RNA ligase compositions and methods of use

Abstract

The present invention provides methods of preparing immobilized RNA ligase polypeptides acting on double stranded nucleic acid substrates (dsRNA ligase), and compositions and methods for using these immobilized enzymes in processes for ligating ligase substrates. Further, 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 provides recombinant single-stranded RNA ligases, polynucleotides encoding the recombinant single-stranded RNA ligases, and compositions of the single-stranded RNA ligases.

Description

RNA LIGASE COMPOSITIONS AND METHODS OF USECROSS-REFERENCE

[0001] This application claims priority to US Provisional Application No. 63 / 789,080 filed April 15, 2025 and US Provisional Application No. 63 / 903,631 filed October 22, 2025, each of which are incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name 5244_287WOl_seqlisting.xml, created on April 14, 2026, with a file size of 413,696 bytes, is part of the specification and is incorporated by reference herein.TECHNICAL FIELD

[0003] The present disclosure provides immobilized RNA ligases that acts on double stranded oligonucleotide or polynucleotide substrates (dsRNA ligase) and use of the immobilized ligase for ligating oligonucleotide and polynucleotide ligase substrates. Further, the present disclosure provides recombinant single-stranded RNA ligases and methods of use.BACKGROUND

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

[0005] 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 intemucleoside 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.

[0006] Modified oligonucleotides are generally synthesized chemically, such as by solid-phase synthesis using phosphoramidite chemistry. However, chemical synthesis has several disadvantages, including low efficiency in synthesis of long oligonucleotides, difficulty in scale-up of manufacturing, and toxic chemical waste and solvent consumption, e.g., toluene and acetonitrile. To circumvent some of the limitations in chemical synthesis. RNA ligase 2 can be used to ligate shorter oligonucleotides to generate the oligonucleotide therapeutic. RNA ligases acting on double stranded oligonucleotide or polynucleotide substrates (dsRNA ligase), also referred to as RNA ligase 2 or RNA ligase II. preferentially join a nick in an RNA duplex. In view of the potential for use of dsRNA ligase for the synthesis of modified oligonucleotides, desirable are methods and compositions of dsRNA ligases that provide efficient processes for ligation of oligonucleotide and polynucleotide substrates and post-ligation processing of products.SUMMARY

[0007] In one aspect, the present disclosure provides RNA ligase polypeptides acting on double stranded nucleic acid substrates (dsRNA ligase), where the dsRNA ligase is immobilized on a solid support. The disclosure further provides methods of using the immobilized dsRNA ligases for ligating oligonucleotide and polynucleotide substrates. The present disclosure also 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.

[0008] In some embodiments, the present disclosure provides a method for preparing an immobilized RNA ligase comprising: (a) contacting an epoxide-functionalized solid support and an RNA ligase polypeptide in solution; (b) incubating the solution under conditions effective to conjugate the RNA ligase polypeptide to the solid support; (c) adding an epoxide quenching reagent to the solution of step (b) to deactivate or cap the epoxide groups on the solid support.

[0009] In some embodiments, the present disclosure provides a method for preparing an immobilized dsRNA ligase comprising: (a) contacting an epoxide-functionalized solid support and a dsRNA ligase polypeptide in solution; (b) incubating the solution under conditions effective to conjugate the dsRNA ligase polypeptide to the solid support; (c) adding an epoxide quenching reagent to the solution of step (b) to deactivate or cap the epoxide groups on the solid support.

[0010] In some embodiments, the method further comprises removing the quenching reagent by washing the immobilized dsRNA ligase on the solid support.

[0011] In some embodiments, the method further comprises adding a second quenching reagent to the solution (b) of the dsRNA ligase and epoxide functionalized solid support.

[0012] In some embodiments, the second quenching reagent is added after completion of treatment with the quenching reagent, i.e., the first quenching reagent. In some embodiments, the solid support is washed to remove the first quenching reagent prior to adding the second quenching reagent. In some aspects, the second quenching agent is different from the first quenching agent.

[0013] In some embodiments, the solution of step (a) comprises a buffered aqueous solution at a pH of about 6.5 to about 8.5 containing the dsRNA ligase polypeptide at a concentration of about 0.1 g / L to about 50 g / L. In some aspects, the buffer in the aqueous solution is selected from borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane- 1,3 -diol (Tris). In some aspects, the buffer concentration in the buffered aqueous solution is 10 mM to 1500 mM.

[0014] In some embodiments, the incubating in (b) is carried out at a temperature of about 20 °C to about 60 °C.

[0015] In some embodiments, the epoxide quenching reagent is selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, (3-mercaptoethanol, TEoA-HCl, and any combinations thereof. In some aspects, the epoxide quenching reagent is selected from L-cysteine, L-lysine, ethanolamine, L-proline, L-alanine, L-glycine, imidazole, glucosamine, sodium thiosulfate, L-glycine benzyl ester, L-glycine methyl ester, L-glycine tert -butyl ester, L-cysteine methyl ester, N-acetyl-cysteine, (3-mercaptoethanol. TEoA-HCl, and any combinations thereof.

[0016] In some embodiments, the second epoxide quenching reagent is selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodimn thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, (3-mercaptoethanol, TEoA-HCl, or any combinations thereof. In some aspects, the secondepoxide quenching reagent is selected from L-cysteine, L-lysine, ethanolamine, L-proline, L-alanine, L-glycine, imidazole, glucosamine, sodium thiosulfate, L-glycine benzy l ester, L-glycine methyl ester, L-glycine tert-butyl ester, L-cysteine methyl ester, N-acetyl-cysteine, (3-mercaptoethanol, TEoA-HCl, and any combinations thereof.

[0017] In some embodiments, the concentration of the epoxide quenching reagent in the solution is about 10 mM to about 3000 mM, from about 20 mM to about 2000 mM, or about 50 mM to about 1000 mM. In some embodiments, the concentration of the second epoxide quenching reagent in the solution is about 10 mM to about 3000 mM, from about 20 mM to about 2000 mM, or about 50 mM to about 1000 mM.

[0018] In some embodiments, the solution of (c) is allowed to incubate until at least 90%, at least 95%, at least 99%, or at least 99.9% of epoxide functional groups are quenched.

[0019] In some embodiments, the epoxide functionalized solid support comprises particles comprising polyacrylate, methacrylate, polymethacrylate, phenolate, polystyrene, polysaccharide, silica or controlled pore glass.

[0020] In some embodiments, the epoxide functionalized solid support comprises particles having a particle size range of about 50 pm to about 1500 pm. about 100 pm to about 1000 pm. about 200 tun to about 700 pm, or about 200 pm to about 500 pm.

[0021] In some embodiments, the epoxide functionalized solid support comprises polymer particles having an average pore diameter of about 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A).

[0022] In some embodiments, the epoxide functionalized solid support comprises polymer particles, wherein the particles comprise a polymer type selected from polyacrylic, methacrylic, poly methacrylic, phenolic, polystyrene, polysaccharide, silica, or controlled pore glass.

[0023] In some embodiments, the immobilized dsRNA ligase polypeptide has an activity capable of ligating a ligase substrate comprising an oligonucleotide acceptor strand and a oligonucleotide donor strand of a double stranded oligonucleotide substrate. In some embodiments, the oligonucleotide or polynucleotide acceptor strand comprises an RNA, and / or the oligonucleotide or polynucleotide donor strand comprises an RNA.

[0024] In some embodiments, the immobilized dsRNA ligase on the solid support retains at least 20%, 30%, 40%, 50%. 60%, 70%, 75%, 80%. 85%, or 90% or greater ligase activity. In some aspects, the percent ligase activity is determined by amount of dsRNA ligase polypeptide bound to resin compared to amount of untreated DNA ligase polypeptide. In some aspects, the percent ligase activity is determined by comparing the ligase activity of dsRNA ligase polypeptide bound to resin compared to the activity of a comparable amount of untreated DNA ligase polypeptide.

[0025] In some embodiments, the immobilized dsRNA ligase on solid support exhibits leaching of dsRNA ligase of less than 2%, 5%, 10%, 15%, 20%. 25% or 30% of measured activity7on the solid support.

[0026] In some embodiments, an immobilized dsRNA ligase prepared by a method of the application is provided.

[0027] An immobilized double stranded RNA ligase (dsRNA ligase) comprising a dsRNA ligase polypeptide and an epoxide-functionalized solid support, wherein the dsRNA ligase polypeptide is attached to the solid support through a covalent linkage comprising a (3-hydroxy -amino, |3-hydroxy ether, [3-hydroxy -carboxyl, and / or (3-hydroxy-thio. wherein at least 90% of the epoxide groups are quenched.

[0028] In another aspect, the present disclosure provide a composition comprising an immobilized dsRNA ligase prepared by method of the present disclosure. In at least one embodiment, the composition is prepared by the method of:(a) contacting an epoxide-functionalized solid support and a dsRNA ligase polypeptide in solution;(b) incubating the solution to conjugate the dsRNA ligase polypeptide to the solid support; (c) adding an epoxide quenching reagent to the solution of step (b).

[0029] In some embodiments, the composition is prepared by adding a second quenching reagent to the solution (c).

[0030] In at least one embodiment, the quenched epoxide groups comprise a covalent linkage with a quenching compound, and optionally, a second quenching compound, selected from cysteine, lysine,etlianolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, 0-mercaptoethanol, TEoA-HCl, and any combinations thereof.

[0031] In some embodiments of the immobilized dsRNA ligase, the epoxide functionalized solid support comprises particles having a particle size range of about 50 pm to about 1500 pm. about 100 pm to about 1000 pm, about 200 pm to about 700 pm, or about 200 pm to about 500 pm.

[0032] In some embodiments, the epoxide functionalized solid support comprises particles having an average pore diameter of about 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A).

[0033] In embodiments of the immobilized dsRNA ligase, the epoxide functionalized solid support comprises polymer particles, wherein the particles comprise a polymer type selected from polyacrylic, methacrylic, polymethacrylic, phenolic, polystyrene, polysaccharide, silica, or controlled pore glass.

[0034] In some embodiments, the epoxide functionalized solid support is a resin selected from HFA (Resindion), HA (Resindion). BU (Resindion), EP (Resindion). EP / S (Resindion), EP403 / M (Resindion). EP600 (Resindion), SP600 (Resindion), HFA403 / S (Resindion), ECR8804F (Purolite), ECR8405F (Purolite), EMC7042 / M (Sunresin). EMC7042 / S (Sunresin), IB-COV-2 (ChiralVision BV), IB-COV-6 (ChiralVision BV), IB-COV-7 (ChiralVision BV), IB-COV-8 (ChiralVision BV), IB-ANI-5 (ChiralVision BV), IB-ANI-7 (ChiralVision BV), IB-ANI-8 (ChiralVision BV), IB-ANI-10 (ChiralVision BV), IB-ANI-13 (ChiralVision BV), EMC7025 (Sunresin), EMC7014 (Sunresin), EMC7032 (Sunresin), EMC7120 / M (Sunresin), Chelex 7350 (Sunresin), EMC7225 / M (Sunresin), EA403 / M (Resindion), HA403 / M (Resindion), IB-ANI-13 (ChiralVision BV), IB-COV-10 (ChiralVision BV), (ChiralVision BV), IB-ANI-3 (ChiralVision BV), FPA51 (Amberlite), ECR1090F (Purolite), ECR1604 (Purolite), ECR1504 (Purolite), ECR1640 (Purolite), CPG-N12 (LGC), CPG-N16 (LGC), CPG-NO cap (LGC), CPG-19 (LGC), CPG-20 (LGC), CPG-21 (LGC), IB-SLC(500A)-MPTMS-P500DGE (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE (ChiralVision BV), IB-SLC(500A)-GPTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P500DGE-MTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE-MTMS (ChiralVision BV), IB-SLC(500A)-GPTMS-MTMS (ChiralVision BV), IB-His-2 COOH (ChiralVision), IB-His-7 COOH (ChiralVision), IB-His-8 COOH (ChiralVision), IB-His-2 Co(II) (ChiralVision), A568 (Duolite), A-7 Freebase (Duolite), and AD7HP (Amberlite).

[0035] In some embodiments, the immobilized dsRNA ligase polypeptide has an activity capable of ligating a ligase substrate comprising an oligonucleotide acceptor strand and an oligonucleotide donor strand of a double stranded oligonucleotide substrate. In various aspects, the oligonucleotide acceptor strand comprises an RNA. In some aspects, the oligonucleotide donor strand comprises RNA.

[0036] In some embodiments, immobilized dsRNA ligase polypeptide or a composition thereof, wherein the dsRNA ligase polypeptide is attached to the solid support through a covalent linkage comprising a 0-hydroxy -amino, 0-hydroxy-ether, 0-hydroxy -carboxyl, and / or 0-hydroxy-thio. and wherein at least 90% of epoxide groups are quenched. In some aspects, the quenched epoxide groups comprise a covalentlinkage with a quenching compound and, optionally, a second quenching compound. The quenching compound and / or the second quenching compound may be selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, f-mercaptoethanol, TEoA-HCl, and any combinations thereof. In some aspects, the quenching compound and / or the second quenching compound may be selected from L-cysteine, L-lysine, L-proline, L-alanine. L-glycine, L-glycine benzy l ester, L-glycine methyl ester. L-glycine tert-butyl ester, L-cysteine methyl ester, N-acetyl-L-cysteine and any combinations thereof.

[0037] In embodiments of the immobilized dsRNA ligase, the epoxide functionalized solid support comprises polymer particles, wherein the particles comprise a polymer type selected from poly acre lie. methacrylic, polymethacrylic, phenolic, polystyrene, polysaccharide, silica, or controlled pore glass. In various aspects of the immobilized dsRNA ligase, the epoxide functionalized solid support comprises polyacrylic, methacrylic, polymethacrylic, phenolic, polystyrene, polysaccharide, silica, or controlled pore glass. In various aspects, the functionalized solid support comprises particles having a particle size range of about 50 pm to about 1500 pm, about 100 pm to about 1000 pm. about 200 pm to about 700 pm, or about 200 pm to about 500 pm. In some aspects, the epoxide functionalized solid support comprises polymer particles having an average pore diameter of about 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A).

[0038] In some embodiments of the immobilized dsRNA ligase, the epoxide functionalized solid support is a resin selected from HFA (Resindion), HA (Resindion), BU (Resindion), EP (Resindion), EP / S (Rcsindion), EP403 / M (Resindion), EP600 (Resindion), SP600 (Resindion), HFA403 / S (Resindion), ECR8804F (Purolite), ECR8405F (Purolite), EMC7042 / M (Sunresin), EMC7042 / S (Sunresin), IB-COV-2 (ChiralVision BV), IB-COV-6 (ChiralVision BV). IB-COV-7 (ChiralVision BV), IB-COV-8 (ChiralVision BV), IB-ANI-5 (ChiralVision BV), IB-ANI-7 (ChiralVision BV), IB-ANI-8 (ChiralVision BV), IB-ANI-10 (ChiralVision BV), IB-ANI-13 (ChiralVision BV), EMC7025 (Sunresin), EMC7014 (Sunresin), EMC7032 (Sunresin), EMC7120 / M (Sunresin), Chelex 7350 (Sunresin), EMC7225 / M (Sunresin), EA403 / M (Resindion), HA403 / M (Resindion). IB-ANI-13 (ChiralVision BV). IB-COV-10 (ChiralVision BV), (ChiralVision BV), IB-ANI-3 (ChiralVision BV), FPA51 (Amberlite). ECR1090F (Purolite). ECR1604 (Purolite), ECR1504 (Purolite), ECR1640 (Purolite), CPG-N12 (LGC). CPG-N16 (LGC), CPG-NO cap (LGC). CPG-19 (LGC). CPG-20 (LGC). CPG-21 (LGC), IB-SLC(500A)-MPTMS-P500DGE (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE (ChiralVision BV). IB-SLC(500A)-GPTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P500DGE-MTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE-MTMS (ChiralVision BV), IB-SLC(500A)-GPTMS-MTMS (ChiralVision BV). IB-His-2 COOH (ChiralVision), IB-His-7 COOH (ChiralVision), IB-His-8 COOH (ChiralVision). IB-His-2 Co(II) (ChiralVision), A568 (Duolite), A-7 Freebase (Duolite), and AD7HP (Amberlite).

[0039] In another aspect, the present disclosure provides a method of ligating dsRNA ligase substrates, comprising contacting an immobilized dsRNA ligase described herein with a ligase substrate comprising an oligonucleotide or polynucleotide acceptor strand and a oligonucleotide or polynucleotide donor strand of a double stranded oligonucleotide or polynucleotide substrate in a reaction solution under reaction conditions suitable for ligation of the oligonucleotide acceptor strand and a oligonucleotide donor strand.

[0040] In some embodiments, the present application provides a method for ligating a dsRNA ligase substrate comprising an oligonucleotide acceptor strand and an oligonucleotide donor strand under reaction conditions suitable for ligation of the oligonucleotide acceptor strand and the oligonucleotide donor strand.

[0041] In some embodiments, the suitable reaction conditions comprise a co-substrate NTP, particularly ATP. In some aspects, the NTP concentration is from 1 mM to 20 mM. In some aspects, the ATP concentration is from 1 mM to 20 mM. In some embodiments, the suitable reaction conditions further include a divalent metal cofactor. In some aspects, the divalent metal is Mg+2.

[0042] In some embodiments, the oligonucleotide or polynucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages or combinations thereof. In some embodiments, the oligonucleotide donor strand or polynucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkage or combinations thereof. In some embodiments, the oligonucleotide or polynucleotide strand complementary to the oligonucleotide or polynucleotide acceptor strand comprises a modified nucleoside and / or modified internucleoside linkage. In some embodiments, the oligonucleotide or poly nucleotide strand complementary to the oligonucleotide or polynucleotide donor strand comprises a modified nucleoside and / or modified intemucleoside linkage.

[0043] In some embodiments, the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages or combinations thereof. In some embodiments, the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkage or combinations thereof. In some embodiments, the oligonucleotide strand complementary to the oligonucleotide acceptor strand comprises a modified nucleoside and / or modified intemucleoside linkage. In some embodiments, the oligonucleotide strand complementary to the oligonucleotide donor strand comprises a modified nucleoside and / or modified intemucleoside linkage.

[0044] In some embodiments, the modified nucleoside comprises a modification of the 2’-position of the sugar moiety and / or a modified nucleobase. In some aspects, the modified nucleoside comprises a locked nucleic acid (LNA) linkage. In some aspects, the modified intemucleoside linkage comprises at least a phosphorothioate linkage.

[0045] In some embodiments, the immobilized RNA ligase comprises a packed resin in a column. In some aspects of the methods, the contacting comprises flowing the reaction solution through a packed resin in a column.

[0046] In some embodiments, the immobilized dsRNA ligase on resin is dispersed or suspended in the reaction solution. In some aspects of the methods, the reaction solution is agitated. In certain aspects, the method further comprises separating the immobilized dsRNA ligase on a resin from the reaction solution.

[0047] In some embodiments, the oligonucleotide or polynucleotide acceptor strand can have modifications at the 5 ’-terminal nucleotide. In some embodiments, the oligonucleotide or polynucleotide donor strand comprises a modified 3 ’-terminal nucleotide.

[0048] In a further aspect, the present disclosure further provides a method of carrying out and processing a dsRNA ligase reaction, comprising(a) contacting a dsRNA ligase with ligase substrate comprising a oligonucleotide acceptor strand and a oligonucleotide donor strand under suitable reaction conditions to ligate the polynucleotide acceptor and polynucleotide donor to form a ligated oligonucleotide product; and(b) passing the reaction solution through a selective filter to separate the dsRNA ligase from the ligase substrate and ligated oligonucleotide product.

[0049] In some embodiments, the present disclosure provides a method of ligating oligonucleotides with a dsRNA ligase, comprising(a) contacting a dsRNA ligase with ligase substrate comprising a oligonucleotide acceptor strand and a oligonucleotide donor strand under suitable reaction conditions to ligate the oligonucleotide acceptor and oligonucleotide donor to form a ligated oligonucleotide product; and(b) passing the reaction solution through a selective filter to separate the dsRNA ligase from the ligase substrate and ligated oligonucleotide product.

[0050] In some aspects of the methods, the filtering is through ultrafiltration (UF) or diafiltration (DF)

[0051] In some embodiments, the filtration is by ultrafiltration (UF) or diafiltration (DF).

[0052] In some embodiments, the selective filter has a molecular weight cutoff of about 100 kDa to about 3 kDa.

