the extended oligonucleotide product is removed or separated from the immobilized single-stranded RNA ligase. In some embodiments, the nucleotide donor comprises a 3’-blocked nucleotide donor to form a 3’-blocked extended oligonucleotide in solution.

[0336] In some embodiments, for ligation reactions using a nucleotide donor comprising a reversible 3’- blocking group, the method further comprises removing or cleaving the 3’-blocking group with a deblocking agent to form an unblocked extended oligonucleotide in solution.

[0337] In some embodiments, the method further comprises inactivating and / or separating the deblocking agent from the unblocked extended oligonucleotide. In some embodiments, where the deblocking agent comprises an enzyme, e.g., phosphatase, the enzyme can be on a support substrate (e.g., immobilizedDocket Number CX10-269WO4 deblocking enzyme) to facilitate reaction with the blocked extended oligonucleotide in solution and its separation from the deblocking agent following the deblocking reaction.

[0338] In some embodiments, the method further comprises degrading the by-products in the reaction containing the extended oligonucleotide in solution. In some embodiments, the degrading of by-products is achieved concurrently or subsequent to the deblocking of the extended oligonucleotide. In some embodiments, the degrading agent, e.g., an enzyme, is immobilized to a support medium to facilitate degradation of by-products in solution and separation of the extended oligonucleotide from the degrading agent.

[0339] In some embodiments, for stepwise, sequential additions or extensions with two or more nucleotide donors, the 3’-blocked extended oligonucleotide in solution is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide, and following the deblocking reaction, the deblocking agent is inactivated or the deblocking agent separated from the unblocked extended oligonucleotide.

[0340] In some embodiments, for each new nucleotide donor in the sequential additions or extensions, the cycle of reaction with the extended oligonucleotide with immobilized single-stranded RNA ligase and deblocking and associated inactivation / separation of deblocking agent and degrading of by-products is repeated for each new nucleotide donor.

[0341] In some embodiments, following the addition of the last nucleotide donor in the stepwise, sequential additions, the final 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to from the unblocked extended oligonucleotide. In some embodiments, use of an immobilized single-stranded RNA ligase and an immobilized deblocking agent, e.g., a phosphatase, provides an efficient process for stepwise sequential addition of nucleotide donors involving extension reaction with the immobilized single-stranded RNA ligase (e.g., in a first column or chamber), separation and transport of the reaction solution to an immobilized deblocking agent (e.g., in a second column or chamber) for deblocking and degradation of by-products of the ligation reaction, and separation and transport of the reaction solution from the deblocking reaction to the extension with the immobilized single-stranded RNA ligase (e.g., in the first column or chamber) for reaction with the next nucleotide donor.

[0342] In some embodiments, the final unblocked extended oligonucleotide can be purified, for example by chromatography (e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography), including HPLP and UPLC; ultrafiltration; and affinity techniques (e.g., capture tags, hybridization, etc.).

[0343] Unlike the process using an oligonucleotide bound to a support medium for the extension reaction, a process using oligonucleotides and nucleotide donors in solution does not require release or cleavage of the desired extended oligonucleotide product from the support medium. Modified Nucleotides

[0344] In some embodiments, in the compositions or methods of using single-stranded RNA ligase for oligonucleotide synthesis, the oligonucleotide acceptor, nucleotide donor, or the combination ofDocket Number CX10-269WO4 oligonucleotide acceptor and nucleotide donor can have various modifications. In some embodiments, the modifications occur on the nucleobase, the sugar moiety, terminal groups, or any combination thereof. The various modifications that can be used are described below. 2’- and 3’-sugar modifications

[0345] In some embodiments, the oligonucleotide acceptor and / or the nucleotide donor comprises a modified nucleoside, wherein the modification is on the sugar moiety of the nucleoside. In some embodiments, the modified sugar moiety is a modified furanosyl sugar moiety, for example ribose or deoxyribose. In some embodiments, the furanosyl sugar moiety is modified or substituted at the 2’, 3’, or a combination of 2’ and 3’ positions. Modifications at the 4’, and / or 5’-positions are described below as “terminal group.”

[0346] In some embodiments, the modification is at the 2’-position of the sugar moiety. In some embodiments, substitutions at the 2’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 2’-alkoxy (e.g., O-methyl, O-ethyl, etc.). In some embodiments, other modifications at the 2’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCH3, SO2CH3, ONO2, NO2, N3, and NH2. In some embodiments, substituent groups at the 2’-position include, among others, O-(C1- C10)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N- alkynyl, O-alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10alkyl or C1-C10alkenyl and alkynyl. In some embodiments, substituent groups at the 2’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl. In some embodiments, the substitution at the 2’-position is a phosphate (see, e.g., Current Protocols in Nucleic Acid Chemistry, 13.1.1- 13.1.31, John Wiley & Sons (2003).

[0347] In some embodiments, the modified 2’-position of the sugar moiety is halo, 2’-O-R’, or 2’-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a C1-C4alkyl. In some embodiments, the modified 2’-position is a 2’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, the 2’-position of the sugar moiety of the nucleoside substrate is -O- R’, wherein R’ is -CH3or -CH2CH3or -CH2CH2OCH3. In some embodiments, the modified 2’-position is 2’- O-(2-methoxyethyl), 2’-O-allyl, 2’-O-propargyl, 2’-O-ethylamine, 2’-O-cyanoethyl, -2’-O-amine, or 2’-O- acetate ester.

[0348] In some embodiments, a modification at the 2’-position comprises a locked nucleoside. In some embodiments, locked nucleosides comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a “2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring (see, e.g., Obika et al., Tetrahedron Letters, 1997, 38(50):8735–8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11):1138–1142). In some embodiments, the ribose moiety of the locked nucleotide is in the C3’-endo (beta-D) or C2’-endo (alpha-L) conformation. In some embodiments, the bridge is a methylene bridge. In some embodiments, the bridge is an ethylene bridge, also referred to as ENA (see, e.g., Morita et al., Bioorg Med Chem Lett., 2002, 12(1):73-6). Other locked nucleoside are described in International patent publication WO 2121249993, incorporated by reference herein.Docket Number CX10-269WO4

[0349] In some embodiments, other locked nucleosides include, among others, 5’-methyl-LNA, 2’-amino- LNA, alpha-L-LNA, and thio-LNA. Structures of certain locked nucleosides are shown below:where R

[0350] In some embodiments, a modification at the 2’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker or a linker, as described herein.

[0351] In some embodiments, the modification is at the 3’-position of the sugar moiety. In some embodiments, in view of the effect of a 3’-modification on ligase activity, and use of the 3’-OH group for internucleoside linkage, the 3’-modification is on the 3’-terminal nucleoside of the nucleotide donor. In some embodiments, the modification at the 3’-position are similar to those at the 2’-position. In some embodiments, substitutions at the 3’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 3’-alkoxy (e.g., O-methyl, O-ethyl, etc.). In some embodiments, other modifications at the 3’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCH3, SO2CH3, ONO2, NO2, N3, and NH2. In some embodiments, substituent groups at the 3’-position include, among others, O-(C1-C10)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O- alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C1-C10 alkenyl and alkynyl. In some embodiments, substituent groups at the 3’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl. In some embodiments, In some embodiments, the substitution at the 3’-position is a phosphate.

[0352] In some embodiments, the modified 3’-position of the sugar moiety is halo, 3’-O-R’, or 3’-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a C1-C4alkyl. In some embodiments, the modified 3’-position is a 3’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, the 3’-position of the sugar moiety of the nucleoside substrate is -O- R’, wherein R’ is -CH3 or -CH2CH3 or -CH2CH2OCH3. In some embodiments, the modified 3’-position is 3’- O-(2-methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O-ethylamine, 3’-O-cyanoethyl, -3’-O-amine, or 3’-O- acetate ester.Docket Number CX10-269WO4

[0353] In some embodiments, the modifications at the 3’-position is a reversible or cleavable 3’-blocking group. In some embodiments, removal or cleaving of the reversible or cleavable 3’-blocking group results in a free 3’-OH group, which in some embodiments can serve as an acceptor for single-stranded RNA ligase or a terminal nucleotidyl transferase. In some embodiments, exemplary reversible or cleavable 3’-blocking groups include, among others, 3’-O-azidomethyl, 3’-O-(2-methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O- ethylamine, 3’-O-cyanoethyl, -3’-O-amine, 3’-O-acetate ester, 3’-phosphate, 3’-diphosphate, or 3’- triphosphate. In some embodiments, the 3’-blocking group is paired with the corresponding deblocking agent used in the deblocking or cleavage of the 3’-blocking group. Other reversible or cleavable 3’-blocking groups are described in International patent publication WO2023183569, incorporated by reference herein.

[0354] In some embodiments, a modification at the 3’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker’ or a linker, as described herein.

[0355] In some embodiments, the modified sugar moiety comprises an unlocked nucleoside. In some embodiments, in the unlocked nucleoside, the furanosyl ring is opened to result in the structure below:where B represents the nucleobase. are described in, among others, International patent publication WO2022 / 098990 and Snead et al., Molecular Therapy-Nucleic Acids, 2013, 2, e103. Modified nucleobases

[0356] In some embodiments, the oligonucleotide acceptor and / or nucleotide donor comprises one or more nucleosides comprising a modified nucleobase. In some embodiments, modified nucleobase that is capable of hydrogen bonding to form Watson and Crick type base pairing is selected. In some embodiments, some positions for possible modifications on naturally occurring nucleobases are indicated below: Nucleobase Modification PositionsDocket Number CX10-269WO4 Nucleobase Modification Positions

[0357] In some embodiments, the nucleobase comprise an inosine nucleoside (i.e., nucleosides comprising a hypoxantine nucleobase). Ln some embodiments, the modified nucleobase is 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2. N-6 and O-6 substituted purines. In some embodiments, the modified nucleobase is 2-aminopropyladenine.5- hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6- N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine.5-propynyl uracil, 5- propynylcytosine.6-azouracil, 6-azocytosine, 6-azothymine.5-ribosyluracil (pseudouracil), 4-thiouracil.8- halo purine, 8-amino purine, 8-thio purine, 8-thioalkyl purine, 8-hydroxy purine, 8-aza purine, 5- bromocytosine.5-trifluoromethylcytosine, 5-halouracil, 5-halocytosine, 7-methylguanine, 7-methyladenine, 2- F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine.3-deazaguanine, 3-deazaadenine, 6-N- benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, and 5-methyl 4-N-benzoyluracil. Further modified nucleobases include tricyclic pyrimidines, e.g., 1,3-diazaphenoxazine-2-one.1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1.3- diazaphenoxazine-2-one (G-clamp).Docket Number CX10-269WO4

[0358] In some embodiments, the modified nucleobase includes, among others, nucleobases based on 2,4- dihalotolene and benzimidazole groups. In some embodiments, the modified nucleobase is 4- methylbenzimidazole, 2,4-difluorotoluene, 9-methylimidazo[(4,5)-b]pyridine, 2,4-dibromotoluene, benzimidazole, 5-nitrobenzimidazole, 6-nitrobenzimidazole, and 5-nitroindole. In some embodiments, the modified nucleobase is 7-azaindole, and isocarbostyril (see, e.g., Berdis et al., Front. Chem.10:1051525). Other modified nucleobases are described in, among others, patent publication WO2021249993.

[0359] In some embodiments, included within modified nucleobase is a nucleobase that does not have a nucleobase, also referred to as an abasic nucleoside. In some embodiments, the abasic nucleoside is present in the internal portion of an oligonucleotide acceptor. In some embodiments, an abasic nucleoside is attached to the 3’- or 5’-terminal end, which is in certain embodiments grouped as a terminal group.

[0360] In some embodiments, the modified nucleobase is present on the 5’-terminal nucleoside of the oligonucleotide acceptor or oligonucleotide donor, 3’-terminal nucleoside of the oligonucleotide acceptor or oligonucleotide donor, and / or present on the internal nucleosides of the oligonucleotide acceptor or oligonucleotide donor. In some embodiments, the blocks or contiguous stretches of nucleosides in the oligonucleotide acceptor or oligonucleotide donor have modified nucleobases. Terminal groups

[0361] In some embodiments, the oligonucleotide acceptor and / or nucleotide donor comprises a terminal group. In some embodiments, the oligonucleotide acceptor comprises a terminal group at the 5’-terminal nucleoside. In some embodiments, the terminal group is attached to the 5’-OH or 4’-carbon atom of the terminal nucleoside.

[0362] In some embodiments, the terminal group comprises a C-4’ modification of the 5’-terminal nucleoside, including among others, 4’-thio-C2’ modifications, 4’-aminoalkyl, C4’-guanidino-C2’- modifications, and C4’-O-methyl (see, e.g., Gangopadhyay et al., RNA Biology, 2022, 19:1, 452-467)

[0363] In some embodiments, the 5’-terminal group is a 5’-phosphate modification. In some embodiments, the 5'-phosphate modification, includes, among others, 5’-C-methyl, particularly S isomer; 5’-(E or Z)- vinylphosphonate, or 5’-methylenephosphonate.

[0364] In some embodiments, the 5’-terminal group comprises an abasic nucleotide attached to the 5’-OH. In some embodiments, the 5’-terminal groups comprises an inverted abasic nucleotide (5’-5’) attached to the 5’-OH of the 5’-end nucleoside.