[0053] In some embodiments, the permeate containing the ligase substrate and / or ligated oligonucleotide product has less than about 20 ng / uL, about 15 ng / uL, about 10 ng / uL, about 9 ng / uL, about 8 ng / uL. about 7 ng / uL, about 6 ng / uL, or 5 ng / uL of protein. In some aspects, the permeate containing the ligase substrate and / or ligated oligonucleotide product has less than about 5 ng / uL of protein.

[0054] In some embodiments of the method, in (a) above, the dsRNA ligase and the ligase substrate are reacted until greater than 50%, 60%, 70%, 80%. 85%. 90%, or 95% of ligase substrate is ligated to ligated product before carrying out (b).

[0055] In some embodiments of the method, the dsRNA ligase in the retentate is reused for reaction with additional ligase substrates.

[0056] In some embodiments, the oligonucleotide or polynucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof, as described herein. In some aspects, the oligonucleotide acceptor strand comprises one or more modifiednucleosides, one or more modified internucleoside linkages, or any combinations thereof, as described herein.

[0057] In some embodiments, the oligonucleotide or polynucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof, as described herein. In some aspects, the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified internucleoside linkages, or any combinations thereof, as described herein.|0058| In some embodiments, an oligonucleotide or polynucleotide strand complementary to the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof. In some aspects, an oligonucleotide strand complementary to the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof.

[0059] In some embodiments, an oligonucleotide strand complementary to the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof.

[0060] In some embodiments, the reaction conditions for carrying out the reaction of dsRNA ligase and ligase substrate can use the conditions described for use of immobilized dsRNA ligase.

[0061] In an embodiment, a compound comprising a ligated oligonucleotide product produced by a method of the application is provided. In certain aspects of the embodiment, the compound comprises a therapeutically effective amount of the ligated oligonucleotide product.

[0062] In some embodiments, the application provides recombinant single-stranded RNA (ssRNA) ligases. The ssRNA ligases comprise 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 terminus of SEQ ID NO:2, 8, 98 or 108 or to reference sequence corresponding to SEQ ID NO:2, 8, 98 or 108. In some aspects, the amino acid sequence of the recombinant single stranded RNA ligase comprises SEQ ID NO:2, 8, 98 or 108. In some aspects, the 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 terminus of SEQ ID NO:2. 8, 98 or 108 or to reference sequence corresponding to SEQ ID NO:2, 8, 98 or 108, comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO:2, 8, 98 or 108 or to reference sequence corresponding to SEQ ID NO:2. 8, 98 or 108. In certain aspects, the 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 carboxyterminus of SEQ ID NO:2, 8, 98 or 108, comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO:2, 8, 98 or 108.

[0063] In some embodiments, the recombinant single stranded RNA ligase comprise at least one substitution at an amino acid position selected from 28, 30, 43. 58, 61, 75, 81. 84, 102, 108, 119. 122, 123, 124, 125, 127. 129, 162, 169, 170, 225, 226, 253, 254. 258, 271, 272. 276, 277, 281, 357, 358. 360, and 366, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO:2. 8, 98 or 108 or relative the reference sequence corresponding to SEQ ID NO:2. 8. 98 or 108.

[0064] In some embodiments, the 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 terminus of SEQ ID NO: 2 or to reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2.

[0065] In some embodiments, the 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 terminus of SEQ ID NO: 8 or to reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 8, or to reference sequence corresponding to SEQ ID NO: 8.

[0066] In some embodiments, the 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 terminus of SEQ ID NO: 98 or to reference sequence corresponding to SEQ ID NO: 98, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 98. or to reference sequence corresponding to SEQ ID NO: 98.

[0067] In some embodiments, the 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 terminus of SEQ ID NO: 108 or to reference sequence corresponding to SEQ ID NO: 108, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 108. or to reference sequence corresponding to SEQ ID NO: 108.

[0068] In some embodiments, the 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 terminus of an even-numbered SEQ ID NO: of SEQ ID NO:4-154 or to a reference sequence correspond to an even-numbered SEQ ID NO: of SEQ ID NO:4-154. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2. or to reference sequence corresponding to SEQ ID NO: 2.

[0069] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 102, 108, 125, 127, 169, 170, 225, 226, 253, 258, 272, 357, 358, and 366 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2. In some aspects, the at least one substitution is selected from the group comprising 102M, 108A. 108C. 125A, 127G, 127L, 127T, 169L, 169V. 170D, 170S, 225D, 226K, 253H.2531, 258H, 258N, 258S. 272S. 357E. 357S. 358E, and 366R or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2.

[0070] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 102, 108, 122, 124, 125, 127, 129, 169, 170, 225, 226, 253, 258, 271, 272, 276, 281, 357. 358, 360, and 366 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2. In some aspects, the at least one substitution is selected from the group comprising 102M, 108A, 108C, 1221, 124L, 125 A, 127G, 127L, 127T, 129G, 169V, 170A, 170D, 170M. 170S. 225D, 226D, 226Y, 2531, 253P, 258H, 258N, 271V, 272S, 272T, 276E, 281P, 357A, 357D, 357E, 357S, 358E, 360M. 360R, 360T, 366A, 366L, and 366S or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2.

[0071] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 43, 122, 253, and 17 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2. In some aspects, the at least one substitution is selected from the group comprising 43L, 122F, 253G, and 277P or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to thesequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, or to reference sequence corresponding to SEQ ID NO: 2.

[0072] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 43 / 170 / 254 / 281, 43 / 281, 170, and 170 / 281 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 8, or to reference sequence corresponding to SEQ ID NO: 8. In some aspects, the at least one substitution is selected from the group comprising 43L / 170S / 254A / 281P, 43L / 281P, 170S, and 170S / 281 P or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 8, or to reference sequence corresponding to SEQ ID NO: 8.

[0073] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 28. 30, 58, 61, 119, 122, 123, 124, 127, and 162 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 98, or to reference sequence corresponding to SEQ ID NO: 98. In some aspects, the at least one substitution is selected from the group comprising 28P, 30T, 58A, 58S, 58V, 61P, 61S, 1191. 122T, 123A, 124L, 124M, 127E, 127K, and 162G or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to tire sequence from residue 12 to the carboxy terminus of SEQ ID NO: 98, or to reference sequence corresponding to SEQ ID NO: 98.

[0074] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 58 / 75, 58 / 75 / 81 / 277, 58 / 75 / 122, 58 / 75 / 277, 75, 75 / 84, and 75 / 277 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 108, or to reference sequence corresponding to SEQ ID NO: 108. In some aspects, the at least one substitution is selected from the group comprising 58A / 75Y, 58A / 75Y / 81I / 277P, 58A / 75Y / 122F, 58A / 75Y / 277P, 75Y, 75Y / 84A, and 75Y / 277P or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 108. or to reference sequence corresponding to SEQ ID NO: 108.

[0075] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution at an amino acid position selected from the group comprising 81. 84, and 84 / 122 / 277 or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 108, or to reference sequence corresponding to SEQ ID NO: 108. In some aspects, the at least one substitution is selected from the group comprising 811, 84A. and 84A / 122F / 277P or anycombinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy tenninus of SEQ ID NO: 108, or to reference sequence corresponding to SEQ ID NO: 108.

[0076] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising at least one substitution provided in Tables 5.2, 6.2. 7.2. 8.2. 9.2, 10.2 and 11.2, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2. 8, 97 or 108, or to reference sequence corresponding to SEQ ID NO: 2. 8. 98 or 108.

[0077] 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 terminus of an even-numbered SEQ ID NO. of SEQ ID NOS: 4-154 or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOS:4-154. In some aspects, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 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 tenninus of an even-numbered SEQ ID NO. of SEQ ID NOS: 4-154 or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOS:4-154.

[0078] In some embodiments of the recombinant single stranded RNA ligase, the amino acid sequence of the recombinant ssRNA ligase comprises at least one substitution selected from the group comprising 28P, 30T, 43L, 58A / S / V. 61P / S, 75Y, 811, 84A, 102M, 108A / C, 1191, 122F / I / T, 123 A, 124L / M, 125 A, 127E / G / K / L / T, 129G, 162G, 169L / V, 170A / D / M / S, 225D, 226D / K / Y, 253G / H / I / P, 254A, 258H / N / S, 271V, 272S / T, 276E, 277P, 281P, 357A / D / E / S, 358E, 360M / R / T, and 366A / L / R / S or any combination thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, 8, 98 or 108. or to reference sequence corresponding to SEQ ID NO: 2, 8, 98 or 108.

[0079] In some embodiments of the recombinant single stranded RNA ligase, the amino acid sequence of the recombinant ssRNA ligase comprises at least one substitution selected from the group comprising 28P, 30T, 43L, 58A / S / V, 61P / S, 75Y, 811, 84A. 102M, 108A / C, 1191, 122F / I / T, 123 A, 124L / M. 125 A, 127E / G / K / L / T, 129G, 162G. 169L / V, 170A / D / M / S. 225D, 226D / K / Y, 253G / H / I / P. 254A, 258H / N / S, 271V, 272S / T, 276E, 277P, 281P, 357A / D / E / S. 358E. 360M / R / T, and 366A / L / R / S or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 2, 8, 98 or 108, or to reference sequence corresponding to SEQ ID NO: 2. 8, 98 or 108.

[0080] In various embodiments the recombinant single stranded RNA ligase exhibits at least one improved property as compared to a reference single stranded RNA ligase. In various aspects, the improved property is selected from the group comprising (i) increased single stranded ligase activity, (ii)increased single stranded ligase activity with a modified oligonucleotide substrate, (iii) increased expression in a host cell, (iv) increased thermostability, and 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 tenninus of SEQ ID NO:2, 8, 98, or 108. or an amino sequence corresponding to SEQ ID NO:2, 8, 98 or 108.

[0081] In some embodiments, the recombinant single stranded RNA ligase further comprises a fusion polypeptide. In some embodiments, the recombinant single stranded RNA ligase is a purified preparation.|0082| In some embodiments, a recombinant polynucleotide comprising a polynucleotide sequence encoding a single stranded RNA ligase is provided. In some aspects of the recombinant polynucleotide, 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’-terminus of SEQ ID NO: 1, 7, 97 or 107 or to a nucleotide sequence corresponding to SEQ ID NO: 1, 7, 97 or 107. In some aspects, the polynucleotide sequence comprises nucleotide residues 34 to the 3’-terminus of SEQ ID NO: 1, 7, 97 or 107. In some aspects of the recombinant polynucleotide, 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 1161 of an odd-numbered SEQ ID NO: of SEQ ID NO:3-153 or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: of SEQ ID NO:3-153, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. In some aspects, the recombination polynucleotide is codon-optimizcd for expression of the encoded recombinant single stranded RNA ligase. In certain aspects, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO: of SEQ ID NO:3-153 or to a polynucleotide sequence comprising an odd-numbered SEQ ID NO: of SEQ ID NO:3-153.

[0083] In some embodiments, an expression vector comprising a recombinant polynucleotide of the application is provided. In various aspects, the expression vector comprises a recombinant polynucleotide operably linked to a control sequence. In some embodiments, host cells comprising an expression vector of the application are provided. In some aspects, the host cell is selected from the group comprising a bacterial cell, a fungal cell, insect cell or mammalian cell.

[0084] In some embodiments, methods of producing a recombinant single stranded RNA ligase polypeptide in a host cell are provided. The methods of producing a recombinant single stranded RNA ligase polypeptide comprise culturing a host cell comprising an expression vector comprising a recombinant polynucleotide of the application under suitable culture conditions such that the recombinant single stranded RNA ligase is produced. In various aspects, the methods further comprise recovering the recombinant single stranded RNA ligase polypeptide from the culture and / or the host cells. In certain aspects, the methods comprise purifying the recombinant single stranded RNA ligase polypeptide. Invarious aspects, compositions comprising a recombinant single stranded RNA ligase polypeptide are provided.

[0085] In some embodiments, a kit comprising at least a recombinant single stranded RNA ligase of the application are provided. In various aspects, the kit further comprises one or more of a buffer, nucleotide cofactor, a ligation enhancer and a single stranded RNA ligase substrate.

[0086] In some embodiments, methods of synthesizing an oligonucleotide are provided. The methods comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in the presence of a recombinant single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor.

[0087] In some embodiments, the nucleotide donor, the oligonucleotide,-,,. or both the nucleotide donor and the oligonucleotide, -., comprise a modified nucleotide.

[0088] In some embodiments, the modified nucleoside comprises a conjugate moiety, reactive group, or linker.

[0089] In some embodiments, the conjugate moiety’ comprises a moiety selected from the group comprisinga carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

[0090] In some embodiments, the reactive group is selected from the group comprising an amino, -CN (cyano), N3(azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

[0091] In some embodiments, the modified nucleoside on the oligonucleotide, -,, is at the 5 ’-terminal nucleotide, an internal nucleotide, or the 3 ’-terminal nucleotide.

[0092] 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).

[0093] 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.

[0094] 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. ’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.

[0095] In some embodiments, the oligonucleotide, -,, comprises the formula (I):Ai[’A2]m«A3-OH (whereineach of Ai, A2and A3is a nucleoside;m is 0-120;OH is at the 3 ‘-position of the sugar moiety; andis an internucleoside linkage.

[0096] In some embodiments, each of A2is the same or different nucleoside. In some embodiments, the oligonucleotide,,, comprises a modified nucleoside of at least one or more of Ai, A2, or A3. In some embodiments, at least one of Ai, A2, or A3is modified with a conjugate moiety or conjugate reactive group.

[0097] In some embodiments, one or more of the intemucleoside linkage is a modified internucleoside linkage. In some embodiments, the modified intemucleoside linkage is a phosphorothioate or a phosphorodithioate.

[0098] In some embodiments, the modified nucleoside on the oligonucleotide< A) comprises the fonnula (II):A-[L]g-[M]h(II)whereinA is a nucleoside;L is a linker;g is 0 or 1;M is a conjugate moiety or reactive group: andh is 0-4;wherein g and h are not simultaneously 0.

[0099] In some embodiments, the oligonucleotide, ',, is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

[0100] 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.

[0101] In some embodiments, the nucleotide donor comprises a nucleotide(D or an oligonucleotide(D). In some embodiments tire nucleotide^; or oligonucleotide< D) comprises a modified nucleoside. In some embodiments, the modified nucleoside of nucleotide^ or oligonucleotide^ comprises a conjugate moiety, a reactive group, or linker.

[0102] 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.

[0103] 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.

[0104] In some embodiments, the modified nucleoside on the oligonucleotide(D; is at the 5 ’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.

[0105] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the modified nucleoside.

[0106] 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.

[0107] In some embodiments, the nucleotide donor comprises the formula (Illa) or (Illb):pD; or (Illa)pDi[-D2]n-D3 (Illb)whereinp is a 5 ’-phosphate group;each of D, Di, D2, and D3is a nucleoside;is an internucleoside linkage; andn is 0-120.

[0108] In some embodiments, the nucleotide donor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.

[0109] 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 Di, D2, or D3is modified. In some embodiments, at least one of Di, D2, or D3is modified with a conjugate moiety, reactive group, or linker.

[0110] In some embodiments, D comprises a modified nucleoside. In some embodiments, D is modified with a conjugate moiety, reactive group, or linker.[oni] In some embodiments, the modified nucleoside on nucleotide®) or oligonucleotide®, has the formula (IV):D-[L]q-[M]r(IV)whereinD is a nucleoside;L is a linker;q is 0 or 1;M is a conjugate moiety or reactive group: andr is 0-4;wherein q and r are not simultaneously 0.

[0112] 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.

[0113] In some embodiments, the nucleotide®) or pD includes a 3 -phosphate (e.g.. pDp).

[0114] In some embodiments, the nucleotide donor (i.e., nucleotide®) or oligonucleotide®)) 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®) 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.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the method further comprises inactivating the deblocking agent or removing or separating the unblocked extended oligonucleotide from the deblocking agent.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor (c.g., nuclcotidc(D) or oligonucleotide^;) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence. In particular, the nuclcotidc,i>, comprises a selected or predetermined nucleotidefD) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence.

[0122] 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.

[0123] In some embodiments of the method, the single-stranded RNA ligase is immobilized on a support medium. In various aspects, any method of immobilizing an RNA ligase or dsRNA ligase provided elsewhere herein may be used to immobilize an engineered single-stranded RNA ligase disclosed herein. In some embodiments, the oligonucleotide acceptor (oligonucleotide,-,,) and nucleotide donor are provided in solution or aqueous phase.

[0124] In some embodiments of the method, the oligonucleotide acceptor (oligonucleotide,,,) 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.

[0125] In some embodiments of the method, the reaction further comprises a pyrophosphatase for cleaving of pyrophosphate product.

[0126] In some embodiments of the method, the reaction further comprises an engineered creatine kinase.

[0127] In some embodiments of the method, the reaction further comprises an ATP recycling system.

[0128] In some embodiments of the method, the suitable reaction conditions comprises one or more of an NTP, a divalent metal ion, and buffer.

[0129] In some embodiments of the method, the suitable reaction conditions comprises a reaction temperature of 5-60 °C.

[0130] In some embodiments of the method, the suitable reaction conditions comprise a reaction pH of about 5-8.

[0131] In some embodiments, engineered creatine kinases are provided. The engineered creatine kinases comprise 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 SEQ ID NO: 164 or SEQ ID NO: 180. In various aspects, the amino acid sequence of the engineered creatine kinase corresponds to an amino acid sequence set forth in the group comprising SEQ ID NO:166. SEQ ID NO:168, SEQ ID NO:170. SEQ ID NO:172, SEQ ID NO: 174, SEQ ID NO: 176. SEQ ID NO: 178 and SEQ ID NO: 182. In some aspects, the engineered creatine kinase 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 SEQ ID NOs: 164 or 180, wherein the amino acid sequence comprises at least one substitution or substitution set relative to the reference sequence corresponding to SEQ ID NO: 164 or 180. In certain aspects, the engineered creatine kinase 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 SEQ ID NO: 164, wherein the amino acid sequence comprises at least one substitution or substitution set relative to the reference sequence corresponding to SEQ ID NO: 164. In other aspects, the engineered creatine kinase 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 SEQ ID NO: 180, wherein the amino acid sequence comprises at least one substitution or substitution set relative to the reference sequence corresponding to SEQ ID NO: 180. DRAWINGS

[0132] Figure 1 (Fig. 1) presents Scheme 4 depicting an ssRNA ligase reaction pathway involving L96.DESCRIPTION OF THE INVENTION

[0133] The present disclosure provides immobilized RNA ligases including both dsRNA ligases and ssRNA ligases. The RNA ligases may be engineered or wildtype. The disclosure further 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 areused to produce modified oligonucleotide, particularly modified oligonucleotides having a conjugate moiety or a reactive group. The disclosure further provides oligonucleotides prepared by the engineered or immobilized RNA ligases.

[0134] 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.

[0135] 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.

[0136] 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 ( / .e., equivalent to the term “including” and its corresponding cognates).

[0137] 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.” Abbreviations

[0138] The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0139] When the three-leter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the acarbon, “D-Ala” and “L-Ala" designate D-alanine and L-alanine, respectively.

[0140] When the one-leter abbreviations are used, upper case leters designate amino acids in the L-configuration about the a-carbon and lower-case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-leter or three-leter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0141] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). These abbreviations are also used interchangeably for nucleosides and nucleotides (nucleosides with one or more phosphate groups). Unless specifically delineated, the abbreviated nucleosides or nucleotides may be either ribonucleosides (or ribonucleotides) or 2 ’-deoxyribonucleosides (or 2’ -deoxyribonucleotides). The nucleosides or nucleotides may also be modified at the 3’ position. The nucleosides or nucleotides may be specified as being either ribonucleosides (or ribonucleotides) or 2 ’-deoxyribonucleosides (or 2’-deoxyribonucleotides) on an individual basis or on an aggregate basis. When nucleic acid 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.Definitions

[0142] In reference to the present invention, 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.

[0143] “ 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.

[0144] “ 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.

[0145] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0146] “NCBI’‘ refers to National Center for Biological Information and the sequence databases provided therein.

[0147] “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).