[0365] In some embodiments, the nucleotide donor comprises a 3’-terminal group. In some embodiments, the 3’-terminal group comprises a 3’-phosphate, which can also function as a reversible blocking group. In some embodiments, the 3’-phosphate is modified, such as with 3’-(E or Z)-vinylphosphonate, or 3’- methylenephosphonate. In some embodiments, the 3’-terminal group on the nucleotide donor comprises an abasic nucleoside. In some embodiments, the 3’-terminal group comprises an inverted abasic nucleotide (3’- 3’). Internucleoside linkagesDocket Number CX10-269WO4

[0366] In some embodiments, the modified oligonucleotide, e.g., oligonucleotide acceptor and / or oligonucleotide donor, comprises at least one modified, non-naturally occurring internucleoside linkage. In some embodiments, the modified oligonucleotide has 1%, 2%, 5%, 10% 20%, 30%, 40%, 50%, or 60% or more modified internucleoside linkages. In some embodiments, all of the internucleoside linkages are modified internucleoside linkages.

[0367] In some embodiments, the modified internucleoside linkage is a phosphorous containing modified internucleoside linkage. Exemplary phosphorous-containing internucleoside linkages include, among others, phosphotriesters, alkylphosphonates (e.g., methyl phosphonate, ethyl phosphonate, etc.), phosphoramidates, phosphorothioate, and phosphorodithioate.

[0368] In some embodiments, the modified internucleoside linkage is a non-phosphorous containing internucleoside linkage. Exemplary non-phosphorous containing internucleoside linkages include, among others, methylenemethylimino (-CH2-N(CH3)-O-CH2), thiodiestcr, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); N,N’-dimethylhydrazine (-CH2-N((CH3)-N((CH3)-); MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacctal (3’-S-CH2-O-5'). In some embodiments, the modified internucleoside linkage is amide linkage, such as those of glycine nucleosides or nucleoside β-amino acids (see, e.g., Banerjee et al., Bioconjugate Chem., 2015, 26, 8, 1737–1742).

[0369] In some embodiments, the modified internucleoside linkages provides for a chiral center. For example, a phosphorothioate or alkylphosphonate internucleoside linkage can be in the Rp or Sp stereomeric configuration. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have a mixture of stereoisomers in the internucleoside linkage. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have greater than 50% of the internucleoside linkages as Rp or Sp configuration. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have at least 60%, 70%, 80%, 90%, or greater of Rp or Sp stereomeric configuration.

[0370] In some embodiments, the modified internucleoside linkages are present in the 5’-terminal region of the oligonucleotide acceptor and / or oligonucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified internucleoside linkages are present at the 5’-terminal region of the oligonucleotide acceptor and / or oligonucleotide donor. In some embodiments at least 1 or 2 phosphorothioate internucleoside linkages are present at the 5’-terminal region of the oligonucleotide acceptor and / or oligonucleotide donor. In some embodiments, the phosphorothioate linkage is a non-bridging phosphorothioate internucleoside linkage.

[0371] In some embodiments, the modified internucleoside linkages are present in the 3’-terminal region of the oligonucleotide acceptor or oligonucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified internucleoside linkages are present at the 3’-terminal region of the oligonucleotide acceptor and / or oligonucleotide donor. In some embodiments, at least 1 or 2 phosphorothioate internucleoside linkages are present at the 3’-terminal region of an oligonucleotide acceptor or oligonucleotide donor.

[0372] In some embodiments, the modified internucleoside linkages are present in the internal portions of the oligonucleotide acceptor or oligonucleotide donor.Docket Number CX10-269WO4

[0373] In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor comprises at least a phosphorothioate internucleoside linkage, where the phosphorothioate linkage is in the Sp configuration, the Rp configuration, or a mixture of Sp and Rp configurations in the population of the oligonucleotide acceptor and / or oligonucleotide donor. Conjugate moiety

[0374] In some embodiments, the oligonucleotide acceptor, or nucleotide donor comprises a conjugate moiety. In some embodiments, the nucleotide donor comprises a conjugate moiety that is compatible with single-stranded RNA ligase activity and that does not interfere with the nucleotide donor acting as a substrate for the single-stranded RNA ligase.

[0375] In some embodiments, the conjugate moiety (i.e., non-nucleotide moiety) includes, among others, carbohydrates (e.g. GalNAc), lipids, sterols, drug substances, hormones, polymers (e.g., polyethylene glycol, etc.), proteins, peptides, toxins (e.g. bacterial toxins, etc.), vitamins (e.g., folate, tocopherol, retinoic acid, etc.), or combinations thereof. In some embodiments, the conjugate moiety is used to affect the pharmacokinetics of the oligonucleotide and / or oligonucleotide cell targeting.

[0376] In some embodiments, the conjugate moiety can be attached to the 5’-terminal nucleotide, the 3’- terminal nucleotide, or in an oligonucleotide an internal nucleotide. In some embodiments, the conjugate moiety is attached the 2’-position of the sugar moiety of a nucleoside, for example, to the 2’-OH. In some embodiments, the conjugate moiety is attached to the 3’-position of the sugar moiety of the nucleoside, for example 3’-OH. In some embodiments, the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res.2019 Feb 20; 47(3): 1082–1096). In some embodiments, the conjugate moiety is attached directly or attached using a linker.

[0377] In some embodiments, the conjugate moiety comprises a C6-C22 alkyl, C6-22 alkenyl, or C6-C22 alkynyl. In some embodiments, the conjugate moiety comprises a C6-alkyl, C7-alkyl, C8-alkyl, C9-alkyl, C10- alkyl, C11-alkyl, C12-alkyl, C13-alkyl, C14-alkyl, C15-alkyl, C16-alkyl, C17-alkyl, C18-alkyl, C19-alkyl, C20-alkyl, C21-alkyl, or C22-alkyl. In some embodiments, the conjugate moiety comprises a C6 alkenyl, C7 alkenyl, C8 alkenyl C9alkenyl, C10alkenyl, C11-alkenyl, C12-alkenyl, C13-alkenyl, C14-alkenyl, C15-alkenyl, C16-alkenyl, C17-alkenyl, C18-alkenyl, C19-alkenyl, C20-alkenyl, C21-alkenyl, or C22-alkenyl. In some embodiments, the conjugate moiety comprises a C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11-alkynyl, C12- alkynyl, C13-alkynyl, C14-alkynyl, C15-alkynyl, C16-alkynyl, C17-alkynyl, C18-alkynyl, C19-alkynyl, C20- alkynyl, C21-alkynyl, or C22-alkynyl.

[0378] In some embodiments, the conjugate moiety comprises a heteroalkyl, heteroalkenyl, or heteroalkynyl. In some embodiments, the heteroalkyl, heteroalkenyl or heteroalkynyl has one or more carbon atoms replaced with a heteroatom, such as O, S, or N.

[0379] In some embodiments, the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group. In some embodiments, the cycloalkyl includes, among others, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. In some embodiments, the heterocycloalkyl includes, among others, 1-(1,2,5,6-tetrahydropyridyfh l-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-Docket Number CX10-269WO4 morpholinyl, tctrahydrofuran-2-yl, tctrahydrofuran-3-yl, tetrahydrothicn-2-yl, tetrahydrothien-3-yl, l- piperazinyl, and 2-piperazinyl.

[0380] In some embodiments, the conjugate moiety comprises an aryl or heteroaryl moiety. In some embodiments, the aryl group includes, among others, phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, anthracenyl, fluorenyl, indenyl, and azulenyl. In some embodiments, a heteroaryl group includes, among others, pyridyl, furanyl, thienyl, pynolyl, oxazolyl, oxadiazolyl, imidazolyl ihiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isoihiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, and indazolyl.

[0381] In some embodiments, the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl-heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, heteroarylalkenyl-, cycloalkylheteroalkenyl-heterocycloalkylheteroalkenyl-, arylheteroalkenyl-, or heteroarylheteroalkenyl-.

[0382] In some embodiments, the conjugate moiety comprises a lipid or lipophilic moiety, for example a fatty acid. In some embodiments, the fatty acid comprises a saturated fatty acid, unsaturated fatty acid, or a polyunsaturated fatty acid. In some embodiments, the fatty acid comprises caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans- vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, eicosapentaenoic acid, decanoic acid, docosahexaenoic acid (DHA), and docosanoic acid (DCA) conjugate moieties (see, e.g., Kubo et al., ACS Chem. Biol., 2021, 16, 150−164; see also, WO2024 / 040041; incorporated herein by reference).

[0383] In some embodiments, the conjugate moiety comprises a sterol. In some embodiments, the sterol comprises cholesterol, alpha-cholesterol, cholesterol ester (e.g., cholesteryl palmitate, etc.), cholesterol sulfate, phytosterol, cholic acid, or lithocholic acid.

[0384] In some embodiments, the conjugate moiety comprises a phospholipid. In some embodiments, the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.

[0385] In some embodiments, the conjugate moiety comprises a carbohydrate, particularly a carbohydrate moiety acting as a ligand for a cellular receptor for cellular targeting of the oligonucleotide. In some embodiments, the carbohydrate moiety comprises galactose or galactose derivatives. In some embodiments, the carbohydrate moiety is attached to the nucleoside via a linker. In some embodiments, exemplary carbohydrates that can be used include the following. ,Docket Number CX10-269WO4 ,

[0386] In some embodiments, the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety. In some embodiments, the oligonucleotide acceptor and / or nucleotide donor may be conjugated to at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the conjugate moiety is a monovalent, divalent, trivalent or tetravalent, GalNAc.

[0387] In some embodiments, the GalNAc moiety has the following structure,where L is a linker, and W is a heteroatom (e.g., O or N). In some embodiments, the W is the 2’-OH of the sugar moiety of a nucleoside. In some embodiments, the L includes a 3’-phosphate at the 3’-terminal nucleoside. An exemplary monovalent GalNAc moiety iswherein the monovalent GalNAc is attached via the linker to the 2’-position of a nucleoside, such as adenine or guanine. These conjugate moieties can be present in contiguous nucleotides in an oligonucleotide (see, e.g., WO2024 / 040041).Docket Number CX10-269WO4

[0388] In some embodiments, the conjugate moiety is a trivalent GalNAc. Tri-valent N-acetylgalactosamine conjugate moieties are described in, for example, WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620. The term “trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker. Exemplary trivalent GalNAc conjugate moiety is depicted below:Docket Number CX10-269WO4

[0389] In some embodiments, the conjugate is GalNAc targeting moiety L96.

[0390] In some embodiments, the conjugate moiety comprises a reporter molecule. Examples of reporter molecules include, among others, fluorescent moieties, such as fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6- carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X- rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), IRDyes (e.g., IRD40, IRD 700, IRD 800). (See, e.g., “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., R.P. Haugland, 2002, Molecular Probes, Inc., Eugene, Oregon)

[0391] In some embodiments, the reporter moiety is a chemiluminescent moiety, for example acridinium esters, ruthenium derivatives (e.g., tris(2,2′-bipyridyl) ruthenium), and dioxetanes.

[0392] In some embodiments, the conjugate moiety comprises an affinity or capture tag. Exemplary affinity or capture tag includes, among others, biotin, desthiobiotin, digoxigenin, 3-amino-3-deoxydigoxigenin, and a hapten (e.g., dinitrophenol, Alexa Fluor 40, Alexa Fluor 488, dansyl, Lucifer yellow, Oregon Green 488, fluorescein).Docket Number CX10-269WO4

[0393] In some embodiments, the conjugate moiety comprises a peptide. In some embodiments, the peptide comprises a cellular targeting peptide and / or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate modified oligonucleotide. In some embodiments, the cell penetrating peptide is attached via a linker, including a cleavable linker. Cell penetrating peptides, include among others, TAT, penetratin, MAP, transportan / TP10, VP22, polyarginine, MPG, Pep-1, pVEC, YTA2, YTA4, M918, and CADY. In some embodiments, the conjugate moiety comprises an RGD (Arg-Gly-Asp) peptide. Sequence of some penetrating peptides are described in Copolovici et al., 2014, 8(3):1972–1994 and some are provided below: CPP Peptide Peptide Sequence TAT GRKKRRQRRRPPQ t ti R IKIWF NRRMKWKK, , , g others, patent publications WO24063570, WO24044663, US2024083949, WO24026141, WO23230600, WO23219933, WO23177261, WO23178327, WO23093960, WO23086342, WO23081893, WO23069332, WO23070108, WO23034515, US2023248630, US2023053924, WO23003380, WO23277628, WO23277575, US2022378946, WO22171972, WO22162200, WO2020144233, WO22180242, WO22132520, WO22129926, WO22125673, WO22120276, WO22101193, US2023287086, US2023357334, US2023144488, and US2023048338; incorporated by reference herein. In some embodiments, the peptide can be attached using a thiol group on the 5’-phosphate of a polynucleotide or oligonucleotide.

[0395] Exemplary nucleotides / nucleosides with conjugate moieties are shown below:Docket Number CX10-269WO4 andwherein R1is H or phosphate group, R2is H or phosphate group, or blocking group, and R3is H, OH, fluoro, or -O-methyl. Reactive Moiety

[0396] In some embodiments, the modification comprises a reactive group that is conjugated to a nucleoside. In some embodiments, the reactive group is attached to the nucleoside via a linker. In some embodiments, the reactive group is a cyano, azido, alkynyl, amino, carboxyl, sulfhydryl, dibenzocyclooctynyl, vinyl, trans-Docket Number CX10-269WO4 cyclooctene, or tetrazine. In some embodiments, the reactive group is those used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below: amino

[0397] Other reactive groups used in click chemistry, particularly for nucleic acids, is described in Fantoni et al., Chem. Rev.2021, 121, 7122−7154, incorporated by reference herein.

[0398] Exemplary single stranded RNA ligase substrates with a reactive moiety is provided below:Docket Number CX10-269WO4wherein R1is H or phosphate; R2is a blocking group or H;; and R3is H, -OR, or halo, e.g., F, Br, or Cl. Linker

[0399] In some embodiments, as described above, the conjugate moiety or reactive moiety is attached to the nucleoside or the terminal group through a linker. Various linkers are known in the art for conjugating chemical groups to nucleosides and phosphate groups.

[0400] In some embodiments, mixtures of linkers are used. In some embodiments, different linker types are connected to form a longer linker or linkers with branched or dendritic structure. For example, an alkylene linker is connected to a polyethylene linker through a functional group, e.g., an amide; an arylene linker is attached to an alkylene linker. As such, different combinations of linker types can be connected to provide for longer linkers and / or branched linkers, for example for attaching multiple conjugate moieties.

[0401] In some embodiments, linkers include, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene, heteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroarylheteroalkylene,Docket Number CX10-269WO4 and heteroarylalkenylene. In some embodiments, the linker comprises substituted or unsubstituted C2-C22 alkylene, heteroalkylene, or polyethylene glycol. In some embodiments, the linkers have functional groups for conjugation.

[0402] In some embodiments, the linker comprises a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms, 1 to 20 carbon atoms, or 1 to 14 carbon atoms, wherein one or more of the carbon atoms in the hydrocarbon chain is optionally replaced by -O-, -NR1- , -NR1-C(=O)-, -C(=O)-NR1, or -S-, and wherein R1is hydrogen or (C1-C6)alkyl, wherein the hydrocarbon chain, is optionally substituted with one or more (e.g.1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0403] In some embodiments, the L is attached to the nucleoside and / or conjugate through -NH-, -O-, -S-, - (C═O)-, -(C═O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C═O)-NH-, or -NH-(SO2)-.

[0404] In some embodiments, the L has the structure below: ,Docket Number CX10-269WO4

[0405] In some embodiments, the linker comprises a substituted or unsubstituted polyethylene glycol linker. In some embodiments, the polyethylene glycol linker has the formula:

[0406] In some embodiments, n is 2-24. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0407] In some embodiments, the polyethylene linker has the structure below: ,,

[0409] In some embodiments, the linker is a cleavable linker in which the linker can be cleaved, for example to detach a conjugate moiety. Example of a cleavable linker includes, by way of example and not limitation, a disulfide linkage, enzymatically cleavable linkers (e.g., peptide linkers), and photocleavable linkers (see, e.g.,Docket Number CX10-269WO4 Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2ndEd., S.S. Mark ed., 2011, Humana Press).

[0410] In some embodiments, bifunctional linkers can be used to attach a conjugate moiety to the linker and attach the linker-conjugate to the nucleoside or vice versa (see, e.g., Hermanson, G., supra; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, supra). In some embodiments, an activating group can be attached to an atom to activate the atom to form a covalent bond with another reactive group. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When an activating group is attached to an oxygen atom of a carboxylic acid, the activating group can be a group that is derivable from a known coupling reagent. Examples of such coupling reagents include, but are not limited to, N,N′- dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N′- ethylcarbonate (EDC), (denzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-1-yl- N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU). Pyrophosphatase coupled reactions and pyrophosphatases

[0411] In some embodiments, the single-stranded RNA ligase reaction further includes a pyrophosphatase for degrading pyrophosphate generated in the single-stranded RNA ligase reaction. In some embodiments, the presence of a pyrophosphatase in the ligase reaction can enhance the forward reaction for attachment of the nucleotide donor to the oligonucleotide acceptor by degrading the pyrophosphate generated in the ligase reaction.

[0412] In some embodiments, the pyrophosphatase is present concurrently with the single-stranded RNA ligase. In some embodiments, where the single-stranded RNA ligase is immobilized on a substrate medium, the pyrophosphatase can be free in solution, co-immobilized with the single-stranded RNA ligase on the substrate medium, or immobilized on a substrate medium separately from the single-stranded RNA ligase. In some embodiments, the pyrophosphatase and the single-stranded ligase can be prepared as a fusion protein, and in some embodiments, immobilized on a support medium.

[0413] In some embodiments, a pyrophosphatase can also be used in conjunction with the attachment of a nucleotide substrate to the oligonucleotide using a terminal nucleotidyl transferase. In some embodiments, the pyrophosphatase can be free in solution in the terminal nucleotidyl transferase reaction, co-immobilized with the terminal nucleotidyl transferase on the substrate medium, or immobilized on a substrate medium separately from the terminal nucleotidyl transferase.

[0414] In some embodiments, a wide variety of pyrophosphatases can be adapted for the coupled reactions. In some embodiments, the pyrophosphatase is a Type 1 group of pyrophosphatases. In some embodiments, the pyrophosphatase is a Type II group of pyrophosphatases. In some embodiments, pyrophosphatases useful in coupled reactions with single-stranded RNA ligase or terminal nucleotidyl transferase reaction are disclosed in U.S. provisional application titled “Uses of Type II Inorganic Pyrophosphatases, filed April 16, 2024, incorporated by reference herein in its entirety.Docket Number CX10-269WO4 ATP Regeneration Systems

[0415] In some embodiments, the RNA ligase reaction further includes a nucleotide substrate regeneration system to regenerate ATP used as a co-factor in the RNA ligase reaction. In some embodiments, the ATP regeneration system is used to convert product AMP to ATP. In some embodiments, the regeneration of ATP is used to increase ligated product formation (e.g., product yield) in the single stranded RNA ligase reaction.

[0416] In some embodiments, the ATP recycling or regeneration system includes a nucleoside monophosphate kinase for converting AMP to ADP in presence of a phosphate donor. Various nucleoside monophosphate kinases can be used for the conversion of AMP to ADP, including various homologs of nucleoside monophosphate kinases. In some embodiments, more than one nucleoside monophosphate kinase can be used in the regeneration system. In some embodiments, the nucleoside monophosphate kinase is an adenosine monophosphate kinase (e.g., adenylate kinase), cytidine monophosphate (CMP) kinase, uridine monophosphate (UMP) kinase, and / or guanylate-monophosphate (GMP) kinase.

[0417] In some embodiments, a nucleoside monophosphate kinase useful in the ATP regeneration reaction is a cytidine monophosphate kinase. Various suitable cytidine monophosphate kinases are known in the art. These include homologs of cytidine monophosphate kinases. In some embodiments, a cytidine monophosphate kinase useful in the regeneration reactions include, among others, the cytidine monophosphate kinase of Thermus thermophilus (Q5SL35), Pyrococcus furiosus (Q8U2L4), Pseudomonas putida (AFO48857.1), Escherichia coli K-12 MG1655 (P0A6I0), Clostridium acetobutylicum (Q97I08), Halobacterium salinarum (Q9HPA5) Bacillus acidicola (WP_066270173), Acetobacter aceti (WP_010667744), Acidithiobacillus thiooxidans (WP_024892761.1), Acidithiobacillus ferrooxidans (WP_064220349.1), Metallosphaera sedula (WP_011921264.1), Amphibacillus xylanus (WP_015009966.1) Thioalkalivibrio denitrificans (WP_077278466.1), Vibrio psychroerythus (Q482G4), Pseudoalteromonas haloplanktis (Q3ILA1), Psychrobacter arcticus (Q4FRL5), Psychromonas ingrahamii (A1SZ01), Pseudomonas syringae (xQ4ZQ97) and Halobacterium salinarum (Q9HPA5).

[0418] In some embodiments, a nucleoside monophosphate kinase useful in the ATP regeneration reactions is a uridine monophosphate kinase. Various suitable uridine monophosphate kinases are known in the art. These include homologs of uridine monophosphate kinases. In some embodiments, a uridine monophosphate kinase useful in the regeneration reactions includes, among others, the uridine monophosphate kinase of Pyrococcus furiosus (Q8U122), Thermus thermophilus (P43891), Pseudomonas putida (I7BW46), Escherichia coli K-12 MG1655 (P0A7E9), Aspergillus niger (A2R195), Saccharomyces cerevisiae (P15700), Clostridium acetobutylicum (Q97I64) ATCC 824 PyrH Halobacterium salinarum (Q9HNN8), Picrophilus torridus (WP_048059653), Metallosphaera sedula (WP_012021705), Thermoplasma acidophilum (WP_010900913), Sulfolobus solfataricus (WP_009992427), Acetobacter aceti (WP_042788648), Thioalkalivibrio sp. HK1 (WP_081759172.1), Amphibacillus xylanus (WP_015010200.1), Vibrio psychroerythus (Q485G8), Pseudoalteromonas haloplanktis (Q3IIX6), Psychrobacter arcticus (Q4FRH5), Psychromonas ingrahamii (ABM04676.1), Pseudomonas syringae (Q4ZWS6), and Halobacterium salinarum (Q9HNN8).

[0419] In some embodiments, the nucleoside monophosphate kinase useful in the ATP regeneration reactions is a guanosine monophosphate kinase (guanylate kinase). Various suitable guanylate kinases are known in theDocket Number CX10-269WO4 art. These include homologs of guanylate kinases. In some embodiments, a guanylate kinase useful in the regeneration reactions includes, among others, the guanylate kinase of Thermotoga maritima (Q9X215), Thermus thermophilus (Q5SI18), Pseudomonas putida (I7C087), Escherichia coli K-12 (P60546), Aspergillus niger (A2QPV2), Saccharomyces cerevisiae (P15454), Clostridium acetobutylicum (Q97ID0), Acidithiobacillus ferrooxidans (WP_064219869.1), Acidithiobacillus thiooxidans (WP_010637919.1), Bacillus acidicola (WP_066264774.1), Acetobacter aceti (WP_018308252.1), Amphibacillus xylanus (WP_015010280.1), Thioalkalivibrio sulfidiphilus (WP_018953989.1), Vibrio psychroerythus (Q47UB3), Pseudoalteromonas haloplanktis (Q3IJH8), Psychrobacter arcticus (Q4FQY7), Psychromonas ingrahamii (A1T0P1), and Pseudomonas syringae (Q4ZZY8).

[0420] In some embodiments, the nucleoside monophosphate kinase useful in the ATP regeneration reactions is an adenosine monophosphate kinase (adenylate kinase). Various suitable adenylate kinases are known in the art. These include homologs of adenylate kinases. In some embodiments, the adenylate kinase is a bacterial, fungal, plant, or animal adenylate kinase. In some embodiments, an adenylate kinase useful in the regeneration reactions includes, among others, adenylate kinases of Thermus thermophilus (Q72125), Pyrococcus furiosus (Q8U207), Pseudomonas putida (17CAA9), Escherichia coli K - 12 W3110 (P69441), Aspergillus niger CBS 513.88 (A2QPN9), Saccharomyces cerevisiae (P07170), Clostridium acetobutylicum (Q97E39), Halobacterium salinarum (Q9HPAT), Acidithiobacillus thiooxidans (WP_024894015.1), Acidithiobacillus ferrooxidans (WP_064218420.1), Bacillus acidicola (WP_066267988.1), Sulfolobus solfataricus (WP_009991241.1), Saccharomyces cerevisiae (P07170), Thermotoga neapolitana (Q8GGL2), Escherichia coli (P69441) and Geobacillus stearothermophilus (WP_049624206.1). In some embodiments, the adenylate kinase is an engineered adenylate kinase described in International patent application No. PCT / US2024 / 051084, filed October 11, 2024, incorporated herein by reference.

[0421] In some embodiments, the ATP regeneration system includes at least an enzyme and a phosphate donor for the conversion of ADP to ATP. In some embodiments, the ATP regeneration system includes, among others, an acetate kinase, adenylate kinase, pyruvate kinase, creatine kinase, or polyphosphate kinase (see, e.g., Endo et al., Adv. Synth. Catal., 2002, 343:521–526; Andexer et al., Chem Bio Chem., 2015, 16:380–386).

[0422] In the ATP regeneration system, the phosphate donor for the conversion of ADP to ATP is selected based on the ATP regenerating enzyme employed. By way of example and not limitation, if acetate kinase enzyme is used for conversion of ADP to ATP, the phosphate donor is acetyl-phosphate. If pyruvate kinase is used for the conversion of ADP to ATP, the phosphate donor is phosphoenolpyruvate. If creatine kinase is used for the conversion of ADP to ATP, the phosphate donor is creatine phosphate. If polyphosphate kinase is used for the conversion of ADP to ATP, the phosphate donor is inorganic polyphosphate.