[0148] “RNA ligase” refers to enzymes that covalently join the 5 '-phosphor l 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 singlestranded 5’-phosphoryl termini of RNA or DNA to single-stranded 3 ’-hydroxyl termini of RNA or DNA. RNA ligase 2, also referred to herein 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 nucleic acid substrates. The phrase “RNA ligase” encompasses engineered RNA ligase variants such as, but not limited to, those described in PCT Application No.PCT / US2023 / 085838, filed December 22. 2023; PCT / US2024 / 056979, filed November 21, 2024 and PCT / US2025 / 010294, filed January 3, 2025, which are incorporated by reference in their entirety. 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.

[0149] “ Protein,” “polypeptide,” and “peptide” are used interchangeably herein 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, phosphor lation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.

[0150] “ Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gin or Q), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine. respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D -configurationabout the a-carbon. For example, “A" designates L-alanine and “a" designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0151] Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0152] “Polynucleotide.” “oligonucleotide.” and “nucleic acid’ ’ are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e.. RNA), wholly comprised of 2’ deoxy ribonucleotides (i.e.. DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo-and / or 2’ deoxyribonucleotides. The polynucleotides may also include modified nucleotides with substitutions, including 2’ substitutions (e.g., 2’-fhioro, 2’-O-methyl, 2’-O-methoxyethyL locked or constrained ethyl modifications, and others known to those skilled in the art). 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 singlestranded or double-stranded, or may include both single-stranded regions and double -stranded regions.

[0153] Nucleosides will be linked together via standard phosphodiester linkages or via one or more nonstandard linkages, including but not limited to phosphorothioate linkages. The polynucleotide may be single-stranded or double -stranded or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobases that are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase. Similarly, the terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are intended to comprise any modified or synthetic structure that is now known or discovered in the future that would be recognized by one of skill in the art as being or having the function of a “polynucleotide,” “oligonucleotide.” or “nucleic acid.” An example of a modified or synthetic structure having the function of a “polynucleotide.” “oligonucleotide.” or “nucleic acid” is PNA or peptide nucleic acid.

[0154] In some embodiments, the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide ornucleic acid or oligonucleotide, which include, among others, nucleosides linked together via other than standard phosphodiester linkages, such as non-standard linkages of phosphoramidates, phosphorothioates, amide linkages, etc.; nucleosides with modified and / or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’-O-alkyl, 2’-halo, 2,3-dideoxy, 2 ’-halo-2 ’-deoxy, (3-D-ribo LNA, a-L-ribo-LNA (e.g., locked nucleic acids), etc.; and / or 5’-phosphate analogs, including, among others, phosphorothioate, phosphoacetate, phosphoramidate, monomethylphosphate, methylphosphonate, or phosphonocarboxylate.[0155| " 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.

[0156] “Nucleoside” refers to a compound comprising a nucleobase and a sugar moiety. The nucleobases and sugar moiety are each, independently, unmodified or modified.

[0157] “ 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 intemucleoside linkages include phosphodiester -O-P(O)2-O- linkages and modified intemucleoside linkages, such as phosphorothioate -O-P-(O, S)-O- and phosphorodithioate -O-P(S)2-O-.

[0158] “ Modified oligonucleotide” refers to an oligonucleotide which contains at least one modified intemucleoside linkage and / or a modified nucleoside, or a modified terminal group.

[0159] “ 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.

[0160] “ 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 tenn “nucleoside analogue.” Nucleosides with an umnodified 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.

[0161] “Modified intemucleoside linkage” refers to as a linkage other than a phosphodiester (PO) linkage that covalently connects tw o nucleosides together. In some embodiments, exemplary modified intemucleoside linkage is a phosphorothioate or phosphorodithioate intemucleoside linkage. Other modified phosphorus-containing intemucleoside linkages include phosphotriesters, methylphosphonates, and phosphoramidates (P-NH2). See, e.g.. Clave et al., RSC Chem Biol., 2021 2(1): 94-150). In some embodiments, the modified intemucleoside linkage is a non-phosphorus containing intemucleoside 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-5'), methoxypropyl, and thiofomiacetal (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.

[0162] “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 intemucleoside linkage or an srP internucleoside linkage. The Sp diastereomer may be referred to as an S-PS internucleoside linkage or ssP intemucleoside linkage. In some embodiments, the oligonucleotide comprises one or more srP intemucleoside linkages. In some embodiments, the oligonucleotide comprises one or more ssP intemucleoside linkages. Where the chirality of a phosphorothioate intemucleoside linkage is not specified, that phosphorothioate intemucleoside linkage may be either an srP linkage or an ssP linkage.

[0163] “Non-bridging phosphorothioate intemucleoside linkage” refers to a phosphorothioate intemucleoside linkage in which the sulfur atom attached to the phosphorous atom is in place of a nonbridging oxygen atom.

[0164] “Non-bridging phosphorodithioate intemucleoside linkage” refers to a modified intemucleoside linkage which is a non-bridging phosphorodithioate intemucleoside linkage. A non-bridging phosphorodithioate intemucleoside 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.

[0165] “ 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.”

[0166] “Inverted nucleoside” refers to a nucleotide having a 3’ to 3’ and / or 5’ to 5’ intemucleoside 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’ intemucleoside linkage.

[0167] “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.

[0168] 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. Chetn. 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.

[0169] “Terminal group” as used herein refers to a group located at the first or last nucleoside in a polynucleotide or oligonucleotide. A 5’-temiinal group refers to the tenninal group bonded to 5'-or d’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.

[0170] “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 ’-terminal nucleoside, a ’-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.

[0171] “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 singlestranded 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’-tenninal nucleoside, particularly the 3’-OH of the 3’-terminal nucleoside.

[0172] “ 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.

[0173] “Enzymatically reversible blocking group” refers to a blocking group that is susceptible to removal or cleaving by an enzyme.

[0174] “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]).

[0175] “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.

[0176] “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.

[0177] “Annealing” or “Hybridization” refers to the base-pairing interactions of one nucleobase polymer (e.g., poly- and oligonucleotides) with another that results in the formation of a double -stranded structure, a triplex structure or a quaternary structure. Annealing or hybridization can occur via Watson-Crick basepairing interactions, but may be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing. In some embodiments, the nucleobase polymer that anneals or hybridizes to another is a single nucleobase polymer while in other embodiments, the nucleobase polymers are separate nucleobase polymers.

[0178] " Polynucleotide acceptor strand,” “oligonucleotide acceptor strand” and “acceptor strand” in context of a ligase substrate are used refer to any oligonucleotide or nucleotide chain or similar moiety with an exposed 3 ’-OH or equivalent thereof that may be recognized by a dsRNA ligase as a substrate for ligation.

[0179] “Polynucleotide donor strand.” “oligonucleotide donor strand” and “donor strand” in context of a ligase substrate are used refer to any oligonucleotide or nucleotide chain or similar moiety with an exposed 5’-phosphate or equivalent thereof that may be recognized by a dsRNA ligase as a substrate for ligation.

[0180] “Nick” or “ligatable nick” refers to a break on a strand of a double stranded oligonucleotide or polynucleotide where there is no phosphodiester bond between adjacent nucleotides of the oligonucleotide or polynucleotide strand. The nucleotide on one side of the nick comprises a 3 ’-OH group or equivalent thereof and the nucleotide on the other side of the nick comprises a 5 ’-phosphate group or equivalent thereof such that a ligase can form a phosphodiester bond to join the nucleotides.

[0181] “Ligation junction” refers to a nick on a double stranded oligonucleotide or polynucleotide. A ligation junction is formed by an oligonucleotide or polynucleotide acceptor strand and an oligonucleotide or polynucleotide donor strand that are hybridized adjacent to each other on a complementary oligonucleotide or polynucleotide strand.

[0182] “Naturally-occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or 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.

[0183] “Recombinant,” “engineered.” and “non-naturally occurring” when used with reference to a cell, nucleic acid, or 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. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or expressed native genes that are otherwise expressed at a different level.

[0184] “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.

[0185] “Fragment” as used herein refers to a polypeptide that has an amino-terminal and / or carboxyterminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%. 98%, and 99% of the full-length RNA ligase polypeptide, for example a DsRNA ligase provided in any one of PCT Application No.PCT / US2023 / 085838, filed December 22, 2023; PCT / US2024 / 056979. filed November 21, 2024 and PCT / US2025 / 010294. filed January 3, 2025 or an ssRNA ligase.

[0186] “Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and / or carboxy -terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full length recombinant ssRNA ligase of the present invention) and that retains substantially all of the activity of the full-length polypeptide. Functional fragments of the single stranded RNA ligases of the present application include, but are not limited to ssRNA ligases corresponding to residues 12 to the carboxy terminus end of a reference sequence set forth in SEQ ID NOS: 2, 8, 98 or 108.

[0187] “ 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 ( / .e., gaps) as compared to the reference sequence for optimal alignment of the tw o 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 Softw are Package), or by visual inspection, as known in the art. Examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length " W” in the query sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary' determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0188] “ 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 primary7sequence.

[0189] " 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 someembodiments, 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.

[0190] “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 single stranded RNA ligase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag).

[0191] “ 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 ammo acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0192] “ 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 adeletion and may be denoted by awhere 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.

[0193] 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 aThe 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.

[0194] “Amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5. 6. 7, 8, 9, 10, 11, 12, 13. 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant single stranded RNA ligase polypeptides listed in any of the Tables in the Examples. In some embodiments, the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set. In the substitution sets, the individual substitutions are separated by a semicolon or slash “ / ”.

[0195] “ 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.

[0196] " 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 aromaticamino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0197] “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 single stranded RNA ligase. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated by and may be present in substitution sets.

[0198] “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.

[0199] “ 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 engineered DsRNA ligase enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as ly sates or isolated preparations. As such, in some embodiments, the engineered DsRNA ligase enzyme can be an isolated polypeptide. The recombinant single stranded RNA ligase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant single stranded RNA ligase polypeptides provided herein are isolated polypeptides.

[0200] “Substantially pure polypeptide” 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 DsRNA ligase composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e.. contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated engineered DsRNA ligasepolypeptide is a substantially pure polypeptide composition. Generally, a substantially pure single stranded RNA ligase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant single stranded RNA ligase polypeptides are substantially pure polypeptide compositions.

[0201] “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 single stranded RNA ligases are codon optimized for optimal production from the host organism selected for expression.

[0202] “ Control sequence” refers herein to include all components that are necessary’ or advantageous for the expression of a polynucleotide and / or poly peptide of the present disclosure. Each control sequence may be native or foreign (c.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.

[0203] “Operably linked” or “operatively linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide of interest, and where appropriate, expression of the encoded polypeptide of interest.

[0204] “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.

[0205] “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). “Suitable reaction conditions” refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which a dsRNA ligase polypeptide is capable of ligating an oligonucleotide or polynucleotide ligase substrate.

[0206] “Conversion” refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a DsRNA ligase polypeptide can be expressed as “percent conversion” of the substrate to the product.

[0207] “Composition” refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances useful for biocatalysis.

[0208] “Loading”, such as in “compound loading” or “enzyme loading” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0209] “ Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst. For example, a dsRNA ligase acts on a nick formed by an oligonucleotide or polynucleotide acceptor strand, and an oligonucleotide or polynucleotide donor strand that hybridize adjacently to each other on an oligonucleotide or polynucleotide strand complementary to the oligonucleotide or polynucleotide acceptor strand and oligonucleotide or polynucleotide donor strand.

[0210] “ Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst. “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the single stranded RNA ligase polypeptide on the substrate.

[0211] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0212] “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.

[0213] “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.

[0214] “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.

[0215] “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.

[0216] “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 single stranded RNA ligase). 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.

[0217] “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.

[0218] “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-l-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-l-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.

[0219] “Alkynyl” refers to a straight or branched chain hy drocarbon having the number of carbon atoms designated, for example 2 to 12 carbon atoms (C2-C12 or C2-12), particularly 2 to 8 carbon atoms (C2-C8 or C2-8), containing at least one triple bond. Exemplary “alkynyl” includes ethynyl, 1-propynyl, 2-propynyl, 1-butynyl. 2-buty nyl, 3-butynyl, 1 -penty nyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5 -hexynyl.

[0220] “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 alky l, alkyloxy,, hydroxyl, carbonyl, carboxyl, halo, nitro, and the like.

[0221] “Lower” in reference to substituents refers to a group having between one and six carbon atoms.

[0222] " Heteroalkyl, “heteroalkenyl.” and heteroalkynyl,” refer respectively, to alkyl, alkenyl and alky nyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to -O-. -S-. -S-O-, -NRg-. -PH-, -S(O)-, -S(O)2-, -S(O) NR8-, -S(O)2NR8, and the like, including combinations thereof, where each R8is independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0223] “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).

[0224] “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.

[0225] “ 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.

[0226] “ 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.

[0227] “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:

[0228] In some embodiments, a locked nucleoside is a bridged bicyclic compound.

[0229] “ 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.

[0230] “Carbonyl” refers to -C(O)-. The carbonyl group may be further substituted w ith 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.

[0231] “Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

[0232] “Haloalkyl” refers to an alkyl substituted with 1 or more halogen atoms. Preferably, the alkyl is substituted with 1 to 3 halogen atoms.

[0233] “Hydroxy” refers to -OH.

[0234] “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.

[0235] “ Acyl” refers to -C(O)R’. where R is hydrogen, or an optionally substituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl as defined herein. Exemplary acyl groups include, but are not limited to, formyl, acetyl, cyclohexy Icarbony 1, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

[0236] “Alkyloxy ” or “alkoxy” refers to -OR’, wherein R’ is an optionally substituted alkyl.

[0237] “Aryloxy” refers to -OR’, wherein R‘ is an optionally substituted ary l.

[0238] “Carboxy” refers to -COO- or COOM, wherein H or a M+ counterion.

[0239] “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.

[0240] “Cyano” refers to -CN.

[0241] “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, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.[0242| " 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. heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. As defined herein, any heterocycloalkyl or heteroaryl group present in a silyl group has from 1 to 3 heteroatoms selected independently from O, N, and S.

[0243] “Thiol” or “sulfhydryl” refers to -SH.

[0244] “ Disulfied” refers to -S-S- groups.

[0245] “Sulfanyl” refers to -SR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. For example, -SR, wherein R is an alkyl is an alkydsulfanyl.

[0246] “ 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: alky l, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0247] “ Amino” or “amine” refers to the group -NH2. Substituted amino refers to the group -NHRh, NRhRh, and NRhRhRh, where each Rhis independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonyl amino, furanyl-oxy-sulfamino, and the like.

[0248] “Aminoalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.

[0249] " Aminocarbonyl” refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NRhRh, where the amino group NRhRhis as defined herein.

[0250] “Alkoxy” or “alkyloxy” are used interchangeably herein to refer to the group -ORZ, wherein Rzis an alkyl group, including optionally substituted alkyl groups.

[0251] “Carboxyalkyl” refers to an alkyl in which one or more of the hydrogen atoms are replaced with one or more carboxy groups.

[0252] “Aminocarbonylalkyr refers to an alkyl substituted with an aminocarbonyl group, as defined herein.

[0253] “Halogen" or “halo" refers to fluoro, chloro, bromo and iodo.

[0254] “Haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl" is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression “(Ci - C2) haloalkyl" includes 1 -fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 1,1 -difluoroethyl. 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.

[0255] “Hydroxy" refers to -OH.

[0256] “Hydroxyalkyl” refers to an alkyl group in which in which one or more of the hydrogen atoms are replaced with one or more hydroxy groups.

[0257] “Thiol” or “sulfanyl” refers to -SH. Substituted thiol or sulfanyl refers to -S-Rh, where Rhis an alkyl, aryl, or other suitable substituent.

[0258] “Alkylsulfonyl" refers to -SCF-R'. where Rzis an alkyl, which can be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0259] “Phosphate” as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups.

[0260] “Phosphory lated” as used herein refers to the addition or presence of one of more phosphory l groups (phosphorous atom covalently linked to the three oxy gen atoms).

[0261] “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.

[0262] “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, ary l, 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, NRR (e.g., primary amine (i.e., NHz), secondary amine, tertian' 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 issubstituted, such substitutions will typically number less than about 10 substitutions, more preferably about 1 to 5, with about 1 or 2 substitutions being preferred.

[0263] “Optionally substituted” as used herein with respect to the foregoing chemical groups means that positions of the chemical group occupied by hydrogen can be substituted with another atom (unless otherwise specified) exemplified by, but not limited to carbon, oxygen, nitrogen, or sulfur, or a chemical group, exemplified by. but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino. amidoximo. hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy. pyrrolidinyl. piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatoms are oxygen, nitrogen, and sulfur. Additionally, where open valences exist on these substitute chemical groups they can be further substituted with alky 1, cycloalkyl, ary 1, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur -bonded substituents, and where multiple such open valences exist, these groups can be joined to fonn a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further contemplated that the above substitutions can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention and is otherwise chemically reasonable. One of ordinary skill in the art would understand that with respect to any chemical group described as optionally substituted, only sterically practical and / or synthetically feasible chemical groups are meant to be included. “Optionally substituted” as used herein refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl.” the “alky 4” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, aryl and heteroaryl.” the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted.

[0264] " Reaction” as used herein refers to a process in which one or more substances or compounds or substrates is converted into one or more different substances, compounds, or processes.

[0265] “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 arenonsuperimposable mirror 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.

[0266] The terms “effective amount”, “therapeutically effective amount” or “pharmaceutically effective amount” as used herein, can refer to a sufficient amount of a ligated oligonucleotide product which will relieve to some extent one or more signs or symptoms of the a disease, disorder or condition of interest. An “effective amount”, "therapeutically effective amount” or "pharmaceutically effective amount” of a pharmaceutical composition may be administered to a subject in need thereof as a unit dose (as described in further detail elsewhere herein). The subject may be a human or non-human mammal.Immobilization of dsRNA ligase Polypeptides

[0267] In one aspect, the present disclosure is directed to the preparation and use of immobilized dsRNA ligase polypeptides. Generally, the immobilization process comprises contacting the dsRNA ligase polypeptide in solution with a solid support under conditions suitable for the formation of a covalent or non-covalent linkage between the polypeptide and a functional group on the solid support. The resulting attachment of dsRNA ligase polypeptide to the support should allow the enzyme to retain its desired activity. The immobilized dsRNA ligase polypeptide can then be used in a ligation reaction while the ligase is retained on the support, and the desired oligonucleotide reaction products and unreacted substrates separated from the immobilized dsRNA ligase.

[0268] In some embodiments, the immobilized dsRNA ligase polypeptide is packed in a column allowing a flow-through ligation of oligonucleotide and polynucleotide dsRNA ligase substrates, wherein the substrates and NTP co-substrate are passed through the column, with the desired products collected at the column output. Where the ligase product is readily separable from the ligase substrates, the ligated product can be removed and the remaining ligase substrates contacted with the immobilized dsRNA ligase to further the formation of ligase products.

[0269] In some embodiments, the immobilized dsRNA ligase is used in batch reaction, where the immobilized dsRNA ligase on the support is mixed in the reaction solution in a vessel or container, such as with an impeller, with ligase substrates until the desired level of ligated product is formed. The immobilized dsRNA ligase can then be separated out from the reaction solution, such as by filtration or centrifugation.

[0270] In some embodiments, the dsRNA ligase can be immobilized non-covalently or covalently on various solid supports or support mediums (e.g.. resins, membranes, beads, glass, etc.). In some embodiments, support medium, including solid supports, useful for immobilizing the dsRNA ligase polypeptides include but are not limited to particles, beads, resins, or membranes, among others, of polyacrylic, methacrylic, polymethacrylic, phenolic, polystyrene, polyacrylamide, polyamino acid or protein (e.g., gelatin), polysaccharide or cellulosic (e.g., agarose, dextran, chitosan, pectin, etc.), controlled pore glass, and silica (e.g., silicon dioxide).

[0271] In some embodiments, the support medium or solid support is in the form of particles, such as beads, having a particle size range of about 1 pm to about 2000 pm. about 10 pm to about 1700 pm, about50 pm to about 1500 gin, about 100 pin to about 1000 pin, about 200 pin to about 700 pin, or about 200 pin to about 500 pin. In some embodiments, the particles have a particle size of about 1 pm, about 10 pm, about 50 pm, about 100 pm, about 200 pm, about 300 pm, about 400 pm. about 500 pm, about 1000 pm, about 1500 pm, 1600 pm, 1700 pm, 1800 pm. 1900 pm, or 2000 pm. In some embodiments, the size of the particles are selected for solution flow properties, amount and density of functional groups, surface area, diffusion of substrates, and the like.