[0423] In some embodiments, the ATP regenerating system includes pyruvate kinase and phosphoenolpyruvate. In some embodiments, the ATP regenerating system includes creatine kinase and creatine phosphate. In some embodiments, the ATP regenerating system includes polyphosphate kinase and inorganic polyphosphate. In some embodiments, the ATP regenerating system includes acetate kinase and acetyl phosphate.Docket Number CX10-269WO4

[0424] In some embodiments, the ATP regenerating system includes acetate kinase and acetyl phosphate, where the acetate kinase is an acetate kinase of Escherichia coli str. K-12 substr. MG1655 (NP_416799.1), Corynebacterium jeikeium K411 (WP_011272972.1), Lactococcus cremoris subsp. cremoris KW2 (WP_011835968.1), Lactococcus lactis (WP_004254593.1), Marinitoga sp.38H-ov (WP_165147355.1), Thermotoga sp. KOL6 (WP_101510533.1), Thermosipho melaniensis (WP_012057479.1), Thermotoga sp. RQ7 (WP_041844042.1), and Thermosipho africanus (WP_004102380.1). In some embodiments, the acetate kinase is an engineered acetate kinase described in, among others, International patent application No. PCT / US2024 / 051118, filed October 11, 2024, incorporated by reference herein. Single-stranded RNA (ssRNA) Ligases, Polynucleotides Encoding the Ligases, and Host Cells Single-stranded RNA (ssRNA) ligase polypeptides

[0425] In some embodiments, the ligation reactions are mediated by a single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligase comprises a viral single-stranded RNA ligase, a bacterial single-stranded RNA ligase, a fungal single-stranded RNA ligase, or a mammalian single-stranded RNA ligase.

[0426] In some embodiments, the single-stranded ligase is a bacteriophage single-stranded RNA ligase. In some embodiments, the bacteriophage single-stranded RNA ligase is single-stranded RNA ligase of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Phage TS2126, or Rhodothermus phage RM378.

[0427] In some embodiments, the single-stranded RNA ligase is a bacterial single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligase is an archael single-stranded RNA ligase (see, e.g., Nucleic Acids Res.2008 Nov; 36(19): 6218–6227). In some embodiments, the single-stranded RNA ligase is a Methanobacterium or Thermococcus single-stranded RNA ligase.

[0428] In some embodiments, exemplary bacterial single-stranded RNA ligase is a single-stranded RNA ligase of Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, or Thermovibrio ammonificans HB-1.

[0429] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

[0430] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.Docket Number CX10-269WO4

[0431] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 4, or to a reference sequence corresponding to SEQ ID NO: 4.

[0432] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 6, or to a reference sequence corresponding to SEQ ID NO: 6.

[0433] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8.

[0434] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 4, or to a reference sequence corresponding to SEQ ID NO: 10.

[0435] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12.

[0436] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

[0437] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 16, or to a reference sequence corresponding to SEQ ID NO: 16.

[0438] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket Number CX10-269WO4 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 18, or to a reference sequence corresponding to SEQ ID NO: 18.

[0439] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 20, or to a reference sequence corresponding to SEQ ID NO: 20.

[0440] In some embodiments, the single-stranded RNA ligase comprises one or more amino acid differences relative to the reference sequence corresponding to a sequence from amino acid residues 12 to the carboxyl terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-20, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-20. In some embodiments, the amino acid differences are based on alignment of the amino acid sequence of the naturally occurring single-stranded RNA ligases, and changing the amino acid residue of one sequence to the different amino acid residue present in the amino acid sequence of another naturally occurring single-stranded RNA ligase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence.

[0441] In some embodiments, the recombinant single-stranded RNA ligase comprises an amino acid sequence comprising amino acid residues 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

[0442] In some embodiments, the single-stranded RNA ligase comprise variants of single-stranded RNA ligase of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1.

[0443] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. In some embodiments, the sequence corresponding to amino acid residues 12 to the carboxy terminal residue is amino acid residues 12 to 387 of the referenced SEQ ID NO.

[0444] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,Docket Number CX10-269WO4 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0445] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0446] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0447] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0448] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167,Docket Number CX10-269WO4 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0449] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G / W, 27A / F / W / Y, 30R, 31I, 32M, 33R, 34G / K / N / R / S / Y, 35A / L / S, 36D / R, 38M / T / V, 40I / L, 41V, 42A, 43G / T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I / L / T / V, 56L / M, 58M, 65P / Q / S, 66A / K / P / Q / R / T, 69A / C, 84L, 88W, 90E / S, 91D / L, 92I, 93A / H / N / P / R, 94D, 97L / P / V, 109G / S, 113D / G, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 123G / S, 125Q / R / T / V, 127I / L / P / Q / V, 130I, 135I / M, 138A, 139R, 141A / D / G / P / R, 144T, 145Y, 146I, 151E / L, 152D, 156A / S, 157L, 160R, 161V, 162S / W, 165F / K / M / P / T, 166H / P / S, 167I / V, 168I / S, 170R, 171P / R / S, 172P, 173E / N, 174P, 177C / I / L / V, 181V, 185E, 190L, 195T, 196D, 197E / T, 198A, 199L / T, 203V, 204E, 205M / T / V, 207E, 212H / S, 213V, 214A / C / E / S, 217G / P, 220A / P / R / S / V, 221P, 222F / G, 223A / G / N / S / T, 224P, 225G / H / P / R / T, 226A / P / Q / T, 229A / L, 230L, 231I, 236H, 237A / G / R, 238G, 240V / W, 246F / I, 248F / H / M, 252R, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 260I / L / R / T / V, 263D / S, 268A / E / R, 269L, 269F / L, 271G / H / M, 272G / L / R / S, 273A / L / S / Y, 274I, 275E / W, 276A / G / H, 277T, 278L / P / R, 279Q / R, 280S, 281A / I / L / M / V / W, 282L, 283K / L / Q / T, 284F / L, 285A / G / P / R / S, 286P / Q, 288T, 289Q, 291L, 295A / R / T, 297C, 299I / R, 302I, 303A / G, 306F / M, 310A / D / G, 311C / F / G / I, 314E / G / L / P / S, 316R, 320A / G, 323D / E / G / N / T / Y, 324T, 325M / P, 326G / M / T / W, 330H / I, 332G / L, 333E, 334I / N / S, 336C / E / N, 337E / K, 340C, 341A / P / S, 343D / P, 345G / T, 346G / P, 347A / E / G / I / L / M / P / R / S / T, 348A / L / S / T, 349S / V, 350A / C / R, 351F / N / Y, 352G / L / M / N / R / S / V, 353T / V, 354E / H / Q / S / V, 355K, 356L / M / V, 357L / M, 358C / D / G / P / R / T / V, 359D / I, 360L / V, 361I, 362A / E / K / Q / R, 363S / V, 365N, 366W, 368G, 369C / L / V, 371R / S, 372A / L / P, 374F / I / L / V, 376E / T, 377L, 380S, 381K / R, or 384C / S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0450] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue V31I, L32M, E33R, R34S / Y, N36D, V38M / T / V, V40L, S41V, Y42A, R43G / T, Y44R, N45Y, A46R, F48W, V49I, F50W, L54T, R56L, V84L, R91D, L92I, R93H / N, G94D, P109G / S, N113G, K115E, L118F / M, Q121K, K122C / I / L / M / S / T / V / Y, G123S, S125Q / R / T / V, P127L / Q / V, E130I, L135I, K138A, N141D, T146I, N151E, K152D, P156A / S, L160R, I161V, S162W, F165F / K / M / P / T, Q166H / P / S, L167I / V, V168I / S, P170R, Y171P / R / S, K173E / N, D174P, F177I / L, I181V, K185E, N196D, K199L, I203V, K204E, I205M / T / V, R207E, R212S, L213V, K214E, L217G / P, I220A / P / R / S / V, E221P, N222F / G, R223A / G / N / S, E224P, S225G / H / P / R / T, I226A / P / Q / T, F229A / L, V230L, L231I, K236H, K237G, E238G, F240V, V246F / I, S248F / H / M, V252R, H254A / D / R / S, F255A / H / M / Q / V / W, Y256L / M / R / V, F258L, T259A / S, Y260I / L / R / T / V, N263D / S, K268A / E / R, F269F / L, N271G / H / M, I272G / L / S,Docket Number CX10-269WO4 F273A / L / S / Y, Q275E, G276A / H, R277T, V278L / P / R, D279Q / R, D280S, I281A / L / V / W, F282L, S283K / L / Q / T, R284L, V285A / G / P / R / S, T286P / Q, E295A / R / T, R297C, K299R, T302I, N303A / G, S310G, L311C / F / G / I, K314G / L / P / S, G320A, K323D / E / T, F324T, E326G / M / T / W, R330H / I, V332L, K333E, R336C, K337E, R341S, F343D / P, K345G / T, L346G / P, K347A / E / G / I / L / M / P / R / S / T, I348A / L / S / T, Q349S, L353T, K354E / H / S / V, A356L / M, V357M, R358C / D / G / P / R / T / V, A360L, K361I, T362A / E / R, G363S, K365N, Y369L, or T380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0451] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0452] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G / K / N / R / S / Y, 35A / L / S, 38M / T / V, 45Y, 48W, 49I, 54I / L / T / V, 66A / K / P / Q / R / T, 90E / S, 93A / H / N / P / R, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 127I / L / P / Q / V, 162S / W, 165F / K / M / P / T, 167I / V, 170R, 177C / I / L / V, 197E / T, 205M / T / V, 213V, 220A / P / R / S / V, 222F / G, 223A / G / N / S / T, 225G / H / P / R / T, 236H, 237A / G / R, 238G, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 269F / L, 271G / H / M, 272G / L / R / S, 275E / W, 276A / G / H, 281A / I / L / M / V / W, 289Q, 316R, 320A / G, 337E / K, 351Y, 354E / H / S / V, 357L, 358C / D / G / P / R / T / V, 359D / I, 362A / E / R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0453] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34N / S / Y, 35L, 35S, 38T / V, 45Y, 48W, 49I, 54T, 66T, 90E / S, 93P, 115S, 118L / M, 121K, 122L / S, 127I / L / V, 162W, 165K, 167V, 170R, 177V, 197E / T, 205M, 213V, 220A / R, 222G, 223G, 225G, 236H, 237G, 238G, 254R, 255A / F / Q, 256R, 258L, 259S, 269L, 271G, 272G, 275W, 276H, 281L, 289Q, 316R, 320A / G, 337E, 351Y, 354E, 357L, 358G, 359D / I, 362E, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0454] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue R34Y, V38T / V, N45Y, F48W, V49I, L54T, L118M, Q121K, G123S, P127L, S162W, F165K, L167V, I205M, L213V, I220A / R, N222G, R223G, S225G, K236H, K237G, H255F?Q, Y256R, F258L, T259S, F269L, I272G, G276H, I281L, G320A / G, K337E, K354E, R358G, T362E, or K365N, or any combinations thereof, wherein the amino acid positions are relative to theDocket Number CX10-269WO4 reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0455] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 121K, 45Y, 41V, 34Y, 269L, or 380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Q121K, N45Y, S41V, R34Y, F269L, or T380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 34 / 45 / 269, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 34Y / 45Y / 269L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set or amino acid residues R34Y / N45Y / F269L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0456] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 162, 236, 237, 320, 337, 358, or 362, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 162W, 236H, 237G, 320A, 337E, 358G, or 362E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue S162W, K236H, K237G, G320A, K337E, R358G, or T362E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments,Docket Number CX10-269WO4 the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 162 / 236 / 237 / 320 / 337 / 358 / 362, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 162W / 236H / 237G / 320A / 337E / 358G / 362E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues S162W / K236H / K237G / G320A / K337E / R358G / T362E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0457] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 123, 256, or 320, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 123S, 256R, or 320G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue G123S, Y256R, or A320G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 123 / 256 / 320, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 123S / 256R / 320G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues G123S / Y256R / A320G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0458] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 38, 48, 49, 118, or 220, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO:Docket Number CX10-269WO4 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 38V, 48W, 49I, 118M, or 220A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue T38V, F48W, V49I, L118M, or R220A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 38 / 48 / 49 / 118 / 220, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 38V / 48W / 49I / 118M / 220A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues T38V / F48W / V49I / L118M / R220A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0459] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 38, 54, 205, or 258, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 38T, 54T, 205M, or 258L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue V38T, L54T, I205M, or F258L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 38 / 54 / 205 / 258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 38T / 54T / 205M / 258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequenceDocket Number CX10-269WO4 corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues V38T / L54T / I205M / F258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0460] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 127, 222, 223, 225, 255, 272, or 276, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 127L, 222G, 223G, 225G, 255Q, 272G, or 276H, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue P127L, N222G, R223G, S225G, F255Q, I272G, or G276H, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 127 / 222 / 223 / 225 / 255 / 272 / 276, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 127L / 222G / 223G / 225G / 255Q / 272G / 276H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues P127L / N222G / R223G / S225G / F255Q / I272G / G276H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0461] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 165, 259, or 281, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 165K, 259S, or 281L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue F165K, T259S, or I281L, or any combinations thereof, wherein theDocket Number CX10-269WO4 amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 165 / 259 / 281, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 165K / 259S / 281L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues F165K / T259S / I281L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0462] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 127, 238, 255, 359, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 127V, 238G, 255A, 359D, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L127V, E238G, Q255A, I359D, or K381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 127 / 238 / 255 / 359 / 381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 127V / 238G / 255A / 359D / 381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues L127V / E238G / Q255A / I359D / K381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0463] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 35, 170, 271, or 357, or any combinations thereof,Docket Number CX10-269WO4 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 35L, 170R, 271G, or 357L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue E35L, P170R, N271G, or V357L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 35 / 170 / 271 / 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 35L / 170R / 271G / 357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues E35L / P170R / N271G / V357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0464] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 34, 35, 118, 127, 275, 351, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 34S, 35S, 118L, 127I, 275W, 351Y, 374L, or 376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L127V, N34S, L35S, M118L, V127I, Q275W, N351Y, F374L, or T376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 34 / 35 / 118 / 127 / 275 / 351 / 374 / 376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence ofDocket Number CX10-269WO4 the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 34S / 35S / 118L / 127I / 275W / 351Y / 374L / 376E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues N34S / L35S / M118L / V127I / Q275W / N351Y / F374L / T376E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0465] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 66, 90, 93, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 66T, 90S, 93P, or 197T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue A19V, K66T, E90S, R93P, or E197T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 19 / 66 / 90 / 93 / 197, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 19V / 66T / 90S / 93P / 197T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues A19V / K66T / E90S / R93P / E197T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0466] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 90, 197, 289, or 316, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 90E, 197E, 289Q, or 316R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxyDocket Number CX10-269WO4 terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue S90E, T197E, N289Q, or S316R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 90 / 197 and / or 289 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 90E / 197E and / or 289Q / 316R wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues S90E / T197E and / or N289Q / S316R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0467] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0468] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0469] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0470] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2,Docket Number CX10-269WO4 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0471] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 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 having a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0472] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0473] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0474] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167,Docket Number CX10-269WO4 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0475] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G / W, 27A / F / W / Y, 30R, 31I, 32M, 33R, 34G / K / N / R / S / Y, 35A / L / S, 36D / R, 38M / T / V, 40I / L, 41V, 42A, 43G / T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I / L / T / V, 56L / M, 58M, 65P / Q / S, 66A / K / P / Q / R / T, 69A / C, 84L, 88W, 90E / S, 91D / L, 92I, 93A / H / N / P / R, 94D, 97L / P / V, 109G / S, 113D / G, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 123G / S, 125Q / R / T / V, 127I / L / P / Q / V, 130I, 135I / M, 138A, 139R, 141A / D / G / P / R, 144T, 145Y, 146I, 151E / L, 152D, 156A / S, 157L, 160R, 161V, 162S / W, 165F / K / M / P / T, 166H / P / S, 167I / V, 168I / S, 170R, 171P / R / S, 172P, 173E / N, 174P, 177C / I / L / V, 181V, 185E, 190L, 195T, 196D, 197E / T, 198A, 199L / T, 203V, 204E, 205M / T / V, 207E, 212H / S, 213V, 214A / C / E / S, 217G / P, 220A / P / R / S / V, 221P, 222F / G, 223A / G / N / S / T, 224P, 225G / H / P / R / T, 226A / P / Q / T, 229A / L, 230L, 231I, 236H, 237A / G / R, 238G, 240V / W, 246F / I, 248F / H / M, 252R, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 260I / L / R / T / V, 263D / S, 268A / E / R, 269L, 269F / L, 271G / H / M, 272G / L / R / S, 273A / L / S / Y, 274I, 275E / W, 276A / G / H, 277T, 278L / P / R, 279Q / R, 280S, 281A / I / L / M / V / W, 282L, 283K / L / Q / T, 284F / L, 285A / G / P / R / S, 286P / Q, 288T, 289Q, 291L, 295A / R / T, 297C, 299I / R, 302I, 303A / G, 306F / M, 310A / D / G, 311C / F / G / I, 314E / G / L / P / S, 316R, 320A / G, 323D / E / G / N / T / Y, 324T, 325M / P, 326G / M / T / W, 330H / I, 332G / L, 333E, 334I / N / S, 336C / E / N, 337E / K, 340C, 341A / P / S, 343D / P, 345G / T, 346G / P, 347A / E / G / I / L / M / P / R / S / T, 348A / L / S / T, 349S / V, 350A / C / R, 351F / N / Y, 352G / L / M / N / R / S / V, 353T / V, 354E / H / Q / S / V, 355K, 356L / M / V, 357L / M, 358C / D / G / P / R / T / V, 359D / I, 360L / V, 361I, 362A / E / K / Q / R, 363S / V, 365N, 366W, 368G, 369C / L / V, 371R / S, 372A / L / P, 374F / I / L / V, 376E / T, 377L, 380S, 381K / R, or 384C / S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0476] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequenceDocket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0477] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G / K / N / R / S / Y, 35A / L / S, 38M / T / V, 45Y, 48W, 49I, 54I / L / T / V, 66A / K / P / Q / R / T, 90E / S, 93A / H / N / P / R, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 127I / L / P / Q / V, 162S / W, 165F / K / M / P / T, 167I / V, 170R, 177C / I / L / V, 197E / T, 205M / T / V, 213V, 220A / P / R / S / V, 222F / G, 223A / G / N / S / T, 225G / H / P / R / T, 236H, 237A / G / R, 238G, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 269F / L, 271G / H / M, 272G / L / R / S, 275E / W, 276A / G / H, 281A / I / L / M / V / W, 289Q, 316R, 320A / G, 337E / K, 351Y, 354E / H / S / V, 357L, 358C / D / G / P / R / T / V, 359D / I, 362A / E / R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0478] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34N / S / Y, 35L, 35S, 38T / V, 45Y, 48W, 49I, 54T, 66T, 90E / S, 93P, 115S, 118L / M, 121K, 122L / S, 127I / L / V, 162W, 165K, 167V, 170R, 177V, 197E / T, 205M, 213V, 220A / R, 222G, 223G, 225G, 236H, 237G, 238G, 254R, 255A / F / Q, 256R, 258L, 259S, 269L, 271G, 272G, 275W, 276H, 281L, 289Q, 316R, 320A / G, 337E, 351Y, 354E, 357L, 358G, 359D / I, 362E, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

[0479] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or to the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0480] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 44- 64, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 44-64,Docket Number CX10-269WO4 wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0481] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34 / 45 / 269, 34 / 45 / 173 / 297, 34 / 173 / 269 / 380, 173 / 269, 156 / 269 / 380, 173 / 269 / 380, 34 / 173, 269, 34 / 380, 34 / 269 / 380, or 173 / 380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0482] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 34Y / 45Y / 269L, 34Y / 45Y / 173E / 297C, 34Y / 173E / 269L / 380S, 173E / 269L, 156S / 269L / 380S, 173E / 269L / 380S, 34Y / 173E, 269L, 34Y / 380S, 34Y / 269L / 380S, or 173E / 380S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0483] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) R34Y / N45Y / F269L, R34Y / N45Y / K173E / R297C, R34Y / K173E / F269L / T380S, K173E / F269L, P156S / F269L / T380S, K173E / F269L / T380S, R34Y / K173E, F269L, R34Y / T380S, R34Y / F269L / T380S, or K173E / T380S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0484] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or to the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0485] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 66- 118, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 66-118, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0486] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94 / 263, 220, 236, 92,Docket Number CX10-269WO4 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0487] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 207E, 237G, 94D / 263D, 220V, 236H, 92I, 91D, 94D, 204E, 185E, 213V, 199L, 152D, 196D, 203V, 141D, 138A, 156A, 93N, or 181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0488] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) R207E, K237G, G94D / N263D, I220V, K236H, L92I, R91D, G94D, K204E, K185E, L213V, K199L, K152D, N196D, I203V, N141D, K138A, P156A, R93N, or I181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0489] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or to the reference sequence corresponding to SEQ ID NO: 100, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.

[0490] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 120- 216, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 120-216, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.

[0491] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283 / 337, 347, 323, 354, 343, 118, 345, 314, 268 / 269, 363, 356, 358, 348, 162, 324 / 330, 346, 160, 362, 361, 341 / 349, 353, 369, 248, 146 / 346, 332, 170, or 269 / 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.

[0492] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 283L / 337E, 347E, 323D, 354V,Docket Number CX10-269WO4 343D, 347P, 118M, 347G, 345G, 347M, 354E, 314G, 345T, 268R / 269F, 363S, 356M, 358P, 343P, 358G, 348S, 348L, 358T, 314S, 162W, 324T / 330H, 354S, 347L, 346P, 323T, 348T, 160R, 362E, 361I, 341S / 349S, 354H, 358D, 347S, 353T, 314P, 369L, 248H, 146I / 346G, 332L, 346G, 314L, 170R, 348A, 269F / 275E, or 268E / 269F, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.

[0493] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) S283L / K337E, K347E, K323D, K354V, F343D, K347P, L118M, K347G, K345G, K347M, K354E, K314G, K345T, K268R / L269F, G363S, A356M, R358P, F343P, R358G, I348S, I348L, R358T, K314S, S162W, F324T / R330H, K354S, K347L, L346P, K323T, I348T, L160R, T362E, K361I, R341S / Q349S, K354H, R358D, K347S, L353T, K314P, Y369L, S248H, T146I / L346G, V332L, L346G, K314L, P170R, I348A, L269F / Q275E, or K268E / L269F, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.

[0494] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or to the reference sequence corresponding to SEQ ID NO: 140, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.

[0495] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 244- 398, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 244-398, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.

[0496] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162 / 337 / 358 / 362, 162 / 236 / 237 / 320 / 337 / 358 / 362, 151 / 231 / 237 / 337, 199 / 231 / 237 / 337, 231 / 237 / 314 / 337, 310 / 314 / 337, 115 / 162 / 310 / 314, 199 / 237 / 337, 151 / 199 / 205 / 310 / 314, 199 / 204 / 205 / 231 / 236 / 310 / 314, 320 / 337 / 358 / 362, 135 / 320 / 337 / 358 / 362, 151 / 212 / 214 / 345 / 347 / 358, 135 / 337 / 358 / 362, 162 / 358 / 362, 337 / 358 / 362, 337 / 358, 92 / 337, 151 / 196 / 199 / 205 / 231 / 237 / 323, 151 / 214 / 347 / 358, 337, 151 / 345 / 347 / 358, 199 / 314, 199 / 205 / 231 / 237 / 323, 151 / 205 / 314, 151 / 212 / 345 / 347, 92 / 214 / 347 / 358, 162 / 204 / 205 / 310 / 314 / 358, 236 / 314, 151 / 345 / 347, 214 / 347 / 358, 151 / 212 / 358, 204 / 283 / 314 / 358, 236 / 237 / 358 / 362, 151 / 199 / 204 / 231 / 236 / 323,Docket Number CX10-269WO4 231 / 236 / 237 / 358 / 362, 162 / 314 / 358, 92 / 151 / 347 / 358, 151 / 236, 212 / 345 / 347, 115 / 314, 345 / 347 / 358, 314, 358 / 362, 115 / 358, 310 / 314, 214 / 347 / 358, 237 / 314, 345 / 347, 151 / 310 / 323 / 343 / 347, 151 / 358, 347 / 358, 93 / 358 / 362, 231 / 236 / 237 / 320 / 358 / 362, 151 / 230 / 345 / 347 / 358, 135 / 231 / 236 / 237 / 358, 310 / 314 / 358 / 362, 151 / 230 / 347 / 358, 231 / 237 / 323, 135 / 358 / 362, 283 / 314 / 358 / 362, 199 / 205, 92 / 151 / 230 / 345 / 347 / 358, 314 / 358 / 362, 199 / 237, or 199 / 204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.

[0497] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 162W / 337E / 358C / 362E, 162W / 236H / 237G / 320A / 337E / 358G / 362E, 151E / 231I / 237G / 337E, 199L / 231I / 237G / 337E, 231I / 237G / 314L / 337E, 310G / 314L / 337E, 115E / 162W / 310G / 314S, 199L / 237G / 337E, 151E / 199L / 205V / 310G / 314L, 199L / 204E / 205V / 231I / 236H / 310G / 314L, 320A / 337E / 358T / 362E, 135I / 320A / 337E / 358T / 362E, 151E / 212S / 214E / 345T / 347L / 358D, 135I / 337E / 358P / 362E, 162W / 358T / 362E, 337E / 358C / 362E, 337E / 358G, 92I / 337E, 151E / 196D / 199L / 205V / 231I / 237G / 323E, 151E / 214E / 347L / 358D, 337E, 337E / 358P / 362E, 162W / 358G / 362E, 151E / 345T / 347A / 358T, 199L / 314L, 199L / 205V / 231I / 237G / 323E, 151E / 345T / 347A / 358D, 151E / 205V / 314L, 151E / 212S / 345T / 347L, 92I / 214E / 347T / 358D, 162W / 204E / 205V / 310G / 314S / 358T, 337E / 358C, 236H / 314L, 151E / 345T / 347P, 337E / 358T, 151E / 345T / 347I, 214E / 347P / 358D, 151E / 212S / 358D, 204E / 283L / 314S / 358T, 236H / 237G / 358T / 362E, 151E / 199L / 204E / 231I / 236H / 323E, 231I / 236H / 237G / 358T / 362E, 162W / 314G / 358T, 92I / 151E / 347I / 358D, 151E / 236H, 212S / 345T / 347I, 115E / 314S, 345T / 347P / 358D, 314L, 358T / 362E, 115E / 358T, 236H / 237G / 358P / 362E, 162W / 358D / 362E, 310G / 314L, 214E / 347I / 358D, 237G / 314L, 345T / 347T, 151E / 310G / 323E / 343D / 347T, 151E / 358D, 345T / 347A / 358D, 347A / 358D, 93H / 358C / 362E, 231I / 236H / 237G / 320A / 358T / 362E, 151E / 230L / 345T / 347A / 358D, 135I / 231I / 236H / 237G / 358C, 310G / 314S / 358D / 362E, 151E / 230L / 347A / 358D, 231I / 237G / 323E, 135I / 358T / 362E, 347I / 358D, 283L / 314S / 358D / 362E, 199L / 205V, 214E / 347R / 358D, 92I / 151E / 230L / 345T / 347L / 358D, 314S / 358D / 362E, 310G / 314S, 199L / 237G, or 199L / 204E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.