[0272] In some embodiments, the particles or resins for immobilization of dsRNA ligase polypeptide comprises an average pore diameter or pore size of about 100 angstroms (A) to about 2000 angstroms (A), 150 angstroms (A) to about 1800 angstroms (A), 200 angstroms (A) to about 1600 angstroms (A), 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A). In some embodiments, the particles comprise an average pore diameter or pore size of about 100 angstroms (A). 150 angstroms (A), 200 angstroms (A). 250 angstroms (A), 300 angstroms (A), about 600 angstroms (A), about 1000 angstroms (A), about 1500 angstroms (A). 1600 angstroms (A). 1700 angstroms (A), 1800 angstroms (A), 1900 angstroms (A), 2000 angstroms (A), or greater.

[0273] Immobilization methods disclosed in the art include, e.g.. Yi et al., Proc. Biochem., 2007,, 42(5):895-898; Martin et al., Appl. Microbiol. Biotechnol.. 2007, 76(4):843-851; Koszelewski et al., J. Mol. Cat. B: Enzymatic. 2010, 63:39-44; Truppo et al., Org. Proc. Res. Dev., published online: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques, 2nded., Academic Press, Cambridge, MA (2008); Mateo et al., Biotechnol. Prog., 2002, 18(3):629-34; and “Bioconjugation Protocols: Strategies and Methods,” In Methods in Molecular Biology. Niemeyer (ed.), Humana Press, New York, NY (2004); the disclosures of each which are incorporated by reference herein.

[0274] A wide range of solid supports useful for immobilizing enzymes are available and it is contemplated that these can be used with dsRNA ligase polypeptides.

[0275] In some embodiments, the support material comprises a polymethacrylate resin. Exemplary polymethacrylate resins include HFA (Resindion). HA (Resindion), BU (Resindion), EP (Resindion), EP / S (Resindion), EP403 / M (Resindion), EP600 (Resindion), SP600 (Resindion), HFA403 / S (Resindion), ECR8804F (Purolite), and ECR8405F (Purolite).

[0276] In some embodiments, the support material comprises a polyacrylic resin. Exemplary polyacrylic resins include IB-COV-2 (ChiralVision BV), IB-COV-6 (ChiralVision BV), IB-COV-7 (ChiralVision BV), IB-COV-8 (ChiralVision BV), IB-ANI-5 (ChiralVision BV), IB-ANI-7 (ChiralVision BV), IB-ANI-8 (ChiralVision BV), IB-ANI-10 (ChiralVision BV), IB-ANI-13 (ChiralVision BV), EMC7025 (Sunresin), EMC7014 (Sunresin), EMC7032 (Sunresin). EMC7120 / M (Sunresin), Chelex 7350 (Sunresin), EMC7225 / M (Sunresin), EMC7042 / M (Sunresin), EA403 / M (Resindion), HA403 / M (Resindion), and EMC7042S (Sunresin).

[0277] In some embodiments, the support material comprises a cellulose resin. Exemplary cellulose resins include IB-ANI-13 (ChiralVision BV) and IB-COV-10 (ChiralVision BV).

[0278] In some embodiments, the support material comprises a polystyrene, styrene, or macroporous styrene resin. Exemplary polystyrene, styrene, or macroporous styrene resins include IB-ANI-2 (ChiralVision BV), IB-ANI-3 (ChiralVision BV), FPA51 (Amberlite), ECR1090F (Purolite), ECR1604 (Purolite). ECR1504 (Purolite), and ECR1640 (Purolite).

[0279] In some embodiments, the support material comprises a controlled pore glass (CPG) resin.Exemplary CPGs include CPG-N12 (LGC). CPG-N16 (LGC), CPG-NO cap (LGC), CPG-19 (LGC), CPG-20 (LGC), and CPG-21 (LGC).

[0280] In some embodiments, the support material comprises silica. Exemplary silica resins include 1B-SLC(500A)-MPTMS-P500DGE (LGC), IB-SLC(500A)-MPTMS-P1000DGE (LGC). IB-SLC(500A)-GPTMS (LGC), IB-SLC(500A)-MPTMS-P500DGE-MTMS (LGC), IB-SLC(500A)-MPTMS-P1000DGE-MTMS (LGC). and IB-SLC(500A)-GPTMS-MTMS.

[0281] In some embodiments, the support material comprises an affinity resin. Exemplary affinity’ resins include IB-His-2 COOH (ChiralVision). IB-His-7 COOH (ChiralVision), IB-His-8 COOH (ChiralVision), and IB-His-2 Co(II) (ChiralVision).

[0282] In some embodiments, some alternative solid supports can be used, including, but not limited to, EnginZyme (including. EziG-1, EziG-2, and EziG-3), chitosan beads. Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120).

[0283] In some embodiments, the support material comprises another organic or inorganic material. Additional exemplar}’ support materials comprise A568 (Duolite), A-7 Freebase (Duolite), and AD7HP (Amberlite).

[0284] Exemplary solid supports useful for immobilizing the engineered dsRNA ligase polypeptides of the present disclosure include those disclosed for use as described in the Examples, and include but are not limited to the supports listed in Table 2 below.

[0285] Covalent Attachment

[0286] In some embodiments, the dsRNA ligase polypeptide is immobilized on the support material by covalent attachment to a reactive chemical group. Exemplary reactive chemical groups include sulfonic, epoxide, epoxy, amino-epoxy, iminodiacetate, amino, primary amine, secondary amine, quaternary amine, tertiary amine, NH2, octadecyl, butyl, high butyl, low butyl, and hydroxyethyl.

[0287] In some embodiments, the dsRNA ligase polypeptide comprises one or more amino acid residues reactive with the reactive chemical group on the support material. In some embodiments, the reactive amino acid is part of the amino acid sequence of the dsRNA ligase, whether the dsRNA ligase is naturally occurring or an engineered dsRNA ligase. In some embodiments, reactive amino acids can be added into the dsRNA ligase amino acid sequence by recombinant methods, such as insertion of cysteine, lysine, aspartic acid, tyrosine, serine, and / or threonine residues. In some embodiments, the dsRNA ligase can befused to polylysine residues (e.g.. 2-12 or more lysine residues), for example, at the amino or carboxy terminus of the dsRNA ligase.

[0288] In some embodiments, the reactive chemical is attached to the support medium via a linker. Any suitable linker may be used. Exemplary linkers include ethylamine. alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, P-hydroxy -amino, P -hydroxy -ether, p-hydroxy-carboxyl, and / or p-hydroxy-thio cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like. In some embodiments the linker comprises a CS-C alkylene or polyethylene linker. In some embodiments, the linker is a silane or silanol linker. In some embodiments, the linker is a poly(ethylene glycol) linker, such as (3-mercaptopropyl)trimethoxysilane, poly(ethylene glycol) diglycidyl ether, or (3-glycidyloxypropyl)trimethoxysilane. Various suitable linkers are known in the art.

[0289] In some embodiments of the method of preparing an immobilized dsRNA ligase, the solid support is an epoxide functionalized solid support. In such an embodiment the engineered dsRNA ligase polypeptide forms a covalent linkage with the solid support. A schematic illustration of immobilization reaction between epoxide-functionalized solid support and a primary amine bearing amino acid residue of an dsRNA ligase polypeptide to form P-hydroxy amino linkage between solid support and protein as shown in Scheme 1 below.Scheme 1

[0290] The four exemplary 0-hydroxy linkages that can form in the reaction betw een an epoxide functionalized solid support and an engineered DsRNA ligase protein are shown in Scheme 2 below.

[0291] In some embodiments, improvement in the performance of the immobilized dsRNA ligase polypeptide occurs when immobilization on an epoxide functionalized solid support includes a step ofquenching the excess epoxide functional group on the support with one or more quenching reagents. Scheme 3 shows an exemplary quenching reaction that use ethanolamine to “quench” the remaining unreacted epoxide groups on a solid support following an epoxide immobilization reaction of dsRNA ligase protein.Scheme 3

[0292] Accordingly, in some embodiments, a method for preparing an immobilized double stranded RNA ligase (dsRNA ligase) comprises:(a) contacting an epoxide-functionalized solid support and a dsRNA ligase polypeptide in solution;(b) incubating the solution under conditions effective to conjugate the dsRNA ligase polypeptide to the solid support; and(c) adding an epoxide quenching reagent to the solution of step (b) to deactivate or cap the epoxide groups on the solid support.

[0293] In some embodiments, a method of preparing an immobilized RNA ligase (DsRNA ligase) comprises:(a) contacting an epoxide functionalized solid support and an dsRNA ligase polypeptide under conditions suitable for reaction of the epoxide with the dsRNA ligase polypeptide; and(b) adding an epoxide quenching reagent to inactivate or cap the unreacted epoxides remaining on the solid support following the reaction of the epoxide functionalized solid support and the dsRNA ligase polypeptide.|0294| In some embodiments, a method of preparing an immobilized dsRNA ligase comprises:(a) preparing a solution comprising an epoxide functionalized solid support and an dsRNA ligase polypeptide;(b) allowing the solution to incubate at a temperature of about 20 °C to about 60 °C for about 1 h to about 6 h; and(c) adding an epoxide quenching reagent to the solution of step (b).

[0295] In at least an embodiment of the method of preparing an immobilized dsRNA ligase, the quenching reagent, or first epoxide quenching reagent, is selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, (3 -mercaptoethanol, or TEoA-HCl, or any combinations thereof.

[0296] In some embodiments of the method of preparing an immobilized dsRNA ligase, the epoxide quenching reagent, or first epoxide quenching reagent, is selected from L-cysteine, L-lysine, ethanolamine, L-proline, L-alanine, L-glycine, imidazole, glucosamine, sodium thiosulfate, L-glycine benzyl ester, L-glycine methyl ester, L-glycine tert-butyl ester, L-cysteine methyl ester, or N-acetyl-L-cysteine, P-mercaptoethanol, or TEoA-HCl, or any combinations thereof.

[0297] In at least one embodiment, the concentration of the epoxide quenching reagent in the solution, is about 10 mM to about 3000 mM, about 50 mM to about 2500 mM, about 100 mM to about 2500 mM, about 200 mM to about 2000 mM, or about 500 mM to about 1000 mM. In some embodiments, the concentration of quenching agent in the solution is about 10 mM, about 50 mM, about 100 mM, about 200 mM, about 500 mM. about 1000 mM, about 1500 mM, about 2000 mM, about 2500 mM, or about 3000 mM. The concentration of the quenching agent can be adjusted depending on the type of quenching agent, the level of quenching desired or targeted, and the number of treatments with quenching agent (e.g., first quenching treatment or second quenching treatment, as further discussed below).

[0298] In some embodiments, as noted above, the reaction or incubation of the dsRNA ligase polypeptide and the epoxide functionalized solid support is carried out at 20 °C to 60 °C.

[0299] In some embodiments, the method further comprises removing the quenching reagent by washing the immobilized dsRNA ligase on the solid support.

[0300] In some embodiments of the method, the solution for reaction of the dsRNA ligase polypeptide and the epoxide functionalized solid support is a buffered aqueous solution at a pH of about 6.5 to about8.5. In some embodiments, the buffer in the aqueous solution is selected from 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).

[0301] In some embodiments, the buffer concentration is from about 10 mM to about 2000 mM, 50 mM to about 1500 mM, about 100 mM to about 1000 mM. about 150 mM to about 800 mM. about 200 mM to about 600 mM, or about 300 to about 500 mM. In some embodiments, the buffer concentration is about 10 mM. 50 mM. 100 mM, 150 mM, 200 mM. 300 mM, 400 mM, 500 mM. 600 mM, 700 mM, 800 mM, 1000 mM, 1500 mM, or 2000 mM.

[0302] In some embodiments, the dsRNA ligase polypeptide is provided at a concentration of about 0.1 g / L to about 100 g / L; about 1 g / L to about 90 g / L, about 2 g / L to about 80 g / L, 3 g / L to about 70 g / L, about 4 g / L to about 60 g / L, about 5 g / L to about 50 g / L, about 10 g / L to about 40 g / L, or about 20 g / L to about 30 g / L. In some embodiments, the dsRNA ligase polypeptide is provided at a concentration of about 0.1 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 5 g / L, about 10 g / L, about 20 g / L. about 30 g / L. about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L. about 80 g / L, about 90 g / L or about 100 g / L.

[0303] In some embodiments, a second epoxide quenching reagent is used. In some embodiments, the method further comprises adding a second quenching reagent to the solution of the dsRNA ligase and epoxide functionalized solid support. In some embodiments, the second epoxide quenching reagent is different from the first epoxide quenching agent. In some embodiments, the second epoxide quenching agent is used after quenching or capping of the epoxide groups using the first quenching reagent. In some embodiments, the second quenching reagent is added after completion of the treatment with the quenching reagent, i.e., first quenching reagent. In some embodiments, the second quenching agent is used after removal, e.g., washing, of the first quenching agent from the immobilized dsRNA ligase on the solid support.

[0304] In some embodiments, the second quenching agent is reacted in a buffered aqueous solution at a pH of about 6.5 to about 8.5. In some embodiments, tire second quenching agent comprises a buffer selected from 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).

[0305] In some embodiments, the buffer concentration for use with the second quenching agent is from about 10 mM to about 2000 mM. 50 mM to about 1500 mM, about 100 mM to about 1000 mM, about 150 mM to about 800 mM. about 200 mM to about 600 mM. or about 300 to about 500 mM. In some embodiments, the buffer concentration is about 10 mM, 50 mM, 100 mM, 150 mM. 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 1000 mM, 1500 mM, or 2000 mM.

[0306] In at least one embodiment of the method of preparing an immobilized dsRNA ligase, the second epoxide quenching reagent is selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester. N-acetyl-cysteine. P-mercaptoethanol, or TEoA-HCl.

[0307] In some embodiments of the method of preparing an immobilized dsRNA ligase, the second epoxide quenching reagent is selected from L-cysteine, L-lysine, ethanolamine, L-proline, L-alanine. L-glycine, imidazole, glucosamine, sodium thiosulfate, L-glycine benzyl ester, L-glycine methyl ester, L-glycine tert-butyl ester, L-cysteine methyl ester, or N-acetyl-L-cysteine, P-mercaptoethanol, or TEoA-HC1, or any combinations thereof.

[0308] In at least one embodiment, the concentration of the second epoxide quenching reagent in the solution is about 10 mM to about 3000 mM, about 50 mM to about 2500 mM, about 100 mM to about 2500 mM, about 200 mM to about 2000 mM, or about 500 mM to about 1000 mM. In some embodiments, the concentration of second quenching agent in the solution is about 10 mM, about 50 mM, about 100 mM, about 200 mM, about 500 mM, about 1000 mM, about 1500 mM, about 2000 mM, about 2500 mM, or about 3000 mM.

[0309] It is to be understood that tire treatment of the support medium with quenching agent following reaction of the dsRNA ligase polypeptide with the epoxide functionalized support is not limited to two treatments. In some embodiments, the additional treatments of the support medium with the quenching reagent can be carried out multiple times to reduce the levels of epoxide groups on the support medium.

[0310] In some embodiments of the method of preparing an immobilized dsRNA ligase, treatment with the quenching is done until at least 80%, 90%. at least 95%, at least 99%, or at least 99.9%, or greater of epoxide functional groups are quenched. In at least one embodiment of the method of preparing an immobilized dsRNA ligase, the solution of step (c) described herein is allowed to incubate until at least 80%, 90%, at least 95%, at least 99%, or at least 99.9%, or greater of epoxide functional groups are quenched.

[0311] In at least one embodiment of the method of preparing an immobilized dsRNA ligase, the epoxide functionalized solid support comprises particles having a particle size range of about 1 pm to about 2000 pm, about 10 pm to about 1700 pm, about 50 pm to about 1 00 pm, about 100 pm to about 1000 pm, about 200 pm to about 700 pm, or about 200 pm to about 500 pm. In some embodiments, the epoxide functionalized particles have a particle size of about 1 pm, about 10 pm, about 50 pm, about 100 pm, about 200 pm. about 300 pm, about 400 pm, about 500 pm, about 1000 pm, about 1500 pm. 1600 pm, 1700 pm, 1800 pm, 1900 pm, or 2000 tun. In some embodiments, the size of the particles are selected for solution flow properties, amount and density of functional groups, surface area, diffusion of substrates, and the like.

[0312] In at least one embodiment of the method of preparing an immobilized dsRNA ligase, the epoxide functionalized solid support comprises particles having an average pore diameter of about 100 angstroms (A) to about 2000 angstroms (A), 150 angstroms (A) to about 1800 angstroms (A), 200 angstroms (A) to about 1600 angstroms (A), 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A). In some embodiments, the particles comprise an average pore diameter or pore size of about 100 angstroms (A). 150 angstroms (A), 200 angstroms (A).250 angstroms (A). 300 angstroms (A), about 600 angstroms (A), about 1000 angstroms (A), about 1500angstroms (A), 1600 angstroms (A), 1700 angstroms (A), 1800 angstroms (A), 1900 angstroms (A), 2000 angstroms (A), or greater.

[0313] In at least one embodiment of the method of preparing an immobilized dsRNA ligase, the epoxide functionalized solid support comprises particles, beads, resins, or membranes, among others, comprised of polyacrylic, methacrylic, polymethacrylic, phenolic, polystyrene, polyacrylamide, polyamino acid or protein (e.g., gelatin), polysaccharide or cellulosic (e.g., agarose, dextran, chitosan, pectin, etc.), controlled pore glass, or silica (e.g., silicon dioxide).|0314| In at least one embodiment of the method of preparing an immobilized dsRNA ligase, the epoxide functionalized solid support is a resin selected from HFA (Resindion), HA (Resindion), BU (Resindion), EP (Resindion), EP / S (Resindion), EP403 / M (Resindion), EP600 (Resindion), SP600 (Resindion), HFA403 / S (Resindion), ECR8804F (Purolite). ECR8405F (Purolite), EMC7042 / M (Sunresin).EMC7042 / S (Sunresin), IB-COV-2 (ChiralVision BV). IB-COV-6 (ChiralVision BV). IB-COV-7 (ChiralVision BV). IB-COV-8 (ChiralVision BV), IB-ANI-5 (ChiralVision BV), IB-ANI-7 (ChiralVision BV), IB-ANI-8 (ChiralVision BV), IB-ANL10 (ChiralVision BV), IB-ANI-13 (ChiralVision BV), EMC7025 (Sunresin), EMC7014 (Sunresin), EMC7032 (Sunresin). EMC7120 / M (Sunresin). Chelex 7350 (Sunresin), EMC7225 / M (Sunresin), EA403 / M (Resindion), HA403 / M (Resindion), IB-ANI-13 (ChiralVision BV), IB-COV-10 (ChiralVision BV), (ChiralVision BV). IB-ANI-3 (ChiralVision BV), FPA51 (Amberlite), ECR1090F (Purolite), ECR1604 (Purolite), ECR1504 (Purolite), ECR1640 (Purolite), CPG-N12 (LGC), CPG-N16 (LGC), CPG-NO cap (LGC), CPG-19 (LGC), CPG-20 (LGC), aCPG-21 (LGC), IB-SLC(500A)-MPTMS-P500DGE (LGC), IB-SLC(500A)-MPTMS-P1000DGE (LGC), IB-SLC(500A)-GPTMS (LGC), IB-SLC(500A)-MPTMS-P500DGE-MTMS (LGC), IB-SLC(500A)-MPTMS-P1000DGE-MTMS (LGC), IB-SLC(500A)-GPTMS-MTMS, IB-His-2 COOH (ChiralVision), IB-His-7 COOH (ChiralVision), IB-His-8 COOH (ChiralVision), IB-His-2 Co(II) (ChiralVision), A568 (Duolite), A- 7 Freebase (Duolite), and AD7HP (Amberlite), as well as any others included in Table 2.

[0315] In some embodiments, the immobilized engineered dsRNA ligase polypeptides can be prepared using dsRNA ligase polypeptide in various forms, for example, such as an isolated preparation, as a substantially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, and / or as cell extracts and / or lysates of such cells. In some embodiments, the final immobilized enzyme preparations can be lyophilized, spray -dried, precipitated, or be in the form of a crude paste, as further discussed below. In some embodiments, the dsRNA ligase polypeptide is simultaneously purified and immobilized on a solid support.