[0498] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) S162W / K337E / R358C / T362E, S162W / K236H / K237G / G320A / K337E / R358G / T362E, N151E / L231I / K237G / K337E, K199L / L231I / K237G / K337E, L231I / K237G / K314L / K337E, S310G / K314L / K337E, K115E / S162W / S310G / K314S, K199L / K237G / K337E, N151E / K199L / I205V / S310G / K314L, K199L / K204E / I205V / L231I / K236H / S310G / K314L, G320A / K337E / R358T / T362E, L135I / G320A / K337E / R358T / T362E, N151E / R212S / K214E / K345T / K347L / R358D, L135I / K337E / R358P / T362E, S162W / R358T / T362E, K337E / R358C / T362E, K337E / R358G, L92I / K337E, N151E / N196D / K199L / I205V / L231I / K237G / K323E, N151E / K214E / K347L / R358D, K337E, K337E / R358P / T362E, S162W / R358G / T362E, N151E / K345T / K347A / R358T, K199L / K314L, K199L / I205V / L231I / K237G / K323E, N151E / K345T / K347A / R358D, N151E / I205V / K314L,Docket Number CX10-269WO4 N151E / R212S / K345T / K347L, L92I / K214E / K347T / R358D, S162W / K204E / I205V / S310G / K314S / R358T, K337E / R358C, K236H / K314L, N151E / K345T / K347P, K337E / R358T, N151E / K345T / K347I, K214E / K347P / R358D, N151E / R212S / R358D, K204E / S283L / K314S / R358T, K236H / K237G / R358T / T362E, N151E / K199L / K204E / L231I / K236H / K323E, L231I / K236H / K237G / R358T / T362E, S162W / K314G / R358T, L92I / N151E / K347I / R358D, N151E / K236H, R212S / K345T / K347I, K115E / K314S, K345T / K347P / R358D, K314L, R358T / T362E, K115E / R358T, K236H / K237G / R358P / T362E, S162W / R358D / T362E, S310G / K314L, K214E / K347I / R358D, K237G / K314L, K345T / K347T, N151E / S310G / K323E / F343D / K347T, N151E / R358D, K345T / K347A / R358D, K347A / R358D, R93H / R358C / T362E, L231I / K236H / K237G / G320A / R358T / T362E, N151E / V230L / K345T / K347A / R358D, L135I / L231I / K236H / K237G / R358C, S310G / K314S / R358D / T362E, N151E / V230L / K347A / R358D, L231I / K237G / K323E, L135I / R358T / T362E, K347I / R358D, S283L / K314S / R358D / T362E, K199L / I205V, K214E / K347R / R358D, L92I / N151E / V230L / K345T / K347L / R358D, K314S / R358D / T362E, S310G / K314S, K199L / K237G, or K199L / K204E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.

[0499] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or to the reference sequence corresponding to SEQ ID NO: 246, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.

[0500] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 400- 490, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 400-490, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.

[0501] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161 / 162, 311 / 320, 123, 320 / 326, 174, 162 / 167, 32, 125, 162 / 166, 320, 283, 330, 278, 303, 281, 333 / 337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.

[0502] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 255F, 255M, 109G, 256R, 260I,Docket Number CX10-269WO4 260R, 273Y, 260V, 46R, 252R, 161V / 162S, 311I / 320G, 123S, 320G / 326G, 174P, 256M, 162S / 167V, 32M, 125V, 311C / 320G, 162S / 166H, 320G, 320G / 326M, 260L, 256V, 283Q, 330I, 278L, 311F / 320G, 320G / 326T, 303A, 125T, 283T, 320G / 326W, 281A, 333E / 337K, 281W, 283K, 277T, 260T, 254R, 278R, 311G / 320G, 255W, 256L, or 173N, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.

[0503] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) H255F, H255M, P109G, Y256R, Y260I, Y260R, F273Y, Y260V, A46R, V252R, I161V / W162S, L311I / A320G, G123S, A320G / E326G, D174P, Y256M, W162S / L167V, L32M, S125V, L311C / A320G, W162S / Q166H, A320G, A320G / E326M, Y260L, Y256V, S283Q, R330I, V278L, L311F / A320G, A320G / E326T, N303A, S125T, S283T, A320G / E326W, I281A, K333E / E337K, I281W, S283K, R277T, Y260T, H254R, V278R, L311G / A320G, H255W, Y256L, or K173N, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.

[0504] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0505] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 492- 510, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 492-510, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0506] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123 / 256 / 320, 260, 256 / 260, 256, 260 / 281, 320, 123 / 260, 123 / 320, 256 / 281, or 260 / 273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0507] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 123S / 256R / 320G, 260I, 256R / 260I, 256R, 260I / 281V, 320G, 123S / 260I, 123S / 320G, 256R / 281V, or 260I / 273Y, wherein the aminoDocket Number CX10-269WO4 acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0508] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G123S / Y256R / A320G, Y260I, Y256R / Y260I, Y256R, Y260I / I281V, A320G, G123S / Y260I, G123S / A320G, Y256R / I281V, or Y260I / F273Y, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0509] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or to the reference sequence corresponding to SEQ ID NO: 492, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.

[0510] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 512- 584, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 512-584, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.

[0511] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118 / 123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.

[0512] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 217P, 220A, 221P, 220S, 220R, 220P, 229L, 246F, 171S, 285R, 217G, 285G, 286Q, 165P, 285P, 226A, 168S, 177L, 226P, 285A, 223N, 224P, 118M / 123G, 285S, 248M, 268A, 286P, 229A, 225P, 171P, 165T, 171R, 226Q, 84L, 226T, 284L, or 225T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.

[0513] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) L217P, I220A, E221P, I220S,Docket Number CX10-269WO4 I220R, I220P, F229L, V246F, Y171S, V285R, L217G, V285G, T286Q, F165P, V285P, I226A, V168S, F177L, I226P, V285A, R223N, E224P, L118M / S123G, V285S, S248M, K268A, T286P, F229A, S225P, Y171P, F165T, Y171R, I226Q, V84L, I226T, R284L, or S225T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.

[0514] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0515] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 586- 602, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 586-602, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0516] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0517] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 254R, 240V, 118M, 347P, 167V, 281V, 303G, 205T, or 50W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0518] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue H254R, F240V, L118M, K347P, L167V, I281V, N303G, I205T, or F50W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.

[0519] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,Docket Number CX10-269WO4 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or to the reference sequence corresponding to SEQ ID NO: 594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.

[0520] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 604- 700, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 604-700, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.

[0521] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118 / 220, 118 / 220 / 303 / 347, 347, 118 / 220 / 254 / 347, 220, 38 / 220, 220 / 254 / 303 / 347, 220 / 254, 220 / 303 / 347, 48 / 220 / 347, 49 / 220, 217 / 220 / 347, 38 / 220 / 254, 49 / 118 / 220 / 254 / 347, 49 / 220 / 254, 38 / 48 / 49 / 118 / 220, 49 / 217 / 220 / 254, 49 / 220 / 347, 49 / 347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248 / 357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.

[0522] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 118M / 220A, 118M / 220A / 303G / K347L, 347L, 118M / 220A / 254S / 347L, 220A, 38V / 220A, 220A / 254S / 303G / 347L, 220A / 254S, 220A / 303G / 347L, 48W / 220A / 347L, 49I / 220A, 217G / 220A / 347L, 38V / 220A / 254S, 49I / 118M / 220A / 254S / 347L, 49I / 220A / 254S, 38V / 48W / 49I / 118M / 220A, 49I / 217G / 220A / 254S, 49I / 220A / 347L, 49I / 347L, 225G, 280S, 118F, 279R, 165K, 273A, 272S, 273S, 166P, 281A, 248F / 357M, 222F, 223S, 281L, 358R, 271H, 168I, 276A, 225R, 36D, 34S, 40L, 263S, 130I, 225H, 356L, 273L, 259S, 358V, or 167I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.

[0523] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) L118M / R220A, L118M / R220A / N303G / K347L, K347L, L118M / R220A / H254S / K347L, R220A, T38V / R220A, R220A / H254S / N303G / K347L, R220A / H254S, R220A / N303G / K347L, F48W / R220A / K347L, V49I / R220A, L217G / R220A / K347L, T38V / R220A / H254S, V49I / L118M / R220A / H254S / K347L, V49I / R220A / H254S, T38V / F48W / V49I / L118M / R220A, V49I / L217G / R220A / H254S, V49I / R220A / K347L, V49I / K347L, S225G, D280S, L118F, D279R, F165K, F273A, I272S, F273S, Q166P, I281A, S248F / V357M, N222F, R223S,Docket Number CX10-269WO4 I281L, G358R, N271H, V168I, G276A, S225R, N36D, Y34S, V40L, N263S, E130I, S225H, A356L, F273L, T259S, G358V, or V167I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.

[0524] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, ...

Claims

Docket Number CX10-269WO4 CLAIMS WHAT IS CLAIMED IS:

1. A method of synthesizing an oligonucleotide, comprising reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor.

2. The method of claim 1, wherein the nucleotide donor, the oligonucleotide acceptor, or both the nucleotide donor and the oligonucleotide(A)comprise a modified nucleotide.

3. The method of claim 1 or 2, wherein the oligonucleotide(A) comprises at least one modified nucleoside, wherein the modified nucleoside comprises a conjugate moiety, reactive group, or linker.

4. The method of claim 3, wherein the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

5. The method of claim 3, wherein the reactive group comprises an amino, -CN (cyano), N3(azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

6. The method of claim 3, wherein the modified nucleoside on the oligonucleotide(A) is at the 5’-terminal nucleotide, an internal nucleotide, or the 3’-terminal nucleotide.

7. The method of any one of claims 3-5, wherein the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A).

8. The method of any one of claims 3-7, wherein the conjugate moiety or the reactive group is attached to the nucleoside via a linker L.

9. The method of claim 8, wherein the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A).

10. The method of any one of claims 1-9, wherein the oligonucleotide acceptor comprises one or more terminal groups.

11. The method of claim 10, wherein the terminal group is at the 5’-terminal nucleotide of the oligonucleotide acceptor.

12. The method of claim 11, wherein the terminal group is a 5’-phosphonate (E or Z vinylphosphonate), 4’-amino, 4’-aminoalkyl, abasic nucleotide, or inverted abasic nucleotide.

13. The method of any one of claims 1-12, wherein the oligonucleotide(A) comprises a 5’-OH or a 5’-blocking group that inhibits ligation by the single stranded RNA ligase.

14. The method of any one of claims 1-13, wherein the oligonucleotide acceptor comprises at least one modified internucleoside linkage.

15. The method of claim 14, wherein the internucleoside linkage comprises a phosphorothioate or phosphorodithioate internucleoside linkage.Docket Number CX10-269WO4 16. The method of claim 15, wherein the phosphorothioate comprises a mixture of Rp and Sp stereoisomers, or is a Rp stereoisomer or an Sp stereoisomer.

17. The method of claim 1, wherein the oligonucleotide(A) comprises the formula (I): A1[•A2]m•A3-OH(I)wherein each of A1, A2and A3is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage.

18. The method of claim 17, wherein m is 2 or greater, each of A2is the same or different nucleoside.

19. The method of claim 17 or 18, wherein when the oligonucleotide(A) comprises a modified nucleotide of at least one or more of A1, A2, or A3.

20. The method of claim 19, wherein at least one of A1, A2, or A3 is modified with a conjugate moiety, reactive group, or linker.

21. The method of any one of claims 17-20, wherein one or more of the internucleoside linkage “•“ comprises a modified internucleoside linkage.

22. The method of claim 21, wherein the modified internucleoside linkage comprises a phosphorothioate or a phosphorodithioate.

23. The method of claim 20, wherein modified nucleoside on the oligonucleotide(A) comprises the formula (II): A-[L]g-[M]h(II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0.

24. The method of any one of claims 1-23, wherein the oligonucleotide acceptor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

25. The method of claim 23 or 24, wherein when g is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside, or wherein when g is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside.Docket Number CX10-269WO4 26. The method of any one of claims 1-25, wherein the nucleotide donor comprises a nucleotide(D) or an oligonucleotide(D).

27. The method of claim 26, wherein the nucleotide(D) or oligonucleotide(D) comprises a modified nucleoside.

28. The method of claim 27, wherein the modified nucleoside of nucleotide(D)or oligonucleotide(D)comprises a conjugate moiety, a reactive group, or linker.

29. The method of claim 28, wherein the conjugate moiety on the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

30. The method of claim 28, wherein the reactive group on the nucleotide donor comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

31. The method of any one of claims 27-30, wherein the modified nucleoside on the oligonucleotide(D)is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.

32. The method of any one of claims 28-31, wherein the conjugate moiety, reactive group or linker is attached to the nucleobase or the sugar moiety of the nucleoside.

33. The method of any one of claims 28-32, wherein the conjugate moiety or the reactive group is attached to the nucleoside via a linker (L).

34. The method of any one of claims 1-33, wherein the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3(IIIb) wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120.

35. The method of any one of claims 1-34, wherein the nucleotide donor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

36. The method of claim 34 or 35, wherein for formula (IIIb), when n is 2 or greater, each of D2is the same or different nucleoside.

37. The method of any one of claims 34-36, wherein the nucleotide donor comprises a modified nucleoside of at least one or more of D1, D2, or D3.

38. The method of claim 37, wherein at least one of DO, D2, or D3 is modified with a conjugate moiety or conjugate reactive group.Docket Number CX10-269WO4 39. The method of any one of claims 34-38, wherein for formula (IIIb) at least one internucleoside linkage “•“ comprises a modified internucleoside linkage.

40. The method of claim 34, for formula (IIIa), D comprises a modified nucleoside.

41. The method of claim 40, wherein D is modified with a conjugate moiety or conjugate reactive group.

42. The method of any one of claims 37-41, wherein the modified nucleoside on nucleotide(D) or oligonucleotide(D)has the formula (IV): D-[L]q-[M]r(IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0.

43. The method of claim 42, wherein when q is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside; or wherein when q is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside.

44. The method of any one of claims 1-43, wherein the nucleotide donor comprises a 3’-blocking group that inhibits ligation by the single stranded RNA ligase to a 3’-OH group of another nucleotide acceptor to produce a 3’-blocked extended oligonucleotide.

45. The method of claim 44, wherein the 3’-blocking group comprises a reversible 3’-blocking group to produce a reversible 3’-blocked extended oligonucleotide.

46. The method of claim 45, further comprising separating the 3’-blocked extended oligonucleotide from the single stranded RNA ligase or inactivating the single stranded RNA ligase.

47. The method of claim 46, further comprising removing or cleaving the 3’-blocking group with a deblocking agent to form an unblocked extended oligonucleotide.

48. The method of claim 47, further comprising inactivating the deblocking agent or removing or separating the unblocked extended oligonucleotide from the deblocking agent.

49. The method of claim 48, further comprising reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single stranded RNA ligase.

50. The method of claim 49, wherein the second nucleotide donor comprises a 3’-blocking group.

51. The method of any one of claims 45-48, further comprising one or more cycles of: extension with a nucleotide donor; separating the 3’-blocked extended oligonucleotide from the single stranded RNADocket Number CX10-269WO4 ligase or inactivating the single stranded RNA ligase; removing or cleaving the reversible 3’-blocking group with a deblocking agent; and separating the unblocked extended oligonucleotide, wherein each cycle uses a new nucleotide donor.

52. The method of claim 51, wherein the nucleotide donor for at least one cycle comprises a mixture of different nucleotide donors.

53. The method of claim 51, wherein the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor nucleotide(D)or oligonucleotide(D)to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence.

54. The method of claim 53, wherein the selected or predetermined nucleotide donor for each cycle is nucleotide(D).

55. The method of any one of claims 1-54, wherein the single stranded RNA ligase comprises RNA ligase 1.

56. The method of any one of claims 1-55, wherein the single stranded RNA ligase comprises a recombinant single stranded RNA ligase of any one of claims 64-125.

57. The method of any one of claims 1-56, wherein the single stranded RNA ligase is immobilized on a support medium and the oligonucleotide acceptor (oligonucleotide(A)) and nucleotide donor are provided in solution or aqueous phase.

58. The method of any one of claims 1-56, wherein the oligonucleotide acceptor is immobilized is attached to a support medium, and the single stranded RNA ligase and the nucleotide donor are provided in solution or aqueous phase.

59. The method of any one of claims 1-58, wherein the reaction with the single stranded RNA ligase further comprises a pyrophosphatase.

60. The method of any one of claims 1-59, wherein the reaction with the single stranded RNA ligase further comprises an ATP recycling system.

61. The method of any one of claims 1-60, wherein the suitable reaction conditions comprises one or more of cofactor ATP, a divalent metal ion, and a buffer.

62. The method of any one of claims 1-61, wherein the suitable reaction conditions comprise a reaction temperature of 5-60 ℃.

63. The method of any one of claims 1-62, wherein the suitable reaction conditions comprise a reaction pH of about 5-8.

64. A recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 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 the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.Docket Number CX10-269WO4 65. The recombinant single stranded RNA ligase of claim 64, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.

66. The recombinant single stranded RNA ligase of claim 64 or 65, wherein the single stranded RNA ligase is the single stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1.

67. The recombinant single stranded RNA ligase of claim 64, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1524, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

68. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

69. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.Docket Number CX10-269WO4 70. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

71. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

72. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

73. The recombinant single stranded RNA ligase of any one of claims 67-72, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G / W, 27A / F / W / Y, 30R, 31I, 32M, 33R, 34G / K / N / R / S / Y, 35A / L / S, 36D / R, 38M / T / V, 40I / L, 41V, 42A, 43G / T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I / L / T / V, 56L / M, 58M, 65P / Q / S, 66A / K / P / Q / R / T, 69A / C, 84L, 88W, 90E / S, 91D / L, 92I, 93A / H / N / P / R, 94D, 97L / P / V, 109G / S, 113D / G, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 123G / S, 125Q / R / T / V, 127I / L / P / Q / V, 130I, 135I / M, 138A, 139R, 141A / D / G / P / R, 144T, 145Y, 146I, 151E / L, 152D, 156A / S, 157L, 160R, 161V, 162S / W, 165F / K / M / P / T, 166H / P / S, 167I / V, 168I / S, 170R, 171P / R / S, 172P, 173E / N, 174P, 177C / I / L / V, 181V, 185E, 190L, 195T, 196D, 197E / T, 198A, 199L / T, 203V, 204E, 205M / T / V, 207E, 212H / S, 213V, 214A / C / E / S,Docket Number CX10-269WO4 217G / P, 220A / P / R / S / V, 221P, 222F / G, 223A / G / N / S / T, 224P, 225G / H / P / R / T, 226A / P / Q / T, 229A / L, 230L, 231I, 236H, 237A / G / R, 238G, 240V / W, 246F / I, 248F / H / M, 252R, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 260I / L / R / T / V, 263D / S, 268A / E / R, 269L, 269F / L, 271G / H / M, 272G / L / R / S, 273A / L / S / Y, 274I, 275E / W, 276A / G / H, 277T, 278L / P / R, 279Q / R, 280S, 281A / I / L / M / V / W, 282L, 283K / L / Q / T, 284F / L, 285A / G / P / R / S, 286P / Q, 288T, 289Q, 291L, 295A / R / T, 297C, 299I / R, 302I, 303A / G, 306F / M, 310A / D / G, 311C / F / G / I, 314E / G / L / P / S, 316R, 320A / G, 323D / E / G / N / T / Y, 324T, 325M / P, 326G / M / T / W, 330H / I, 332G / L, 333E, 334I / N / S, 336C / E / N, 337E / K, 340C, 341A / P / S, 343D / P, 345G / T, 346G / P, 347A / E / G / I / L / M / P / R / S / T, 348A / L / S / T, 349S / V, 350A / C / R, 351F / N / Y, 352G / L / M / N / R / S / V, 353T / V, 354E / H / Q / S / V, 355K, 356L / M / V, 357L / M, 358C / D / G / P / R / T / V, 359D / I, 360L / V, 361I, 362A / E / K / Q / R, 363S / V, 365N, 366W, 368G, 369C / L / V, 371R / S, 372A / L / P, 374F / I / L / V, 376E / T, 377L, 380S, 381K / R, or 384C / S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

74. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

75. The recombinant single stranded RNA ligase of any one of claims 67-71 or 74, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G / K / N / R / S / Y, 35A / L / S, 38M / T / V, 45Y, 48W, 49I, 54I / L / T / V, 66A / K / P / Q / R / T, 90E / S, 93A / H / N / P / R, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 127I / L / P / Q / V, 162S / W, 165F / K / M / P / T, 167I / V, 170R, 177C / I / L / V, 197E / T, 205M / T / V, 213V, 220A / P / R / S / V, 222F / G, 223A / G / N / S / T, 225G / H / P / R / T, 236H, 237A / G / R, 238G, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 269F / L, 271G / H / M, 272G / L / R / S, 275E / W, 276A / G / H, 281A / I / L / M / V / W, 289Q, 316R, 320A / G, 337E / K, 351Y, 354E / H / S / V, 357L, 358C / D / G / P / R / T / V, 359D / I, 362A / E / R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

76. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

77. The recombinant single stranded RNA ligase of any one of claims 67-71 or 76, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or aminoDocket Number CX10-269WO4 acid residue 121K, 45Y, 41V, 34Y, 269L, or 380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

78. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

79. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

80. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 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 a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:

14.

81. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

82. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 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 sequenceDocket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

83. The recombinant single stranded RNA ligase of claims 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

84. The recombinant single stranded RNA ligase of any one of claims 81-83, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G / W, 27A / F / W / Y, 30R, 31I, 32M, 33R, 34G / K / N / R / S / Y, 35A / L / S, 36D / R, 38M / T / V, 40I / L, 41V, 42A, 43G / T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I / L / T / V, 56L / M, 58M, 65P / Q / S, 66A / K / P / Q / R / T, 69A / C, 84L, 88W, 90E / S, 91D / L, 92I, 93A / H / N / P / R, 94D, 97L / P / V, 109G / S, 113D / G, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 123G / S, 125Q / R / T / V, 127I / L / P / Q / V, 130I, 135I / M, 138A, 139R, 141A / D / G / P / R, 144T, 145Y, 146I, 151E / L, 152D, 156A / S, 157L, 160R, 161V, 162S / W, 165F / K / M / P / T, 166H / P / S, 167I / V, 168I / S, 170R, 171P / R / S, 172P, 173E / N, 174P, 177C / I / L / V, 181V, 185E, 190L, 195T, 196D, 197E / T, 198A, 199L / T, 203V, 204E, 205M / T / V, 207E, 212H / S, 213V, 214A / C / E / S, 217G / P, 220A / P / R / S / V, 221P, 222F / G, 223A / G / N / S / T, 224P, 225G / H / P / R / T, 226A / P / Q / T, 229A / L, 230L, 231I, 236H, 237A / G / R, 238G, 240V / W, 246F / I, 248F / H / M, 252R, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 260I / L / R / T / V, 263D / S, 268A / E / R, 269L, 269F / L, 271G / H / M, 272G / L / R / S, 273A / L / S / Y, 274I, 275E / W, 276A / G / H, 277T, 278L / P / R, 279Q / R, 280S, 281A / I / L / M / V / W, 282L, 283K / L / Q / T, 284F / L, 285A / G / P / R / S, 286P / Q, 288T, 289Q, 291L, 295A / R / T, 297C, 299I / R, 302I, 303A / G, 306F / M, 310A / D / G, 311C / F / G / I, 314E / G / L / P / S, 316R, 320A / G, 323D / E / G / N / T / Y, 324T, 325M / P, 326G / M / T / W, 330H / I, 332G / L, 333E, 334I / N / S, 336C / E / N, 337E / K, 340C, 341A / P / S, 343D / P, 345G / T,Docket Number CX10-269WO4 346G / P, 347A / E / G / I / L / M / P / R / S / T, 348A / L / S / T, 349S / V, 350A / C / R, 351F / N / Y, 352G / L / M / N / R / S / V, 353T / V, 354E / H / Q / S / V, 355K, 356L / M / V, 357L / M, 358C / D / G / P / R / T / V, 359D / I, 360L / V, 361I, 362A / E / K / Q / R, 363S / V, 365N, 366W, 368G, 369C / L / V, 371R / S, 372A / L / P, 374F / I / L / V, 376E / T, 377L, 380S, 381K / R, or 384C / S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

85. The recombinant single stranded RNA ligase of any one of claims 81-83, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

86. The recombinant single stranded RNA ligase of any one of claims 81-83, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G / K / N / R / S / Y, 35A / L / S, 38M / T / V, 45Y, 48W, 49I, 54I / L / T / V, 66A / K / P / Q / R / T, 90E / S, 93A / H / N / P / R, 115E / S, 118F / L / M, 121K, 122A / C / I / L / M / S / T / V / Y, 127I / L / P / Q / V, 162S / W, 165F / K / M / P / T, 167I / V, 170R, 177C / I / L / V, 197E / T, 205M / T / V, 213V, 220A / P / R / S / V, 222F / G, 223A / G / N / S / T, 225G / H / P / R / T, 236H, 237A / G / R, 238G, 254A / D / R / S, 255A / F / M / Q / S / V / W, 256L / M / R / V, 258L, 259A / N / S, 269F / L, 271G / H / M, 272G / L / R / S, 275E / W, 276A / G / H, 281A / I / L / M / V / W, 289Q, 316R, 320A / G, 337E / K, 351Y, 354E / H / S / V, 357L, 358C / D / G / P / R / T / V, 359D / I, 362A / E / R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

87. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34 / 45 / 269, 34 / 45 / 173 / 297, 34 / 173 / 269 / 380, 173 / 269, 156 / 269 / 380, 173 / 269 / 380, 34 / 173, 269, 34 / 380, 34 / 269 / 380, or 173 / 380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.Docket Number CX10-269WO4 88. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94 / 263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO:

44.

89. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283 / 337, 347, 323, 354, 343, 118, 345, 314, 268 / 269, 363, 356, 358, 348, 162, 324 / 330, 346, 160, 362, 361, 341 / 349, 353, 369, 248, 146 / 346, 332, 170, or 269 / 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO:

100.

90. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162 / 337 / 358 / 362, 162 / 236 / 237 / 320 / 337 / 358 / 362, 151 / 231 / 237 / 337, 199 / 231 / 237 / 337, 231 / 237 / 314 / 337, 310 / 314 / 337, 115 / 162 / 310 / 314, 199 / 237 / 337, 151 / 199 / 205 / 310 / 314, 199 / 204 / 205 / 231 / 236 / 310 / 314, 320 / 337 / 358 / 362, 135 / 320 / 337 / 358 / 362, 151 / 212 / 214 / 345 / 347 / 358, 135 / 337 / 358 / 362, 162 / 358 / 362, 337 / 358 / 362, 337 / 358, 92 / 337, 151 / 196 / 199 / 205 / 231 / 237 / 323, 151 / 214 / 347 / 358, 337, 151 / 345 / 347 / 358, 199 / 314, 199 / 205 / 231 / 237 / 323, 151 / 205 / 314, 151 / 212 / 345 / 347, 92 / 214 / 347 / 358, 162 / 204 / 205 / 310 / 314 / 358, 236 / 314, 151 / 345 / 347, 214 / 347 / 358, 151 / 212 / 358, 204 / 283 / 314 / 358, 236 / 237 / 358 / 362, 151 / 199 / 204 / 231 / 236 / 323, 231 / 236 / 237 / 358 / 362, 162 / 314 / 358, 92 / 151 / 347 / 358, 151 / 236, 212 / 345 / 347, 115 / 314, 345 / 347 / 358, 314, 358 / 362, 115 / 358, 310 / 314, 214 / 347 / 358, 237 / 314, 345 / 347, 151 / 310 / 323 / 343 / 347, 151 / 358, 347 / 358, 93 / 358 / 362, 231 / 236 / 237 / 320 / 358 / 362, 151 / 230 / 345 / 347 / 358, 135 / 231 / 236 / 237 / 358, 310 / 314 / 358 / 362, 151 / 230 / 347 / 358, 231 / 237 / 323, 135 / 358 / 362, 283 / 314 / 358 / 362, 199 / 205, 92 / 151 / 230 / 345 / 347 / 358, 314 / 358 / 362, 199 / 237, or 199 / 204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO:

140.

91. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161 / 162, 311 / 320, 123, 320 / 326, 174, 162 / 167, 32, 125, 162 / 166, 320, 283, 330, 278, 303, 281, 333 / 337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO:

246.

92. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123 / 256 / 320, 260, 256 / 260, 256, 260 / 281, 320, 123 / 260, 123 / 320, 256 / 281, or 260 / 273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to theDocket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO:

400.

93. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118 / 123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO:

492.

94. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO:

520.

95. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118 / 220, 118 / 220 / 303 / 347, 347, 118 / 220 / 254 / 347, 220, 38 / 220, 220 / 254 / 303 / 347, 220 / 254, 220 / 303 / 347, 48 / 220 / 347, 49 / 220, 217 / 220 / 347, 38 / 220 / 254, 49 / 118 / 220 / 254 / 347, 49 / 220 / 254, 38 / 48 / 49 / 118 / 220, 49 / 217 / 220 / 254, 49 / 220 / 347, 49 / 347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248 / 357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO:

594.

96. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 54 / 127 / 205 / 254, 165 / 255 / 258 / 259, 205 / 254 / 258, 33 / 38 / 127 / 165 / 254, 38 / 54 / 205 / 258, 127, 127 / 165 / 258, 127 / 205 / 254 / 255 / 258 / 259, 205 / 258, 33 / 38 / 254 / 255, 127 / 254 / 258 / 259, 127 / 165, 38 / 127 / 165 / 259, 38 / 127 / 258 / 259, 38 / 254 / 255, 127 / 205 / 254 / 258 / 259, 127 / 165 / 258 / 259, 165 / 205 / 258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO:

634.

97. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127 / 222 / 223 / 225 / 255 / 272 / 276, 127 / 255 / 259, 276, 255 / 259, 127 / 255, 127 / 162 / 223 / 255, 127 / 162 / 255 / 259 / 272 / 276, or 259 / 272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO:

710.

98. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set atDocket Number CX10-269WO4 amino acid position(s) 165 / 259 / 281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO:

738.

99. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279 / 281, 303, 165 / 166, 281 / 282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO:

756.

100. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44 / 118, 44 / 118 / 246 / 272 / 276, 44 / 118 / 246 / 280, 44 / 118 / 246 / 280 / 302, 44 / 118 / 272 / 276, 44 / 246, 44 / 246 / 272 / 276 / 302, 44 / 246 / 276 / 279 / 280, 44 / 246 / 276 / 280, 44 / 272 / 276 / 280, 44 / 272 / 279, 44 / 272 / 280, 44 / 276, 44 / 276 / 279, 44 / 276 / 280 / 295 / 302, 44 / 280, 44 / 302, 118 / 246 / 276 / 280 / 302, 118 / 246 / 280 / 295, 118 / 272 / 276, 118 / 280 / 302, 246 / 272 / 276 / 279 / 280 / 302, 246 / 272 / 279 / 280, 246 / 276, 246 / 276 / 302, 246 / 279 / 280, 246 / 279 / 280 / 302, 246 / 280, 272 / 276 / 279 / 280 / 302, 272 / 280, 272 / 280 / 302, 276 / 279 / 280 / 302, 276 / 280, 279 / 280 / 302, or 280, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO:

768.

101. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO:

844.

102. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38 / 127 / 238 / 284, 38 / 127 / 255, 38 / 127 / 255 / 359, 38 / 127 / 284, 38 / 238 / 255, 38 / 238 / 255 / 359, 38 / 238 / 255 / 359 / 381, 38 / 238 / 284 / 359, 127, 127 / 212 / 238 / 284 / 359, 127 / 238 / 255, 127 / 238 / 255 / 359 / 381, 127 / 238 / 284 / 359, 127 / 238 / 284 / 359 / 381, 127 / 238 / 359, 127 / 255 / 359 / 381, 127 / 255 / 381, 238, 238 / 255, 238 / 255 / 359, 238 / 255 / 381, 238 / 284, 238 / 359, 255, 255 / 284, 255 / 359, 255 / 359 / 362 / 381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO:

882.

103. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34 / 35 / 38, 34 / 38 / 170, 34 / 135 / 170 / 271 / 357, 34 / 170 / 271, 34 / 271, 35 / 38 / 170 / 357, 35 / 38 / 271 / 357, 35 / 170 / 271 / 357, 38 / 170, 38 / 170 / 271, 38 / 170 / 357, 56 / 135 / 170 / 357, 56 / 135 / 271 / 357, 135 / 170 / 271, 170, 170 / 271 / 272, 170 / 271 / 272 / 357, 170 / 271 / 357, 170 / 357, 254 / 260, 254 / 281, 271, or 357,Docket Number CX10-269WO4 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO:

936.

104. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9 / 369, 34 / 38 / 357, 34 / 135 / 170 / 271 / 357, 34 / 271, 56 / 135 / 170 / 357, 56 / 135 / 271 / 357, 113, 115, 141, 144, 145, 177, 214, 254 / 260, 254 / 281, 260, 274, 340, 341, 350, 354 / 359, 359 / 360, 359 / 362, 368, 369, 371, 377 / 381, or 381 / 384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO:

936.

105. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113 / 115, 113 / 115 / 177 / 254 / 281, 113 / 115 / 177 / 254 / 281 / 350 / 359, 113 / 115 / 177 / 254 / 350, 113 / 115 / 177 / 254 / 359, 113 / 115 / 177 / 281 / 359, 113 / 115 / 254, 113 / 115 / 254 / 281, 113 / 115 / 254 / 350, 113 / 115 / 254 / 359, 113 / 115 / 350, 113 / 115 / 359, 113 / 177 / 254 / 350, 113 / 177 / 281 / 359, 113 / 177 / 350 / 354 / 359, 113 / 254, 113 / 254 / 281 / 350 / 359, 113 / 254 / 281 / 359, 113 / 254 / 350 / 354 / 359, 113 / 254 / 354 / 359, 113 / 254 / 359, 113 / 359, 115 / 177, 115 / 177 / 254, 115 / 177 / 254 / 359, 115 / 177 / 359, 115 / 254, 115 / 254 / 350 / 354 / 359, 115 / 254 / 359, 115 / 350, 144 / 145 / 260, 144 / 145 / 260 / 341, 144 / 145 / 260 / 341 / 366 / 371, 144 / 260 / 341 / 366, 144 / 260 / 366 / 369, 145 / 260 / 263, 145 / 260 / 341, 145 / 260 / 341 / 366 / 369, 145 / 341, 145 / 341 / 371, 145 / 371, 177, 177 / 254, 177 / 254 / 281 / 359, 177 / 254 / 354 / 359, 177 / 254 / 359, 177 / 359, 254, 254 / 281 / 354 / 359, 254 / 350, 254 / 354 / 359, 254 / 359, 260 / 366, 341, or 359, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO:

992.

106. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.

107. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27 / 127 / 374, 27 / 127 / 374 / 376, 27 / 275 / 356, 27 / 351, 34 / 35 / 118 / 127 / 275 / 351 / 374 / 376, 122, 127 / 275 / 374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

108. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative toDocket Number CX10-269WO4 the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

109. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

110. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.

111. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19 / 21 / 65, 19 / 21 / 65 / 66, 19 / 21 / 65 / 66 / 90 / 93, 19 / 21 / 65 / 66 / 93, 19 / 21 / 65 / 93, 19 / 21 / 65 / 190, 19 / 21 / 65 / 197, 19 / 21 / 66, 19 / 21 / 190, 19 / 65, 19 / 65 / 66, 19 / 65 / 66 / 90 / 93 / 190, 19 / 65 / 66 / 93, 19 / 65 / 66 / 190, 19 / 65 / 66 / 197, 19 / 66, 19 / 66 / 90 / 93 / 197, 25, 25 / 54, 25 / 122, 65, 65 / 66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.

112. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351 / 352, 351 / 353, or 351 / 358, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.

113. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351 / 352, 351 / 353, 351 / 355, or 372 / 374 / 376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.

114. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21 / 54, 21 / 65 / 66 / 151, 21 / 90 / 93 / 122, 21 / 93, 54 / 58, 65 / 93, 65 / 151, 66 / 90 / 151 / 190 / 197, 90 / 93 / 122, 90 / 190 / 197, 90 / 197, 93, or 93 / 151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.Docket Number CX10-269WO4 115. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289 / 316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.

116. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.

117. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

118. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

119. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 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 a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.Docket Number CX10-269WO4 120. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524.

121. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

122. The recombinant single stranded RNA ligase of claim 67, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprising residues 12 to the carboxy terminal of an even- numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524.

123. The recombinant single stranded RNA ligase of any one of claims 67-122, having single stranded RNA ligase activity and exhibits at least an improved property as compared to a reference single stranded RNA ligase, wherein the improved property is selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), wherein the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.

124. The recombinant single stranded RNA ligase of any one of claims 64-123, wherein the recombinant single stranded RNA ligase further comprises a fusion polypeptide.

125. The recombinant single stranded RNA ligase of any one of claims 64-124, wherein the recombinant single stranded RNA ligase is a purified preparation.

126. A recombinant polynucleotide comprising a polynucleotide sequence encoding a single stranded RNA ligase of any one of claims 64-124.

127. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises at least 60%, 65%, 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 the sequence from nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11,Docket Number CX10-269WO4 13, 15, 17, or 19, or to a reference nucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

128. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, or comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

129. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises at least 60%, 65%, 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 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, wherein the recombinant polynucleotide encodes a single stranded RNA ligase.

130. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises at least 60%, 65%, 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 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, wherein the recombinant polynucleotide encodes a single stranded RNA ligase.

131. The recombinant polynucleotide of any one of claims 126-130, wherein the polynucleotide sequence is codon-optimized for expression of the encoded recombinant single stranded RNA ligase.

132. The recombinant polynucleotide of claim 126, comprising a polynucleotide sequence comprising nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243- 911, and 933-1523, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523.

133. An expression vector comprising a recombinant polynucleotide of any one of claims 126- 132.

134. The expression vector of claim 133, wherein the polynucleotide is operably linked to a control sequence.

135. A host cell comprising an expression vector of claim 133 or 134.

136. The host cell of claim 135, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

137. A method of producing a recombinant single stranded RNA ligase polypeptide in a host cell, comprising culturing a host cell of any one of claims 135 or 136, under suitable culture conditions such that the recombinant single stranded RNA ligase is produced.Docket Number CX10-269WO4 138. The method of claim 137, further comprising recovering the recombinant single stranded RNA ligase polypeptide from the culture and / or host cells.

139. The method of claim 137 or 138, further comprising purifying the recombinant single stranded RNA ligase polypeptide.

140. A composition comprising a single stranded RNA ligase of any one of claims 64-125.

141. A kit comprising at least an RNA ligase of any one of claims 64-125.

142. The kit of claim 141, further comprising one or more of a buffer, nucleotide cofactor, a ligation enhancer, and a single stranded RNA ligase substrate.

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