[0316] In some embodiments of the methods of preparing the immobilized dsRNA ligase as described herein, the dsRNA ligase polypeptide may be added to the reaction mixture comprising the functionalized solid support in the form of a purified enzyme, partially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, as cell extracts and / or lysates of such cells, and / or as an enzyme immobilized on a solid support. Whole cells transformed with gene(s) encoding the dsRNA ligaseenzyme or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety of different fomrs, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like, followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, etc.). Any of the enzy me preparations (including whole cell preparations) may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like).[0317| In at least one embodiment, a purified or substantially purified dsRNA ligase polypeptide preparation can be used in the immobilization process and the immobilized enzyme retained as a slurry in an aqueous buffer. Such a resin slurry can then be used to pack a column for use in flow-through oligonucleotide or polynucleotide ligation process.

[0318] In some embodiments of the method, the immobilized dsRNA ligase on the support retains at least 20%. 30%, 40%, 50%, 60%. 70%, 75%, 80%, 85%. or 90% or greater ligase activity. In some embodiments, the % ligase activity retained on the support is determined by amoimt of dsRNA ligase polypeptide activity on the support compared to amount of untreated dsRNA ligase polypeptide.

[0319] In some embodiments of the method, the immobilized dsRNA ligase on the support has leaching of dsRNA ligase of less than 2%, 5%, 10%, 15%, 20%, 25% or 30% of measured activity on support.

[0320] In another aspect, the present disclosure further provides a composition comprising an immobilized dsRNA ligase prepared by any of the methods described herein.

[0321] In some embodiments, the composition of immobilized dsRNA ligase comprises a dsRNA ligase polypeptide and an cpoxidc-functionalizcd support, wherein the dsRNA ligase polypeptide is attached to the solid support through a covalent linkage comprising a |3-hydroxy-amino, P-hydroxy -ether, P-hydroxy -carboxyl, and / or P-hydroxy-thio, and wherein at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 99% of epoxide groups are quenched.

[0322] In some embodiments, the compositions comprises a dsRNA ligase immobilized on an epoxide functionalized resin, wherein the epoxide groups on the resin following immobilization of the dsRNA ligases are quenched or capped to at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or at least 99.9%.

[0323] In some embodiments, the epoxide groups are quenched or capped with a quenching compound or reagent, and, optionally, a second quenching compound or reagent, selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, P-mercaptoethanol, TEoA-HCl, and any combinations thereof.

[0324] In some embodiments, the epoxide groups are quenched or capped with a quenching compound or reagent, and. optionally, a second quenching compound or reagent, selected from L-cysteine. L-lysine, L-proline, L-alanine, L-glycine. L-glycine benzyl ester. L-glycine methyl ester, L-glycine tert -butyl ester, L-cysteine methyl ester, N-acetyl-L-cysteine, and any combinations thereof.

[0325] In some embodiments, the immobilized dsRNA ligase polypeptides of the present disclosure can be in the fonn of a composition.

[0326] In some embodiments, the composition comprises the immobilized dsRNA ligase, and at least one oligonucleotide or polynucleotide substrate. In some embodiments, the composition further comprises cosubstrate NTP, particularly ATP. In some embodiments, the composition further comprises a suitable cofactor. In some embodiments, the suitable cofactor is Mg+2, or any other suitable divalent metal cofactor.

[0327] In some embodiments, the immobilized dsRNA ligase polypeptides described herein are provided in the form of kits. The immobilized enzymes in the kits may be present individually or as a plurality of immobilized enzymes. The kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzymes, as well as reagents for detecting the products. The kits can also include reagent dispensers and instructions for use of the kits.

[0328] In some embodiments, the kits of the present invention include arrays comprising a plurality of different immobilized dsRNA ligase polypeptides at different addressable positions, wherein the different immobilized polypeptides are different variants of a reference sequence each having at least one different improved enzyme property. In some embodiments, a plurality of polypeptides immobilized on solid supports are configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and / or instruments. The array can be used to test a variety of substrate compounds for conversion by the dsRNA ligase polypeptides. Such arrays comprising a plurality of engineered polypeptides and methods of their use are known in the art (See e.g., W02009 / 008908A2).

[0329] dsRNA Ligase Polypeptides

[0330] In some embodiments, the dsRNA ligase for immobilization and die methods herein can be any wild-type or engineered dsRNA ligase polypeptide.

[0331] In some embodiments, the wild-tj pe dsRNA ligase is from a phage or bacterial encoded dsRNA ligase. Exemplary wild-type dsRNA ligase include, among others, T4 RNA ligase 2, vibriophage KV40, Shigella phage vB, E. coli phage vB, E. coli phage RB69, Escherichia phage EcoM_JS09, Escherichia phage Bp7, Escherichia phage PSD2002, Enterobacter phage PG7, Klebsiella phage KPI 79, Salmonella phage vB_SenM-S16, Serratia phage X20, Acinetobacter phage ZZ1. Brucella melitensis, and Klebsiella pneumoniae, as disclosed in PCT publication W02024138200. incorporated herein by reference.

[0332] In some embodiments, the dsRNA ligase is an engineered dsRNA ligases. In some embodiments, the engineered dsRNA ligases comprises one or more amino acid residue differences relative to the wildtype polypeptide that result in improved properties. In some embodiments, the engineered dsRNA ligase display improved properties, including, among others, increased soluble expression, increased activity, increased thermal stability, and increased activity on oligonucleotide substrates having modified nucleosides and / or modified internucleoside linkages. In some embodiments, engineered ssRNA ligase polypeptides with improved properties for immobilization are described in PCT publication W02024138200. PCT application No. PCT / 2024 / 056979; PCT publication WO2024 / 134502, PCTpublication WO2024 / 134505; and U. S. patent publication US2021301280; all of which are incorporated herein by reference.

[0333] In some embodiments, the dsRNA ligase polypeptides useful in the immobilized methods and compositions of the present invention are fusion polypeptides in which the engineered dsRNA ligase polypeptide is fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g.. His tags for binding to metals), lysine residues (e.g.. for conjugation), cell localization signals (e.g., secretion signals), and polypeptides with enzymatic activity. Thus, the dsRNA ligase polypeptides described herein can be used with or without fusions to other polypeptides.

[0334] Methods of Using Immobilized dsRNA ligase Polypeptides

[0335] In some embodiments, the immobilized dsRNA ligase enzymes described herein find use in processes for ligation of oligonucleotide or polynucleotide ligase substrates.

[0336] In some embodiments, a method for ligating a dsRNA ligase substrate comprises contacting an immobilized dsRNA ligase described herein with a ligase substrate comprising an oligonucleotide or polynucleotide acceptor strand, and an oligonucleotide or polynucleotide donor strand in a reaction solution under reaction conditions suitable for ligation of the oligonucleotide or polynucleotide acceptor strand and an oligonucleotide or polynucleotide donor strand. In some embodiments, the acceptor strand comprises a ligatable 3’-end and donor strand comprises a 5’-end ligatable to the 3’-end of the acceptor strand.

[0337] In some embodiments of the method, the 3 ’-terminal nucleotide of the acceptor strand has a free 3 ’-OH, or equivalent thereof. In some embodiments of the method, the 5 ’-terminal nucleotide of the donor strand has a 5 '-phosphate, or equivalent thereof.

[0338] In some embodiments of the method, the ligase substrate comprises an oligonucleotide or polynucleotide strand complementary to the oligonucleotide or polynucleotide acceptor strand, and complementary to the oligonucleotide or polynucleotide donor strand, wherein the oligonucleotide acceptor or polynucleotide acceptor strand, and the oligonucleotide or polynucleotide donor strand base pair or hybridize adjacently on the complementary oligonucleotide or polynucleotides strand to fonn a ligatable nick.

[0339] In some embodiments, the oligonucleotide or polynucleotide strand complementary to the oligonucleotide or polynucleotide acceptor strand and the oligonucleotide or polynucleotide donor strand can act as a splint or bridging oligonucleotide or polynucleotide to provide for formation of a nick between the hybridized acceptor strand and the hybridized donor strand.

[0340] In some embodiments, the ligase substrate comprises a first oligonucleotide or polynucleotide acceptor strand, at least a first oligonucleotide or polynucleotide donor strand, at least a second oligonucleotide or polynucleotide acceptor strand, and at least a second oligonucleotide or polynucleotide donor strand, where the first oligonucleotide or polynucleotide acceptor strand base pair with the second oligonucleotide or polynucleotide donor strand to form a first double stranded fragment having a singlestranded end, and the first oligonucleotide or polynucleotide donor strand base pair with the second oligonucleotide or polynucleotide acceptor strand to form a second double stranded fragment having a single stranded end, where the single stranded terminal region of the first double stranded fragment can base pair with the second doubles stranded fragment to fonn a ligase substrate having a nick on one strand of the ligase substrate and a second nick on the other strand of the ligase substrate. In such embodiments, the ligase substrates comprise double stranded oligonucleotide or polynucleotide fragments, where the double stranded oligonucleotide or polynucleotide fragments have single stranded cohesive or sticky ends of sufficient length that base pair to form suitable substrates for the dsRN A ligase. In some embodiments, the complementary single stranded end comprises at least 1-50, 2-40. 3-35. 4-30, 5-25, 6-20. or 8-15 or more nucleotides in length. In some embodiments, the complementary single stranded end is from 1-10, 2-8, or 4-6 nucleotides in length. In some embodiments, the complementary single stranded end is 1. 2, 3, 4, 5. 6. 8, 10. 12. 14, 16, 18, 20. or more nucleotides in length.

[0341] In some embodiments of the method, the ligase substrates comprise at least 2, 3, 4. 5, or 6 or more double stranded fragments, where each double stranded fragment has a complementary end that can base pair with at least one other double stranded fragment with a complementary end to form substrates for the RNA ligase. In some embodiments, the double stranded fragments have complementary or cohesive ends, e.g.. at the 5’ and 3’ ends, such that the double stranded fragments can be ligated to form concatemers. In some embodiments, where the number of double stranded fragments is 3, at least one of the double stranded fragments has complementary’ ends at the 5’ and 3’ ends of the fragment, where double stranded fragment base pairs with two other different double stranded fragments to form a substrate that can be ligated to a product containing the three double stranded fragments. It is to be understood that where 3 or more double stranded fragments are ligated, the complementary’ ends of the double stranded fragments can be designed for ligation of all of the different double stranded fragments.

[0342] In some embodiments, the oligonucleotide or polynucleotide acceptor strand comprises a modified nucleoside, a modified internucleoside linkage, a modified 3 ’-terminal group, a modified 5’-temiinal group, or any combination thereof. In some embodiments, the 3 ’-terminal terminal group is selected to retain the 3 ’-terminal nucleoside OH group for ligation to the donor strand.

[0343] In some embodiments, the oligonucleotide or polynucleotide donor strand comprises a modified nucleoside, a modified intemucleoside linkage, a modified 3 ’-terminal group, a modified 5 ’-terminal group, or any combinations thereof. In some embodiments, the modified 5’ -terminal group is selected to retain the 5 ’-terminal phosphate group for ligation to the acceptor strand.

[0344] In some embodiments, the oligonucleotide or polynucleotides strand complementary’ to the oligonucleotide or polynucleotide acceptor strand comprises a modified nucleoside, a modified intemucleoside linkage, a modified 3 ’-terminal group, a modified 5 ’-terminal group, or any combination thereof.

[0345] In some embodiments, the oligonucleotide or polynucleotides strand complementary to the oligonucleotide or polynucleotide donor strand comprises a modified nucleoside, a modifiedinternucleoside linkage, a modified 3 ’-terminal group, a modified 5 ’-terminal group, or any combination thereof.

[0346] In some embodiments of the method, the modified nucleoside comprises a modified sugar moiety. In some embodiments, the modified nucleoside sugar residue comprises a 2’-modified sugar moiety. In some embodiments, the 2’ -modification is a 2’-O-alkyl, a 2’-halo. In some embodiments, the modified nucleoside sugar residue is, among others, a 2’-O-methyl, 2’-0-ethyl, 2’-O-propyl, or 2’-O-methoxyethyl. In some embodiments, the modified nucleoside sugar residue is 2 ’-fluoro, 2 ’-bromo, or 2 ’-chloro, preferably 2’ -fluoro. In some embodiments, the sugar residue is modified with a conjugate, such as a targeting moiety, for example, GalNAc or lipid moieties.

[0347] In some embodiments, the modified sugar may have other modifications at other positions, such as locked nucleotides or constrained ethyl nucleotides, as is known in the art. In some embodiments, “locked nucleoside” or “locked nucleotide” or “locked nucleic acid” (LNA) refers to a nucleoside, nucleotide or nucleic acid, respectively, in which the ribose moiety is modified with a bridge connecting the 2’ ox gen and 4’ carbon. The LNA may be either in the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation (see. e.g., Obika et al., Tetrahedron Letters, 1997, 38(50):8735-8738; (Drum et al.. Current Pharmaceutical Design, 2008, 14(11): 1138-1142). Typically, the bridge is a methylene bridge. LNA may confer additional stability to a polynucleotide.

[0348] In some embodiments, the modified nucleoside can comprise a glycol nucleic acid (GNA) modification, wherein the sugar moiety’ of the NTP is replaced by propylene glycol. GNA may confer additional stability to a polynucleotide.

[0349] In some embodiments of the method, the modified nucleoside residues having a modified nucleobase or nucleobase analog. In some embodiments of the method, the first polynucleotide and / or second polynucleotide comprises one or more modified nucleotide residues having a modified nucleobase or nucleobase analog. In some embodiments, the nucleobase analog is, among others, xanthine, hypoxanthine, inosine, 6-methyladenine, 7-methylguanine. 2,6-diaminopurine, 5 -methylcytosine, 5-hydroxycytosine, 5 -bromocytosine. 5-iodocytosine, 2-thiothymine, 5 -fluorouracil, 5-bromouracil, 8-bromoguanine, 8-aminoguanine, or 8-aza-7-deazaguanine. In some embodiments, the nucleobase is modified with a conjugate, such as a targeting moiety, for example, GalNAc and lipid moieties.

[0350] In some embodiments of the method, the modified intemucleoside linkage comprises a nonstandard internucleoside linkage, i.e.. intemucleoside linkages other than phosphate linkages. In some embodiments, the modified intemucleoside linkage is a phosphorothioate, phosphorodithioate. phosphoacetate, phosphoramidate, methylphosphonate, or phosphonocarboxylate. In some embodiments, at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the intemucleoside linkages are non-standard intemucleoside linkages. In some embodiments, the non-standard intemucleoside linkages are present 1. 2, 3, 4 or 5 nucleotides of the 5’ and / or 3 ’-terminus of the oligonucleotide or polynucleotide substrates.

[0351] In some embodiments of the method, the oligonucleotide or polynucleotide comprises a modified 3 ’-hydroxyl group. In some embodiments, 3 ‘-end of a polynucleotide substrate is attached to a matrix or surface, such as a solid matrix. In such instances, the polynucleotide substrate attached to a matrix has a ligatable 5 ’-end. In some embodiments, the 3 ’-end of a oligonucleotide or polynucleotide substrate is modified with an amine, halo, phosphate, phosphate analog, -O-alkyl, lipid moiety, or a detectable label.

[0352] In some embodiments, the oligonucleotide or polynucleotide comprises a C-4’ modification, including among others, 4’-thiol-C2’ modification, 475’ aminoalkvl / C2 modification, C4’-Guanidino-C2’ -modification, and C4’-O-Me / C2’ -modification (see. e.g., Gangopadhyay et al., RNA Biology. 2022.19:1, 452-467).

[0353] In some embodiments, the oligonucleotide or polynucleotide substrate comprises a 2 ’-modified sugar moiety on the 3 ’-terminal nucleotide and / or 5 ’-terminal nucleotide, where the 3 ’-terminal nucleotide on the oligonucleotide or polynucleotide acceptor strand is being ligated to the 5 ’-terminal nucleotide of the oligonucleotide or polynucleotide donor strand substrates.

[0354] In some embodiments, the oligonucleotide or polynucleotide substrate comprises a modified nucleoside at the 3’-terminal nucleoside of the oligonucleotide or polynucleotide acceptor strand. In some embodiments, the oligonucleotide or polynucleotide acceptor strand comprises a modified 3 ’-terminal nucleoside of 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.

[0355] In some embodiments, the oligonucleotide or poly nucleotide substrate comprises a modified nucleoside at the 5 ’-terminal nucleoside of the oligonucleotide or polynucleotide donor strand. In some embodiments, an oligonucleotide or polynucleotide donor strand comprises a modified 5 ’-terminal nucleoside of 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.

[0356] In some embodiments, the oligonucleotide or polynucleotide substrate comprises an oligonucleotide or polynucleotide acceptor strand having a modified 3 ’-terminal nucleoside of 2 ’-fluoroadenosine, 2 ’-fluoro -guanosine, 2’ -fluoro-cytidine, 2 ’-fluoro-uridine. 2’-fluoro-thyniidine. 2’-O-methyl-adenosine, 2’-O-methyl-guanosine. 2’-O-methyl-cytidine, 2’-O-methyl-uridine. or 2’-O-methyl-thymidine, and an oligonucleotide or polynucleotide donor strand having a modified 5 ’-terminal nucleoside of 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.

[0357] In some embodiments, the oligonucleotide or polynucleotide substrate comprises an oligonucleotide or polynucleotide acceptor strand having a modified 3 ’-terminal nucleoside of 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; a polynucleotide donor strand having a modified 5 ‘-terminal nucleoside of 2 ’-fluoroadenosine, 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; and an oligonucleotide or polynucleotide complementary to the oligonucleotide or polynucleotide acceptor strand and oligonucleotide or polynucleotide donor strand, wherein the oligonucleotide or polynucleotide complementary to the oligonucleotide or polynucleotide acceptor strand and oligonucleotide or polynucleotide donor strand comprises a complementary 2’ -fluoro or 2’-O-methyl nucleoside at the position complementary to the 3 ’-terminal nucleoside of the oligonucleotide or polynucleotide acceptor strand, and / or a complementary 2-fluoro or 2-O-methyl nucleoside at the position complementary to the 5 ’-terminal nucleoside of the oligonucleotide or polynucleotide donor strand.

[0358] In some embodiments, the 5 ’-terminal nucleotide of the oligonucleotide or polynucleotide acceptor strand comprises a 5 '-blocked nucleotide that prevents the 5 ’-terminal nucleotide from acting as a donor in the ligase reaction.

[0359] In some embodiments, the 3 ’-terminal nucleotide of the oligonucleotide or polynucleotide donor strand comprises a 3’-blocked nucleotide that prevents the 3’-terminal nucleotide from acting as an acceptor in the ligase reaction.

[0360] In some embodiments, the suitable reaction condition comprise a co-substrate NTP. In some embodiments, the co-substrate NTP is ATP. In some embodiments, the co-substrate NTP, particularly ATP, is present at about 1 mM to about 30 mM, about 2 mM to about 25 mM, about 5 mM to about 20 mM, or about 10 mM to about 15 mM. In some embodiments, the co-substrate NTP is present at about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM.

[0361] In some embodiments, the suitable reaction conditions further comprises a divalent metal, particularly Mg+2. In some embodiments, the divalent metal can be provided in different salt forms, as is known in the art. In some embodiments, the divalent metal is provided at a concentration of about 1 mM to about 30 mM, about 2 mM to about 25 mM, about 5 mM to about 20 mM, or about 10 mM to about 15 mM. In some embodiments, the co-substrate NTP is present at about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM. In some embodiments the Mg+2is provided in molar excess of co-substrate ATP. In some embodiments, the Mg+2to ATP ratio is about 1.1:1, 1.2:1, 1.5:1, 2:1. 2.5:1 orabout3:l.

[0362] 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).

[0363] In some embodiments, ligation reaction is carried out at a suitable temperature and reaction time period. In some embodiments, the ligation reaction temperature is from about 2° C to about 60° C. In some embodiments, the 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 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 ligation reaction temperature can use different temperatures, for example a temperature at which stable hybrids are formed between polynucleotide substrate(s) followed by a higher temperature to promote completion of ligation reaction.

[0364] In some embodiments, the ligation reaction time can be a sufficient time for ligation of oligonucleotide or polynucleotide substrate(s) with the immobilized dsRNA ligase. 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.

[0365] In some embodiments, the ligase reaction is carried out at a suitable pH. In some embodiments, the pH of the ligase reaction is about pH 5-9, about pH 5.5-8.5, about pH 6-8, or about pH 6.5-7.5. In some embodiments, the pH of the ligase reaction is about pH 5. about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8. about pH 8.5 or about pH 9. In some embodiments, pH of the reaction is adjusted or maintained using a suitable buffer. Suitable buffers to provide desired pH ranges are known in the art and include, by way of example and not limitation, borate, phosphate, 2-(N-morpholinojethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris), and the like. In some embodiments, the reaction conditions comprise water as a suitable solvent with no buffer present.

[0366] In some embodiments, the dsRNA ligase substrate concentration is at about 0.01 to about 20 mM, about 0.1 to about 15 mM, about 1 to about 10 mM, about 2 to about 8 mM, or about 4 to about 6 mM. In some embodiments, the engineered RNA ligase exhibits increased product yield at about 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, or 20 mM.

[0367] In some embodiments, the immobilized dsRNA ligase polypeptide is present at a molar ratio of engineered polypeptide to substrate of about 50 to 1, 25 to 1, 10 to 1, 5 to 1, 1 to 1, 1 to 5, 1 to 10, 1 to 25 or 1 to 50. 1 to 100, 1 to 500. 1 to 1000. or 1 to 2000. In some embodiments, the immobilized engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate from a range of about 50 to 1 to a range of about 1 to 2000.

[0368] In some embodiments, the immobilized dsRNA ligase polypeptide is present at 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.

[0369] In some embodiments, the immobilized dsRNA ligase polypeptide is present at about 0.01 g / L.0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L. 20 g / L. 25 g / L, 30 g / L, 35 g / L. 40 g / L, or 50 g / L.

[0370] In some embodiments, the reaction conditions comprise a surfactant for stabilizing or enhancing the reaction. Surfactants can comprise non-ionic, cationic, anionic and / or amphiphilic surfactants.Exemplary surfactants, include by way of example and not limitation, nonyl phenoxypolyethoxylethanol (NP40). TRITON™ X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitan monostearate, hexadecyl dimethylamine, etc. Any surfactant that may stabilize or enhance the reaction may be employed. The concentration of the surfactant to be employed in the reaction may be generally from 0.1 to 50 mg / mL, particularly from 1 to 20 mg / mL.

[0371] In some embodiments, the reaction conditions include an antifoam agent, which aids in reducing or preventing formation of foam in the reaction solution, such as when the reaction solutions are mixed or sparged. Anti-foam agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkyl amides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary anti-foam agents include Y-30® (Dow Coming), poly-glycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the anti-foam can be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the anti-foam agent can be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v). or about 5% (v / v) or more as desirable to promote the reaction.

[0372] In the various embodiments herein, the synthesis processes of the present invention are generally allowed to proceed until further conversion of substrate to product docs not change significantly with reaction time (e.g., less than 10% of substrate being converted, or less than 5% of substrate being converted). In some embodiments, the reaction is allowed to proceed mitil there is complete or near complete conversion of substrate to product. Transformation of substrate to product can be monitored using known methods by detecting substrate and / or product, with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS. and the like. In some embodiments, after suitable conversion to product, the reactants are separated from the oligo acceptor substrate extension product and additional reactants are added to the oligo acceptor substrate extension product to further extend the growing polynucleotide chain. The processes of the present invention may be used to iteratively extend the oligo acceptor extension product until a polynucleotide of a defined sequence and length is synthesized.

[0373] Processing of dsRNA ligase and Products by Filtration

[0374] In a further aspect, the present disclosure further provides a process for separating dsRNA ligase, unreacted substrates, and ligase products. While immobilized dsRNA ligase provides a facile method for separating unreacted substrates and ligation products, in some instances, it may be desirable to provide alternatives to use of immobilized dsRNA ligases or as an adjunct to use of immobilized dsRNA ligases, such as where additional separation from the dsRNA ligase is desired.

[0375] Accordingly, in some embodiments, the present disclosure provides a method of ligating oligonucleotides with a dsRNA ligase, the method comprising(a) contacting a dsRNA ligase with ligase substrate comprising a oligonucleotide acceptor strand and a oligonucleotide donor strand under suitable reaction conditions to ligate the polynucleotide acceptor and polynucleotide donor to form a ligated oligonucleotide product; and(b) passing the reaction solution through a selective filter to separate the dsRNA ligase from the ligase substrate and ligated oligonucleotide product.|0376| It is to be understood that use of filtration can be used with dsRNA ligase provided in solution, or in some embodiments, following use of dsRNA ligase immobilized on a support.

[0377] In some embodiments, the selective filter can be based on modified (e.g., stabilized) cellulose, polyethersulfone, regenerated cellulose membrane, charged polyacrylonitrile, polyvinylidene difluoride (PVDF). nitrocellulose and derivatives thereof, and the like. Consideration for selection of filter material include, among others, flow rate and performance, degree of protein binding, chemical resistance, and pH compatibility.

[0378] In some embodiments, the filtering is through or by tangential flow filtration. In some embodiments, the tangential flow filtration uses flat plates (e.g., as a cassette or capsule), hollow fiber, or spiral wound filtration modules.

[0379] In some embodiments, the selective filter has a molecular weight cutoff of about 100 kDa to about 3 kDa. In some embodiments, the selective filter has a molecular weight cutoff about 30 kDa to about 5 kDa.

[0380] In some embodiments, the permeate containing the ligase substrate and / or ligated oligonucleotide product has less than about 25 ng / L, less than about 20 ng / uL, less than about 15 ng / uL, less than about 10 ng / uL, less than about 9 ng / uL, less than about 8 ng / uL, less than about 7 ng / uL, less than about 6 ng / uL, or less than about 5 ng / uL of protein.

[0381] In some embodiments, in (a), the dsRNA ligase and the ligase substrate are reacted until greater than 50%. 60%, 70%, 80%, 85%. 90%, or 95% of ligase substrate is ligated to oligonucleotide product before carrying out (b).

[0382] In some embodiments of the method, the dsRNA ligase in the retentate is reused for reaction with additional ligase substrates.

[0383] In some embodiments, the oligonucleotide or polynucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof, as described herein

[0384] In some embodiments, the oligonucleotide or polynucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof, as described herein.

[0385] In some embodiments, an oligonucleotide or polynucleotide strand complementary to the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof.

[0386] In some embodiments, an oligonucleotide strand complementary to the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified internucleoside linkages, or any combinations thereof.

[0387] In various embodiments, the modified nucleoside and the modified intemucleoside linkages can comprise those described herein and known in the art.

[0388] In some embodiments of the method, the reaction conditions for the ligation of oligonucleotide or polynucleotide ligase substrate can use the conditions described herein, whether the dsRNA ligase is immobilized on a support or in solution.

[0389] Ligation of Oligonucleotides with single-stranded RNA Ligases

[0390] In one aspect, the present disclosure provides a method obligating 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.

[0391] In some embodiments, a method of synthesizing an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide^)) 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.

[0392] In some embodiments, the oligonuclcotidc(A), the nucleotide donor, or both the oligonuclcotidc(A) and the nucleotide donor comprise a modified nucleotide.

[0393] 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.

[0394] Oligonucleotide Acceptor

[0395] 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.

[0396] 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 morenucleotides in length. In some embodiments, the oligonucleotides is up to 200 or more nucleotides in length.

[0397] 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.

[0398] 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.

[0399] 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.

[0400] 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 intemucleoside linkage, a modified nucleoside, a 5 ’-terminal group, or any combinations thereof.

[0401] In some embodiments, a modified nucleoside on the oligonucleotide acceptor is at the 5 ‘-terminal nucleoside, an internal nucleoside, or the 3’-tenninal 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.

[0402] 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’-tenninal 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.

[0403] 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.

[0404] In some embodiments, the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.

[0405] 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.

[0406] 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.

[0407] 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).

[0408] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the 2’-position of the sugar moiety.

[0409] 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.

[0410] 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.

[0411] 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.), ’amino, 4’ -aminoalkyl, abasic nucleoside, or inverted abasic nucleoside.

[0412] In some embodiments, the oligonucleotide acceptor comprises one or more modified intemucleoside 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.

[0413] 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 intemucleoside linkages.

[0414] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 3 ’-terminal region with a modified intemucleoside 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 intemucleoside linkages.

[0415] In some embodiments, the oligonucleotide acceptor (oligonucleotide,..,) comprises the formula (I):Ai[’A2]m«A3-OH(I)whereineach of Ai, A2 and A3 is a nucleoside;m is 0-120;OH is at the 3 ’-position of the sugar moiety; andis an intemucleoside linkage.

[0416] 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.

[0417] In some embodiments, at least one or more of Ai, A2, or A3 comprises a modified nucleoside. In some embodiments, Ai 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 A2is modified. In some embodiments, A3is modified. In some embodiments, at least one of Ai, A2, or A3is modified with a conjugate moiety, reactive group, or linker.

[0418] In some embodiments, A3 comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, A3comprises 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.

[0419] In some embodiments, at least one intemucleoside linkagecomprises a modified intemucleoside linkage. In some embodiments, the intemucleoside linkage is a phospho containing intemucleoside linkage or a non-phospho intemucleoside linkage. In some embodiments, the intemucleoside linkage is a phosphorothioate or phosphorodithioate intemucleoside linkage. In some embodiments, the intemucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp or Rp stereoisomer

[0420] 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, lieterocycloalky I. arylene, or heteroarylene based linkers, and the like. In some embodiments the linker comprises an C2-C2oalkylene or polyethylene linker. Various linkers suitable for the purpose herein are described herein and also known in the art.

[0421] In some embodiments, the modified nucleoside on the oligonucleotide acceptor comprises the formula (II):A-[L]g-[M]h(II)whereinA is a nucleoside;L is a linker;g is 0 or 1;M is a conjugate moiety or reactive group; andh is 0-4;wherein g and h are not simultaneously 0.

[0422] In some embodiments, the oligonucleotide acceptor is 2, 3, 4. 5, or 6 or more up to 122 nucleotides in length.

[0423] 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 Substrate

[0424] 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.

[0425] In some embodiments, the nucleotide donor comprises a single nucleotide donor, also referred to herein as nucleotide^) or D, or an oligonucleotide donor, also referred to as oligonucleotide^).

[0426] 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.

[0427] In some embodiments, the oligonucleotide donor is 2 nucleotide s 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.

[0428] 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.

[0429] In some embodiments, the modified nucleotide on nucleotide(D) comprises a modified nucleoside, a 3 ’-terminal group, or any combinations thereof.

[0430] In some embodiments, the modified nucleotide on an oligonucleotide(D) comprises a modified intemucleoside linkage, a modified nucleoside, a 3 ’-terminal group, or any combinations thereof.

[0431] 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 theoligonucleotide donor, as limited by the length of the oligonucleotide donor. In some embodiments, all nucleosides of an oligonucleotide donor comprises a modified nucleoside.

[0432] 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.

[0433] In some embodiments, the modified nucleoside of nucleotide donor nucleotide(D) or oligonucleotide(D) comprises a conjugate moiety, a reactive group, or a linker.

[0434] 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.

[0435] 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.

[0436] 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 lieteroarvlene based linkers, and the like. In some embodiments the linker comprises a C2-C20alkylene or poly ethy lene linker. As noted above, various linkers suitable for the modified nucleoside of the nucleotide donor arc described herein and also known in the art.

[0437] 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’-temiinal 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.

[0438] 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.

[0439] 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.

[0440] 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’- orthe 3‘-position of the sugar moiety, or a 3’-terminal group, or any combinations thereof, of the 3’-terminal nucleotide.

[0441] 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.

[0442] 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’-phosphate 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.

[0443] 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 intemucleoside linkage or a non-phospho intemucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the intemucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or mixtures thereof.

[0444] In some embodiments, the oligonuclcotidc(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 intemucleoside linkages.

[0445] In some embodiments, the oligonucleotide(D) has at least 1 nucleotide at the 3 ’-terminal region with a modified intemucleoside 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 intemucleoside linkages.

[0446] 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.

[0447] In some embodiments, the nucleotide donor comprises the formula (Illa) or (IITb):pD; or (Hla)pDl[«D2]n«D3 (Illb)whereinp is a 5 ‘-phosphate group;each of D, DI, D2, and D3 is a nucleoside;is an intemucleoside linkage; andn is 0-120.

[0448] In some embodiments, for formula (Illb), 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, Z1, 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.

[0449] In some embodiments, for formula (Illa), D comprises a modified nucleoside. In some embodiments, D is modified with a conjugate moiety, reactive group, or a linker.

[0450] In some embodiments, for formula (Illb), at least one or more of DI, D2, or D3 comprises a modified nucleoside. In some embodiments, DI is modified. In some embodiments, at least one of D2 is modified. In some embodiments, at least 2. 3, 4, 5, 6 or all of D2 is modified. In some embodiments, D3 is modified. In some embodiments, at least one of DI. D2. or D3 is modified with a conjugate moiety, reactive group, or linker.

[0451] In some embodiments, DI comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments. DI comprises a 2‘ -fluoro-adenosine, 2‘-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine. 2’-O-methyl-adenosine, 2 -O-mcthyl-guanosinc, 2’-O-mcthyl-cytidinc, 2‘-O-mcthyl-uridinc, or 2’-O-mcthyl-thymidinc.

[0452] In some embodiments, for formula (Illb) 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 intemucleoside linkage. In some embodiments, the intemucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp and Rp stereoisomers.

[0453] In some embodiments, the modified nucleoside on the nucleotide donor, e.g., of formula (Illa) or formula (Illb). comprises the formula (IV):D-[L]q-[M]r (IV)whereinD is a nucleoside;L is a linker;q is 0 or 1;M is a conjugate moiety or reactive group; andr is 0-4;wherein q and r are not simultaneously 0.

[0454] 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.

[0455] 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 (Illa). L can be attached to 2 '-position or 3 ’-position of the sugar moiety. For formula (Illb), where D is DI 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.[0456| In some embodiments. D or D3 further 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.

[0457] In some embodiments, for formula (Illa), 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, the 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.

[0458] In some embodiments, for formula (Illb), the nucleotide donor comprises the formula pDl [-D2]n-D3p, wherein the pDl represents the 5 ’-phosphate on the 5 -terminal nucleoside DI, and D3p represents the 3 ’-phosphate on the 3 ’-terminal nucleoside D3.

[0459] In some embodiments, for formula (Illa), D further comprises a 3 ’-blocking group. In some embodiments, for formula (Illb), D3 further 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.

[0460] Reiterative Extensions or Additions with single -stranded RNA ligase and Optionally Terminal Nucleotidyl Transferase

[0461] 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 donorallows 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.

[0462] In some embodiments, the 3 ’-blocking group present on the nucleotide donor is a reversible 3’-blocking group, thereby resulting in fonnation 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.

[0463] 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.

[0464] In some embodiments, optionally the method further comprises degrading or inactivating the byproducts (e.g., unreacted nucleotide donor, pyrophosphate, etc.) of the single-stranded RNA ligase reaction.

[0465] 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 tire deblocking reaction, the deblocking agent is inactivated or the unblocked extended oligonucleotide is separated from the deblocking agent.

[0466] 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.

[0467] 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.

[0468] 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 someembodiments, where the nucleotide donor is oligonucleotide(D), repeating each cycle with new oligonucleotide(D) allows for step-wise extensions with oligonucleotide (D).

[0469] 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.

[0470] In some embodiments, each nucleotide donor is a selected or predetermined nucleotide donor for producing an extended oligonucleotide with a defined nucleotide sequence.

[0471] 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.

[0472] 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.

[0473] 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.

[0474] In some embodiments, the method further comprises removing or cleaving the 3 ’-blocking group to fonn 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.

[0475] 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.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] Reaction Conditions and Process

[0481] 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.

[0482] Scheme 4 shows an ssRNA Ligase Mechanism in Fig. 1.

[0483] 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.

[0484] 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, KC1. ammonium salts (e.g.. NH4C1), 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.

[0485] 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).

[0486] 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.

[0487] 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.

[0488] 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 about 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.

[0489] 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.

[0490] 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 intemucleoside linkages, sequence of the acceptor, presence of secondary' structure. 3’-tenninal properties, etc.), and the efficiency of the nucleotide donor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified intemucleoside 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.

[0491] 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, 1-8 mM. 1-3 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.

[0492] 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, 1-5 mM, 2-3 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, 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.

[0493] 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.

[0494] 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.

[0495] Oligonucleotide Synthesis Using Oligonucleotide Bound to Support Medium

[0496] 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 someembodiments, the oligonucleotide is conjugated to the support medium via the 5 ’-terminal nucleotide to provide for a ligase accessible free 3‘-OH.

[0497] In some embodiments, the single-stranded RNA ligase, any unreacted nucleotide donor, and byproducts 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.

[0498] 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.

[0499] 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.

[0500] 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.

[0501] 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.

[0502] 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.

[0503] 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.

[0504] 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.).

[0505] Oligonucleotide Synthesis Using Immobilized Enzymes

[0506] 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 singlestranded 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 singlestranded 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’-blockcd extended oligonucleotide in solution.

[0507] 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.

[0508] 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., immobilized deblocking enzyme) to facilitate reaction with the blocked extended oligonucleotide in solution and its separation from the deblocking agent following the deblocking reaction.

[0509] 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.

[0510] 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 agentto 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.

[0511] 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.[0512| 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.

[0513] 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.).

[0514] 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.

[0515] Modified Nucleotides

[0516] 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 of 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.

[0517] 2 - and 3 ’-sugar modifications

[0518] 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’, ora combination of 2’ and 3’ positions. Modifications at the 4‘, and / or 5 ‘-positions are described below as “terminal group.”

[0519] 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-Cl())alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl. N-alkenyl, O-alkvnyl. 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 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).

[0520] 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 hcteroarylalky I. In some embodiments, R’ is a Cl-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 -CH3 or -CH2CH3 or -CH2CH2OCH3. In some embodiments, the modified 2’-position is 2'-O-(2-niethoxyethyl). 2’-O-allyl, 2’-O-propargyl, 2‘-O-ethylamine, 2’-O-cyanocthyl, -2’-0-aminc, or 2’-O-acctatc ester.

[0521] 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; (Drum 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(l):73-6). Other locked nucleoside are described in International patent publication WO 2121249993. incorporated by reference herein.

[0522] 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 in the above is alkyl or acyl.

[0523] In some embodiments, a modification at the 2’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety7attached via a linker or a linker, as described herein.

[0524] 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 intemucleoside linkage, the 3 ’-modification is on the 3 ’-terminal nucleoside of the nucleotide donor. In sonic embodiments, the modification at the 3 ’-position arc 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-(Cl-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 Cl -CIO alkyl or Cl -CIO 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.

[0525] In some embodiments, the modified 3 ’-position of the sugar moiety is halo, 3 -O-R’, or 3 -0-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl. arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a Cl-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 -methoxy ethyl), 3’-O-allyl, 3 -0-propargyl. 3’-O-ethylamine. 3’-O-cyanoethyl, -3’-0-amine. or 3’-O-acetate ester.

[0526] 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 -0-propargyl, 3’-O-ethylamine, 3 ’-O-cy anoethyl. -3’-O-amine, 3’-0-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.

[0527] In some embodiments, a modification at tire 3 ’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker’ or a linker, as described herein.

[0528] 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:.0.<where B represents the nuclcobasc. Unlocked nucleosides arc described in, among others, International patent publication W02022 / 098990 and Snead et al., Molecular Therapy-Nucleic Acids, 2013, 2, e!03.

[0529] Modified nucleobases

[0530] 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 fonn Watson and Crick type base pairing is selected. In some embodiments, some positions for possible modifications on naturally occurring nucleobases are indicated below:

[0531] 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 0-6 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 modifiednucleobases include tricyclic pyrimidines, e.g., l,3-diazaphenoxazine-2-one. l,3-diazaphenothiazine-2-one, and 9-(2 -aminoethoxy)-!.3 -diazaphenoxazine -2-one (G-clamp).

[0532] 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 -nitro indole. 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.

[0533] 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.

[0534] 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.

[0535] Terminal groups

[0536] 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.

[0537] 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)

[0538] 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.

[0539] 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.

[0540] 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’).

[0541] Internucleoside linkages

[0542] In some embodiments, the modified oligonucleotide, e.g., oligonucleotide acceptor and / or oligonucleotide donor, comprises at least one modified, non-naturally occurring intemucleoside linkage. In some embodiments, the modified oligonucleotide has 1%, 2%, 5%, 10% 20%, 30%, 40%. 50%, or 60% or more modified intemucleoside linkages. In some embodiments, all of the intemucleoside linkages are modified intemucleoside linkages.

[0543] In some embodiments, the modified intemucleoside linkage is a phosphorous containing modified intemucleoside linkage. Exemplary phosphorous-containing intemucleoside linkages include, among others, phosphotriesters, alkylphosphonates (e.g., methyl phosphonate. ethyl phosphonate. etc.), phosphoramidates, phosphorothioate, and phosphorodithioate.

[0544] In some embodiments, the modified intemucleoside linkage is a non-phosphorous containing intemucleoside linkage. Exemplary non-phosphorous containing intemucleoside 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 intemucleoside linkage is amide linkage, such as those of glycine nucleosides or nucleoside (3-amino acids (see, e.g., Banerjee etal., Bioconjugate Chem., 2015, 26, 8, 1737-1742).

[0545] In some embodiments, the modified intemucleoside linkages provides for a chiral center. For example, a phosphorothioate or alkylphosphonate intemucleoside linkage can be in the Rp or Sp stcrcomcric configuration. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have a mixture of stereoisomers in the intemucleoside linkage. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have greater than 50% of the intemucleoside 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.

[0546] In some embodiments, the modified intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside linkage.

[0547] In some embodiments, the modified intemucleoside 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 intemucleoside linkages are present at the 3’-terminal region of the oligonucleotide acceptorand / or oligonucleotide donor. In some embodiments, at least 1 or 2 pliosphorothioate internucleoside linkages are present at the 3 ’-terminal region of an oligonucleotide acceptor or oligonucleotide donor.

[0548] In some embodiments, the modified intemucleoside linkages are present in the internal portions of the oligonucleotide acceptor or oligonucleotide donor.

[0549] In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor comprises at least a phosphorothioate intemucleoside 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.

[0550] Conjugate moiety

[0551] 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.

[0552] 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.

[0553] In some embodiments, the conjugate moiety can be attached to the 5 ’-terminal nucleotide, the 3’-tenninal 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, tire 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.

[0554] 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, ClO-alkyl, Cll-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 C9 alkenyl. CIO alkenyl, Cll-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, CIO alkynyl. Cl 1 -alkynyl, C12-alkynyl, C13-alkynyL C14-alkynyl, C15-alkynyL C16-alkynyl. C17-alkynyl, C18-alkynyl. C19-alkynyl, C20-alkynyl. C21-alkynyl, or C22-alkynyl.

[0555] 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.

[0556] In some embodiments, the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group. In some embodiments, the cycloalkyl includes, among others, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3 -cyclohexenyl, and cycloheptyl. In some embodiments, the heterocycloalkyl includes, among others, l-(1.2,5,6-tetrahydropyridyfh 1-piperidinyl, 2-piperidinyl. 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tctrahydrofuran-2-yl. tctrahydrofuran-3-yl, tetrahydrothicn-2-yl, tetrahydrothien-3-yl. 1-piperazinyl, and 2-piperazinyl.

[0557] 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.

[0558] In some embodiments, the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl-heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, lieteroar lalkenyl-. cycloalkylheteroalkenyl-heterocycloalkylheteroalkenyl-, ar lheteroalkeny 1-. or heteroarylheteroalkenyl-.

[0559] 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 capry lic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans-vacccnic 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, W02024 / 040041; incorporated herein by reference).

[0560] 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.

[0561] In some embodiments, the conjugate moiety comprises a phospholipid. In some embodiments, the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.

[0562] 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.

[0563] 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.

[0564] In some embodiments, the GalNAc moiety has the following structure,

[0565] 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 is

[0566] wherein 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., W02024 / 040041).

[0567] 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:

[0568] In some embodiments, the conjugate is GalNAc targeting moiety L96.

[0569] 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., methoxy coumarin, 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)

[0570] 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.

[0571] 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).

[0572] 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 cellulardelivery 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 / TPIO, 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:

[0573] Other cell penetrating peptides, including those conjugated to nucleic acids, are disclosed in, among others, patent publications WO24063570, WO24044663, US2024083949, WO24026141, W023230600. WO23219933, WO23177261, WO23178327, WO23093960, WO23086342, WO23081893. WO23069332, W023070108, WO23034515, US2023248630, US2023053924.W023003380. WO23277628, WO23277575, US2022378946, WO22171972, W022162200, 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.

[0574] Exemplar^' nucleotides / nucleosides with conjugate moieties are shown below:wherein R1is H or phosphate group, R2is H or phosphate group, or blocking group, and R3is H, OH. fluoro, or -O-methyl.

[0575] Reactive Moiety

[0576] 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-cyclooctene. or tetrazine. In some embodiments, the reactive group isthose used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below:

[0577] 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.

[0578] Exemplary single stranded RNA ligase substrates with a reactive moiety is provided below:wherein R1is H or phosphate; R2is a blocking group or H;; and R3is H, -OR, or halo, e.g., F, Br, or Cl.

[0579] Linker

[0580] 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.

[0581] In some embodiments, mixtures of linkers are used. In some embodiments, different linker types are comiected to form a longer linker or linkers with branched or dendritic structure. For example, an alk lene 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.

[0582] In some embodiments, linkers include, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene,lieteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroar lheteroalkylene, 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.

[0583] 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-, -NR -C('=O)-. -C(=O)-NR1. or -S-, and wherein R1is hydrogen or (Ci-Ce)alkyl, wherein the hydrocarbon chain, is optionally substituted with one or more (e.g. 1. 2, 3, or 4) substituents selected from (C 1 -C, (alkoxy. (Cs-Cejcycloalkyl, (Ci-Cejalkanoyl, (Cj-Cejalkanoyloxy. (Ci-Ce)alkoxy carbonyl, (Ci-Csjalkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0584] 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)-.

[0585] In some embodiments, the L has the structure below:

[0586] In some embodiments, the linker comprises a substituted or unsubstituted polyethylene glycol linker. In some embodiments, the polyethylene glycol linker has the formula:

[0587] 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.

[0588] In some embodiments, the polyethylene linker has the structure below:

[0101] In some embodiments, the polyethylene linker has the structure below:

[0589] 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., 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).

[0590] 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-l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-l-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU).

[0591] Pyrophosphatase coupled reactions and pyrophosphatases

[0592] 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.

[0593] 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.

[0594] 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.

[0595] 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.

[0596] ATP Regeneration Systems

[0597] 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.

[0598] 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 guanylatc-monophosphatc (GMP) kinase.

[0599] 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 Thennus 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).

[0600] 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 uridinemonophosphate 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). Themioplasma acidophiluni (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 (Q311X6). Psychrobacter arcticus (Q4FRH5), Psychromonas ingrahamii (ABM04676.1), Pseudomonas syringae (Q4ZWS6). and Halobacterium salinarum (Q9HNN8).

[0601] 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 the 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 (Pl 5454), 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 psychroery thus (Q47UB3), Pseudoalteromonas haloplanktis (Q3IJH8). Psy chrobacter arcticus (Q4FQY7), Psychromonas ingrahamii (A1T0P1), and Pseudomonas syringae (Q4ZZY8).

[0602] 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.

[0603] 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). Creatine kinases (CK) or creatine phosphokinases catalyze the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Without being limited by mechanism, a creatine kinase may transfer a phosphoryl group (see e.g. Bonilla et al 2021 Nutrients 13(4): 1238; McLeish et al 2005 Crit Rev Biochem Mol Biol 40: 1-20, herein incorporated by reference in their entirety). In some embodiments, the ATP regenerating system includes creatine kinase and creatine phosphate, where the creatine kinase is a creatine kinase of Gallus gallus, Xenopus laevis, Oryctolagus cuniculus, or Perkinsus marinus. See also Forstner et al 1998 Biophysical J. 75:1016-1023, herein incorporated by reference in its entirety.

[0604] 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.

[0605] 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.

[0606] 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.

[0607] Single-stranded RNA (ssRNA) Ligases. Polynucleotides Encoding the Ligases, and Host Cells

[0608] Single-stranded RNA (ssRNA) ligase polypeptides

[0609] 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 singlestranded RNA ligase.

[0610] 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 Rhodothemius phage RM378.

[0611] 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.

[0612] 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.

[0613] 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 terminus of SEQ ID NO: 2, 8, 98 or 108 or to a reference sequence corresponding to SEQ ID NO: 2, 8, 98 or 108.

[0614] 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 terminus of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.

[0615] 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 terminus of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8.

[0616] 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 terminus of SEQ ID NO: 98, or to a reference sequence corresponding to SEQ ID NO: 98.

[0617] 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 terminus of SEQ ID NO: 108, or to a reference sequence corresponding to SEQ ID NO: 1 8.

[0618] 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: 4-154, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154. 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 singlestranded RNA ligase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence.

[0619] In some embodiments, the recombinant single-stranded RNA ligase comprises an amino acid sequence comprising amino acid residues 12 to the carboxy terminus of SEQ ID NO: 4-154.

[0620] 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.

[0621] 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’ terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 98 or 108, or relative to the reference sequence corresponding to SEQ ID NO: 2, 8, 98 or 108. In some embodiments, the sequence corresponding to amino acid residues 12 to the carboxy’ terminus residue is amino acid residues 12 to 387 of the referenced SEQ ID NO.

[0622] 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 terminus of SEQ ID NO: 1. 8. 98, or 108, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 1, 8, 98, or 108. or relative to the reference sequence corresponding to SEQ ID NO: 1, 8, 98. or 108.

[0623] 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 terminus of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 8, wherein the amino acid sequence comprises oneor more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminus of SEQ ID NO: 8. or relative to tire reference sequence corresponding to SEQ ID NO: 8.

[0624] 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 terminus of SEQ ID NO: 8. 98 or 108. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 98. or relative to the reference sequence corresponding to SEQ ID NO: 98.

[0625] 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 terminus of SEQ ID NO: 8, 98 or 108, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

[0626] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 28, 30, 43, 58. 61, 75, 81, 84, 102, 108, 119, 122, 123, 124, 125, 127, 129, 162, 169, 170, 225, 226, 253, 254, 258, 271, 272, 276, Til. 281, 357, 358, 360, and 366 or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0627] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue selected from 28P, 30T, 43L, 58A / S / V, 61P / S, 75Y, 811, 84A, 102M, 108A / C. 1191, 122F / I / T, 123A, 124L / M, 125 A, 127E / G / K / L / T, 129G, 162G, 169L / V, 170A / D / M / S. 225D, 226D / K / Y, 253G / H / I / P, 254A, 258H / N / S, 271V, 272S / T, 276E, 277P, 281P. 357A / D / E / S, 358E, 360M / R / T. and 366A / L / R / S or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0628] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 28P, 30T, 43L, 58A / S / V. 61P / S, 75Y. 811, 84A.102M. 108A / C, 1191, 122F / I / T. 123 A, 124L / M, 125 A. 127E / G / K / L / T, 129G, 162G. 169L / V, 170A / D / M / S, 225D, 226D / K / Y. 253G / H / I / P, 254A, 258H / N / S. 271V. 272S / T. 276E, 277P, 281P, 357A / D / E / S. 358E. 360M / R / T, and 366A / L / R / S or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0629] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Y28P. K30T, R43L, L58A / S / V, I61P / S. F75Y, L81I, V84A, L102M, H108A / C, LI 191, L122F / I / T, S123A, F124L / M, S125A, I127E / G / K / L / T, T129G, W162G, I169L / V, R170A / D / M / S, G225D, I226D / K / Y. E253G / H / I / P, R254A, L258H / N / S, G271V, L272S / T, G276E, R277P, L281P, L357A / D / E / S, G358E, A360M / R / T, and Y366A / L / R / S or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0630] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 102, 108, 125, 127, 169, 170, 225, 226, 253, 258, 272, 357, 358, and 366 or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 8, or relative to the reference sequence corresponding to SEQ ID NO: 8.

[0631] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 102M. 108A, 108C, 125 A, 127G, 127L, 127T. 169L, 169V. 170D, 170S, 225D, 226K, 253H. 2531, 258H, 258N, 258S, 272S, 357E, 357S, 358E, and 366R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 8. or relative to the reference sequence corresponding to SEQ ID NO: 8.

[0632] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L102M, H108A, H108C, S125A, I127G, I127L, I127T, I169L, I169V, R170D, R170S, G225D, I226K, E253H, E253I, L258H, L258N, L258S, L272S, L357E, L357S, G358E, and Y366R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 8. or relative to the reference sequence corresponding to SEQ ID NO: 8.

[0633] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 28, 30, 58, 61, 119, 122, 123, 124, 127, and 162, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 98, or relative to the reference sequence corresponding to SEQ ID NO: 98.

[0634] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 28P, 30T, 58A, 58S, 58V, 61P, 61S, 1191, 122T, 123 A, 124L, 124M, 127E, 127K, and 162G or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 98. or relative to the reference sequence corresponding to SEQ ID NO: 98.

[0635] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Y28P, K30T, L58A, L58S, L58V. I61P, 161 S,LI 191, L122T, S123A, F124L, F124M, I127E, I127K, and W162G or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 98, or relative to the reference sequence corresponding to SEQ ID NO: 98.

[0636] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 58 / 75. 58 / 75 / 81 / 277, 58 / 75 / 122, 58 / 75 / 277, 75, 75 / 84, 75 / 277. 81, 84, and 84 / 122 / 277 or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

[0637] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 58A / 75Y, 58A / 75Y / 81I / 277P, 58A / 75Y / 122F, 58A / 75Y / 277P, 75Y, 75Y / 84A, 75Y / 277P, 811, 84A, and 84A / 122F / 277P or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

[0638] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L58A / F75Y. L58A / F75Y / L81I / R277P, L58A / F75Y / L122F, L58A / F75Y / R277P, F75Y, F75Y / V84A, F75Y / R277P, L81I, V84A, and V84A / L122F / R277P or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

[0639] 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 5.2, 6.7, 7.2, 8.2, 10.2, and 11.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 2. or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0640] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 5.2, 6.7, 7.2, 8.2, 10.2, and 11.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 8, or relative to the reference sequence corresponding to SEQ ID NO: 8.

[0641] 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 5.2, 6.7, 7.2, 8.2, 10.2, and 11.2, wherein the amino acid positions are relative to the reference sequence corresponding...

Claims

CLAIMSWhat is claimed is:

1. A method for preparing an immobilized double stranded RNA ligase (dsRNA ligase) comprising:(a) contacting an epoxide-functionalized solid support and a dsRNA ligase polypeptide in solution;(b) incubating the solution to conjugate the dsRNA ligase polypeptide to the solid support; and (c) adding an epoxide quenching reagent to the solution of step (b) to deactivate or cap the epoxide groups on the solid support.

2. The method of claim 1. further comprising removing the quenching reagent by washing the immobilized dsRNA ligase on the solid support.

3. The method of claim 1, further comprising adding a second quenching reagent to the solution (b) of the dsRNA ligase and epoxide functionalized solid support.

4. The method of claim 3, wherein the second quenching is added after completion of treatment with the first quenching reagent.

5. The method of claim 4, wherein the solid support is washed to remove the first quenching agent prior to adding the second quenching reagent.

6. The method of any one of claims 3-5, wherein the second quenching agent is different from the first quenching agent.

7. The method of any one of claims 1-6. wherein the incubating in (b) is carried out at a temperature of about 20 °C to about 60 °C.

8. The method of any one of claims 1-7, wherein the solution of (a) comprises a buffered aqueous solution at a pH of about 6.5 to about 8.5 containing the dsRNA ligase polypeptide at a concentration of about 0.1 g / L to about 50 g / L.

9. The method of claim 8, wherein the buffer in the aqueous solution is selected from borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris).

10. The method of claim 9. wherein the buffer concentration is 10 mM to 1500 mM.

11. The method of any one of claims 1-10, wherein the epoxide quenching reagent and, optionally, the second epoxide quenching, is selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester, N-acetyl-cysteine, P-mercaptoethanol, TEoA-HCl, and any combinations thereof.

12. The method of any one of claims 1-11, wherein the epoxide quenching reagent and, optionally, the second epoxide quenching, is selected from L-cysteine, L-lysine, ethanolamine, L-proline, L-alanine,L-glycine. imidazole, glucosamine, sodium thiosulfate, L-glycine benzyl ester, L-glycine methyl ester, L-glycine tert-butyl ester, L-cysteine methyl ester, N-acetyl-L-cysteine, p-mercaptoethanol, TEoA-HCl, and any combinations thereof.

13. The method of any one of claims 1-12, wherein the concentration of the epoxide quenching reagent or the second epoxide quenching reagent (if present) in the solution is about 10 mM to about 3000 mM.

14. The method of any one of claims 1-13, wherein the solution of step (c) is allowed to incubate until at least 90%. at least 95%, at least 99%, or at least 99.9% of epoxide functional groups are quenched.

15. The method of any one of claims 1-14, wherein the epoxide functionalized solid support comprises particles comprising polyacrylate, methacrylate, polymethacrylate, phenolate, polystyrene, polysaccharide, silica, or controlled pore glass.

16. The method of any one of claims 1-15, wherein the epoxide functionalized solid support comprises polymer particles having a particle size range of about 50 pm to about 1500 pm, about 100 pm to about 1000 pm, about 200 pm to about 700 pm. or about 200 pm to about 500 pm.

17. The method of any one of claims 1-16, wherein the epoxide functionalized solid support comprises polymer particles having an average pore diameter of about 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A).

18. The method of any one of claims 1-16, wherein the epoxide functionalized solid support is a resin selected from HFA (Resindion), HA (Resindion), BU (Resindion), EP (Resindion), EP / S (Resindion), EP403 / M (Resindion), EP600 (Resindion), SP600 (Resindion). HFA403 / S (Resindion), ECR8804F (Purolite), ECR8405F (Purolite), EMC7042 / M (Sunresin), EMC7042 / S (Sunresin), IB-COV-2 (ChiralVision BV), IB-COV-6 (ChiralVision BV), IB-COV-7 (ChiralVision BV), IB-COV-8 (ChiralVision BV), IB-ANI-5 (ChiralVision BV), IB-ANI-7 (ChiralVision BV). IB-ANI-8 (ChiralVision BV), IB-ANI-10 (ChiralVision BV), IB-ANI-13 (ChiralVision BV), EMC7025 (Sunresin), EMC7014 (Sunresin), EMC7032 (Sunresin), EMC7120 / M (Sunresin), Chelex 7350 (Sunresin), EMC7225 / M (Sunresin), EA403 / M (Resindion), HA403 / M (Resindion), IB-ANI-13 (ChiralVision BV), IB-COV-10 (ChiralVision BV), (ChiralVision BV), IB-ANI-3 (ChiralVision BV), FPA51 (Amberlite), ECR1090F (Purolite), ECR1604 (Purolite). ECR1504 (Purolite), ECR1640 (Purolite), CPG-N12 (LGC). CPG-N16 (LGC), CPG-NO cap (LGC). CPG-19 (LGC), CPG-20 (LGC), CPG-21 (LGC), IB-SLC(500A)-MPTMS-P500DGE (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE (ChiralVision BV), IB-SLC(500A)-GPTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P500DGE-MTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE-MTMS (ChiralVision BV), IB-SLC(500A)-GPTMS-MTMS (ChiralVision BV), IB-His-2 COOH (ChiralVision). IB-His-7 COOH (ChiralVision), IB-His-8 COOH (ChiralVision), IB-His-2 Co(Il) (ChiralVision), A568 (Duolite), A- 7 Freebase (Duolite). and AD7HP (Amberlite).

19. The method of any one of claims 1-18, wherein the immobilized dsRNA ligase polypeptide has an activity capable of ligating a ligase substrate comprising an oligonucleotide acceptor strand and a oligonucleotide donor strand of a double stranded oligonucleotide substrate.

20. The method of claim 19, wherein the oligonucleotide acceptor strand comprises an RNA, and / or the oligonucleotide donor strand comprises an RNA.

21. The method of any one of claims 1-20, wherein the immobilized dsRNA ligase on the solid support retains at least 20%, 30%. 40%, 50%, 60%, 70%. 75%. 80%, 85%, or 90% or greater ligase activity.

22. The method of claim 21, wherein the % ligase activity retained is determined by amount of dsRNA ligase polypeptide bound to resin compared to amount of untreated dsRNA ligase polypeptide.

23. The method of any one of claims 1-22, wherein the immobilized dsRNA ligase on solid support exhibits leaching of dsRNA ligase of less than 2%, 5%, 10%, 15%, 20%, 25% or 30% of measured activity on the solid support.

24. An immobilized dsRNA ligase prepared by the method of any one of claims 1-23.

25. An immobilized double stranded RNA ligase (dsRNA ligase) comprising a dsRNA ligase polypeptide and an epoxide-functionalized solid support, wherein the dsRNA ligase polypeptide is attached to the solid support through a covalent linkage comprising a -hydroxy -amino, -hydroxy -ether, P-hydroxy -carboxyl, and / or P-hydroxy-thio, and wherein at least 90% of epoxide groups are quenched.

26. The immobilized dsRNA ligase of claim 25, wherein tire quenched epoxide groups comprise a covalent linkage with a quenching compound and. optionally, a second quenching compound, selected from cysteine, lysine, ethanolamine, proline, alanine, glycine, imidazole, glucosamine, sodium thiosulfate, glycine benzyl ester, glycine methyl ester, glycine tert-butyl ester, cysteine methyl ester. N-acetyl-cysteine. P-mercaptoethanol. TEoA-HCl, and any combinations thereof.

27. The immobilized dsRNA ligase of claim 26. wherein the quenched epoxide groups comprise a covalent linkage with a quenching compound and. optionally, a second quenching compound, selected from L-cysteine. L-lysine, L-proline, L-alanine, L-glycine. L-glycine benzyl ester, L-glycine methyl ester, L-glycine tert-butyl ester, L-cysteine methyl ester. N-acetyl-L-cysteine. and any combinations thereof.

28. The immobilized dsRNA ligase of any one of claims 25-27, wherein the epoxide functionalized solid support comprises polyacrylate, methacrylate, polymethacrylate, phenolate, polystyrene, polysaccharide, silica, or controlled pore glass.

29. The immobilized dsRNA ligase of any one of claims 25-28, wherein the epoxide functionalized solid support comprises particles having a particle size range of about 50 pm to about 1500 pm, about 100 pm to about 1000 pm, about 200 pm to about 700 pm, or about 200 pm to about 500 pm.

30. The immobilized dsRNA ligase of any one of claims 25-29, wherein the epoxide functionalized solid support comprises polymer particles having an average pore diameter of about 250 angstroms (A) to about 1500 (A), about 300 angstroms (A) to about 1000 (A), or about 300 angstroms (A) to about 600 (A).

31. The immobilized dsRNA ligase of any one of claims 25-30, wherein the epoxide functionalized solid support is a resin selected from HFA (Resindion), HA (Resindion), BU (Resindion), EP (Resindion), EP / S (Resindion), EP403 / M (Resindion), EP600 (Resindion), SP600 (Resindion), HFA403 / S (Resindion), ECR8804F (Purolite), ECR8405F (Purolite), EMC7042 / M (Sunresin), EMC7042 / S (Sunresin), IB-COV-2 (ChiralVision BV), IB-COV-6 (ChiralVision BV), IB-COV-7 (ChiralVision BV), IB-COV-8 (ChiralVision BV), IB-ANI-5 (ChiralVision BV), IB-ANI-7 (ChiralVision BV), IB-ANI-8 (ChiralVision BV), IB-ANI-10 (ChiralVision BV), IB-ANI-13 (ChiralVision BV), EMC7025 (Sunresin), EMC7014 (Sunresin), EMC7032 (Sunresin), EMC7120 / M (Sunresin), Chelex 7350 (Sunresin), EMC7225 / M (Sunresin), EA403 / M (Resindion), HA403 / M (Resindion). IB-ANI-13 (ChiralVision BV). IB-COV-10 (ChiralVision BV), (ChiralVision BV), IB-ANI-3 (ChiralVision BV), FPA51 (Amberlite). ECR1090F (Purolite). ECR1604 (Purolite), ECR1504 (Purolite), ECR1640 (Purolite), CPG-N12 (LGC), CPG-N16 (LGC), CPG-NO cap (LGC). CPG-19 (LGC). CPG-20 (LGC). CPG-21 (LGC), IB-SLC(500A)-MPTMS-P500DGE (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE (ChiralVision BV). IB-SLC(500A)-GPTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P500DGE-MTMS (ChiralVision BV), IB-SLC(500A)-MPTMS-P1000DGE-MTMS (ChiralVision BV), IB-SLC(500A)-GPTMS-MTMS (ChiralVision BV). IB-His-2 COOH (ChiralVision), IB-His-7 COOH (ChiralVision), IB-His-8 COOH (ChiralVision). IB-His-2 Co(II) (ChiralVision), A568 (Duolite), A-7 Freebase (Duolite), and AD7HP (Amberlite).

32. A method for ligating a dsRNA ligase substrate comprising:contacting an immobilized dsRNA ligase of any one of claims 24-31. with a ligase substrate comprising an oligonucleotide acceptor strand and a oligonucleotide donor strand under reaction conditions suitable for ligation of the oligonucleotide acceptor strand and the oligonucleotide donor strand.

33. The method of claim 32, wherein the reaction conditions comprise co-substrate ATP.

34. The method of claim 33, wherein the ATP concentration is from 1 mM to 20 rnM.

35. The method of any one of claims 32-34, wherein the suitable reaction conditions further include a divalent metal.

36. The method of claim 35, wherein the divalent metal is Mg2+.

37. The method of any one of claims 32-36, wherein the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or combinations thereof.

38. The method of any one of claims 32-37. wherein the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified internucleoside linkages, or combinations thereof.

39. The method of any one of claims 32-38, wherein an oligonucleotide strand complementary to the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof.

40. The method of any one of claims 32-39, wherein an oligonucleotide strand complementary to the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof.

41. The method of any one of claims 37-40, wherein the modified nucleoside comprises a modification at the 2 ’-position of the sugar moiety and / or a modified nucleobase.

42. The method of any one of claims 37-41. wherein the modified nucleoside comprises at least a locked nucleic acid (LNA) linkage.

43. The method of any one of claims 37-42, wherein the modified intemucleoside linkage comprises at least a phosphorothioate linkage.

44. The method of any one of claims 37-43, wherein the immobilized RNA ligase comprises a packed resin in a column.

45. The method of claim 44, wherein the contacting comprises flowing the reaction solution through the packed resin in the column.

46. The method of any one of claims 37-43. wherein the immobilized dsRNA ligase on resin is dispersed or suspended in the reaction solution.

47. The method of claim 46, wherein the reaction solution is agitated.

48. The method of claim 46 or 47, further comprising separating the immobilized dsRNA ligase on resin from the reaction solution.

49. A method of ligating oligonucleotides with a dsRNA ligase, comprising(a) contacting a dsRNA ligase with ligase substrate comprising a oligonucleotide acceptor strand and a oligonucleotide donor strand under suitable reaction conditions to ligate the polynucleotide acceptor and polynucleotide donor to form a ligated oligonucleotide product; and(b) passing the reaction solution through a selective filter to separate the dsRNA ligase from the ligase substrate and ligated oligonucleotide product.

50. The method of claim 49, wherein the filtering is through ultrafiltration (UF) or diafiltration (UD).

51. The method of claim 49 or 50, wherein the selective filter has a molecular weight cutoff of 100 kDa to 3kDa.

52. The method of any one of claims 49-51. wherein the permeate containing the ligase substrate and / or ligated oligonucleotide product has less than 5 ng / uL of protein.

53. The method of any one of claims 49-52, wherein (a) the dsRNA ligase and the ligase substrate is reacted until greater than 50%, 60%, 70%, 80%, 85%, 90%, or 95% of ligase substrate are ligated to oligonucleotide product before carry ing out (b).

54. The method of any? one of claims 49-53, wherein the dsRNA ligase in the retentate is reused for reaction with additional ligase substrate.

55. The method of any one of claims 49-54. wherein the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combination thereof.

56. The method of any one of claims 49-55. wherein the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified internucleoside linkages, or any combination thereof.

57. The method of any one of claims 49-56, wherein an oligonucleotide strand complementary to the oligonucleotide acceptor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combination thereof.

58. The method of any one of claims 49-57. wherein an oligonucleotide strand complementary to the oligonucleotide donor strand comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or any combinations thereof.

59. A compound comprising a ligated oligonucleotide product produced by the method of any one of claims 1-58. optionally wherein the compound comprises a therapeutically effective amount of the ligated oligonucleotide product.

60. 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 terminus of SEQ ID NO: 2, 8. 98, or 108 or to a reference sequence corresponding to SEQ ID NO: 2, 8, 98, or 108.

61. The recombinant single stranded RNA ligase of claim 60, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises SEQ ID NO:

2. 8, 98. or 108.

62. The recombinant single stranded RNA ligase of claim 60, 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 terminus of SEQ ID NOs: 2, 8, 98, or 108, or to a reference sequence corresponding to SEQ ID NOs: 2, 8, 98, or 108, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxyterminus of SEQ ID NO: 2, 8, 98, or 108, or relative to the reference sequence corresponding to SEQ ID NO: 2, 8, 98, or 108.

63. The recombinant single stranded RNA ligase of any of claims 60-62, 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 terminus of SEQ ID NO: 2, 8, 98, or 108, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 98, or 108.

64. The recombinant single stranded RNA ligase of any of claims 60-63, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 28, 30, 43, 58, 61, 75, 81, 84. 102, 108, 119, 122, 123, 124, 125, 127. 129, 162, 169, 170, 225, 226, 253, 254, 258, 271, 272, 276, 277, 281, 357, 358, 360, and 366, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 98, 108, or relative to the reference sequence corresponding to SEQ ID NO: 2, 8, 98, 108.

65. The recombinant single stranded RNA ligase of any of claims 60-64, 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 the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO: 8, 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 terminus of SEQ ID NO: 8, or relative to the reference sequence corresponding to SEQ ID NO: 8.

66. The recombinant single stranded RNA ligase of any of claims 60-65, 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 terminus of SEQ ID NO: 98, or to the reference sequence corresponding to SEQ ID NO: 98, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy' terminus of SEQ ID NO: 98, or relative to the reference sequence corresponding to SEQ ID NO: 98.

67. The recombinant single stranded RNA ligase of any of claims 60-66, 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 terminus of SEQ ID NO: 108, or to the reference sequence corresponding to SEQ ID NO: 108, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

68. The recombinant single stranded RNA ligase of any of claims 60-67, 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 terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154. wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy tenninus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

69. The recombinant single stranded RNA ligase of any one of claims 60-68, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 102, 108, 125, 127, 169, 170, 225, 226, 253, 258, 272. 357, 358, and 366 or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

70. The recombinant single stranded RNA ligase of any one of claims 60-69, wherein the at least a one substitution is selected from the group comprising 102M, 108A, 108C, 125 A, 127G, 127L, 127T, 169L, 169V, 170D, 170S, 225D, 226K, 253H, 2531, 258H. 258N, 258S, 272S, 357E, 357S, 358E, and 366R or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

71. The recombinant single stranded RNA ligase of any one of claims 60-70, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 102, 108, 122, 124, 125, 127, 129, 169, 170, 225, 226, 253, 258, 271, 272, 276, 281, 357, 358, 360, and 366 or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

72. The recombinant single stranded RNA ligase of any one of claims 60-71, wherein the at least a one substitution is selected from the group comprising 102M, 108A, 108C, 1221, 124L, 125 A, 127G, 127L, 127T, 129G, 169V, 170A, 170D, 170M, 170S, 225D, 226D, 226Y, 2531, 253P, 258H, 258N, 271V, 272S, 272T, 276E, 281P. 357A, 357D, 357E, 357S, 358E, 360M, 360R, 360T, 366A, 366L, and 366S or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy tenninus of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

73. The recombinant single stranded RNA ligase of any one of claims 60-72. wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acidposition 43, 122, 253, and 277, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

74. The recombinant single stranded RNA ligase of any one of claims 60-73, wherein the at least a one substitution is selected from the group comprising 43L, 122F, 253G, and 277P, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

75. The recombinant single stranded RNA ligase of any one of claims 60-74. wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 43 / 170 / 254 / 281, 43 / 281, 170, and 170 / 281, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 8, or relative to the reference sequence corresponding to SEQ ID NO: 8.

76. The recombinant single stranded RNA ligase of any one of claims 60-75, wherein the at least a one substitution is selected from the group comprising 43L / 170S / 254A / 281P. 43L / 281P, 170S, and 170S / 281P, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 8, or relative to the reference sequence corresponding to SEQ ID NO: 8.

77. The recombinant single stranded RNA ligase of any one of claims 60-76, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 28, 30, 58, 61, 119, 122, 123, 124, 127. and 162, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 98, or relative to the reference sequence corresponding to SEQ ID NO: 98.

78. The recombinant single stranded RNA ligase of any one of claims 60-77, wherein the at least a one substitution is selected from the group comprising 28P, 30T, 58A, 58S, 58V, 61P, 61S, 1191, 122T, 123 A, 124L, 124M, 127E, 127K, and 162G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy tenninus of SEQ ID NO: of 98, or relative to the reference sequence corresponding to SEQ ID NO: 98.

79. The recombinant single stranded RNA ligase of any one of claims 60-78, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 58 / 75, 58 / 75 / 81 / 277, 58 / 75 / 122, H I n, 75, 75 / 84, and 75 / 277, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

80. The recombinant single stranded RNA ligase of any one of claims 60-79, wherein the at least a one substitution is selected from the group comprising 58A / 75Y, 58A / 75Y / 81I / 277P, 58A / 75Y / 122F, 58A / 75Y / 277P, 75Y, 75Y / 84A, and 75Y / 277P, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy tenninus of SEQ ID NO: of 108. or relative to the reference sequence corresponding to SEQ ID NO: 108.

81. The recombinant single stranded RNA ligase of any one of claims 60-80, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 81, 84, and 84 / 122 / 277, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

82. The recombinant single stranded RNA ligase of any one of claims 60-81, wherein the at least a one substitution is selected from the group comprising 811, 84A, and 84A / 122F / 277P, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: of 108, or relative to the reference sequence corresponding to SEQ ID NO: 108.

83. The recombinant single stranded RNA ligase of any one of claims 60-82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 5.2, 6.2, 7. 8.2, 9.2 10.

2. and 11.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, 8, 98, or 108, or relative to the reference sequence corresponding to SEQ ID NO: 2, 8, 98, or 108.

84. The recombinant single stranded RNA ligase of any of claims 60-83, 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 terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154.

85. The recombinant single stranded RNA ligase of any of claims 60-84. comprising an amino acid sequence having at least 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 terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 4-154.

86. The recombinant single stranded RNA ligase of any one of claims 60-85, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution selected from the group comprising 28P, 30T, 43L, 58A / S / V, 61P / S, 75Y, 811, 84A, 102M, 108A / C, 1191, 122F / I / T,123A, 124L / M, 125 A, 127E / G / K / L / T, 129G, 162G, 169L / V, 170A / D / M / S, 225D, 226D / K / Y, 253G / H / I / P. 254A, 258H / N / S, 271V, 272S / T, 276E. 277P. 281P, 357A / D / E / S, 358E, 360M / R / T, and 366A / L / R / S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, 8. 98, or 108, or relative to the reference sequence corresponding to SEQ ID NO:

2. 8, 98. or 108.

87. The recombinant single stranded RNA ligase of any one of claims 60-86, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution selected from the group comprising 28P, 30T, 43L, 58A / S / V, 61P / S, 75Y, 811, 84A, 102M, 108A / C, 1191, 122F / I / T, 123A, 124L / M, 125 A, 127E / G / K / L / T, 129G, 162G, 169L / V, 170A / D / M / S, 225D, 226D / K / Y, 253G / H / I / P, 254A, 258H / N / S, 271V, 272S / T, 276E. 277P. 281P. 357A / D / E / S, 358E, 360M / R / T, and 366A / L / R / S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 2, 8. 98, or 108, or relative to the reference sequence corresponding to SEQ ID NO:

2. 8, 98. or 108.

88. The recombinant single stranded RNA ligase of any one of claims 60-87, exhibits at least an improved property as compared to a reference single stranded RNA ligase, wherein the improved property is selected from i) increased single stranded RNA ligase activity, ii) increased single stranded ligase activity with a modified oligonucleotide substrate, iii) increased expression in a host cell, 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 terminus of SEQ ID NO: 2, 8, 98, or 108. or an amino acid sequence corresponding to SEQ ID NO: 2, 8. 98, or 108.

89. The recombinant single stranded RNA ligase of any one of claims 60-88, wherein the recombinant single stranded RNA ligase further comprises a fusion polypeptide.

90. The recombinant single stranded RNA ligase of any one of claims 60-89, wherein the recombinant single stranded RNA ligase is a purified preparation.

91. A recombinant polynucleotide comprising a polynucleotide sequence encoding a single stranded RNA ligase of any one of claims 60-90.

92. The recombinant polynucleotide of claim 91, 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, 7, 97 or 107, or to a reference nucleotide sequence corresponding to SEQ ID NO: 1, 7, 97 or 107.

93. The recombinant polynucleotide of claim 91, wherein the polynucleotide sequence comprises nucleotide residues 34 to the 3‘-tenninal of SEQ ID NO: 1, 7, 97 or 107, or comprising SEQ ID NO: 1, 7, 97 or 107.

94. The recombinant polynucleotide of claim 91, 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: 1, 7. 97 or 107, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 7. 97 or 107. wherein the recombinant polynucleotide encodes a single stranded RNA ligase.

95. The recombinant polynucleotide of claim 91, 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: 3-153, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-153, wherein the recombinant polynucleotide encodes a single stranded RNA ligase.

96. The recombinant polynucleotide of any one of claims 91-95, wherein die polynucleotide sequence is codon-optimized for expression of the encoded recombinant single stranded RNA ligase.

97. The recombinant polynucleotide of claim 91, comprising a polynucleotide sequence comprising nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-153, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 3-153.

98. An expression vector comprising a recombinant polynucleotide of any one of claims 90-97.

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

100. A host cell comprising an expression vector of claim 98 or 99.

101. The host cell of claim 100, selected from the group comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

102. 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 100 or 101. under suitable culture conditions such that the recombinant single stranded RNA ligase is produced.

103. The method of claim 102, further comprising recovering the recombinant single stranded RNA ligase polypeptide from the culture and / or host cells.

104. The method of claim 102 or 103, further comprising purifying the recombinant single stranded RNA ligase polypeptide.

105. A composition comprising a recombinant single stranded RNA ligase of any one of claims 60-90.

106. A kit comprising at least a recombinant single stranded RNA ligase of any one of claims 60-90.

107. The kit of claim 106, further comprising one or more of a buffer, nucleotide cofactor, a ligation enhancer, and a single stranded RNA ligase substrate.

108. A method of synthesizing an oligonucleotide, comprising reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of an engineered single strand RNA ligase of any one of claims 60-90 under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor.

109. The method of claim 108. wherein the nucleotide donor, the oligonucleotide acceptor, or both the nucleotide donor and the oligonucleotide(A) comprise a modified nucleotide, wherein the modified nucleoside comprises a conjugate moiety, reactive group, or linker, and wherein the conjugate moiety comprises a moiety selected from the group comprising a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof; and wherein the reactive group is selected from the group comprising an amino, -CN (cyano), N3(azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.

110. The method of any one of claims 108-109. wherein the single stranded RNA ligase is immobilized on a support medium.

111. The method of any one of claims 108-110, wherein the reaction with the single stranded RNA ligase further comprises an engineered creatine kinase.

112. The method of any one of claims 108-111, wherein the reaction with the single stranded RNA ligase further comprises an ATP recycling system.

113. An engineered creatine kinase 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 SEQ ID NO: 164 or 180.

114. The engineered creatine kinase of claim 113. wherein the amino acid sequence of the engineered creatine kinase comprises an amino acid sequence set forth in the group comprising SEQ ID NO:

166. SEQ IDNO:168, SEQ ID NOT70, SEQ IDNO:172, SEQIDNOT74, SEQ ID NO: 176, SEQ ID NO: 178 and SEQ ID NO: 182.

115. The engineered creatine kinase of claim 113, 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 SEQ ID NOs: 164 or 180. wherein the amino acid sequence comprises at least one substitution or substitution set relative to the reference sequence corresponding to SEQ ID NO: 164 or 180.

116. The engineered creatine kinase of claim 113, 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 toSEQ ID NO: 164, wherein the amino acid sequence comprises at least one substitution or substitution set relative to the reference sequence corresponding to SEQ ID NO: 164.

117. The engineered creatine kinase of claim 113, 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 SEQ ID NO: 180, wherein the amino acid sequence comprises at least one substitution or substitution set relative to the reference sequence corresponding to SEQ ID NO: 180.

Images