Uses of purine nucleosidase and amp phosphorylase

Abstract

The present disclosure relates to methods of using purine nucleosidase and / or AMP phosphorylase for increasing product formation in enzymatic reactions that generate reaction product nucleoside-5'-monophosphate (NMP), particularly AMP.

Description

Docket Number CX10-274WO1USES OF PURINE NUCEEOSIDASE AND AMP PHOSPHORYEASECROSS-REFERENCEThis application is a PCT filing and claims priority to U.S. Provisional Application No. 63 / 718748, filed 11 November 2025, which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0001] The Sequence Listing concurrently submitted herewith as file name 5244.274WOl.xml, created on November 5, 2025, with a file size of 65,536 bytes, is part of the specification and is incorporated by reference herein.BACKGROUND

[0002] Several enzymatic reactions generate nucleotide-5 ’-monophosphate (NMP) as a reaction product, including DNA and RNA ligases. For example, double stranded DNA (dsDNA) ligase uses co-factor ATP as an adenylyl donor to form an intermediate in which AMP is covalent attached to the 5 ’-phosphate on the donor DNA substrate. Subsequent nick sealing is achieved by DNA ligase catalyzed attack of the 3 ’-OH of the acceptor DNA substrate by the adenylated 5 ’-phosphate to generate a phosphodiester bond and release of reaction product AMP. The reaction generally requires a divalent metal, e.g., Mg+2. Similar reaction mechanisms based on an AMP intermediate are used by other ligases, such as double stranded RNA ligase, single stranded RNA ligase, and RNA splicing ligase.

[0003] In these nucleic acid ligase reactions, the efficiency of ligation can vary, particularly for different types of ligase substrates. For example, blunt ended dsDNA ligase substrates are less efficiently ligated than dsDNA ligase substrates that have cohesive ends. Furthermore, modifications to the nucleotides on the ligase substrates, such as 2’-fluoro and 2’-O-methyl modification on the sugar moiety, can also affect the efficiency of ligation. Desirable are approaches to increase product formation in enzymatic reactions that produce reaction product NMP.SUMMARY

[0004] The present disclosure provides use of purine nucleosidase and / or AMP phosphorylase for increasing product formation in enzymatic reactions that produce nucleoside-5’ -monophosphate (NMP) as a reaction product of the enzymatic reaction. The purine nucleosidase converts product NMP to its constituent nucleobase and ribose-5-phosphate while the AMP phosphorylase converts the AMP in presence of a phosphate to adenine and ribose-1, 5, -bisphosphate, thereby reducing the concentration of reaction product NMP.Docket Number CX10-274WO1

[0005] In some embodiments, a method of increasing product formation in an enzymatic reaction producing NMP as a reaction product comprises carrying out the enzymatic reaction in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for cleavage of the NMP to the corresponding nucleoside and ribose-5 -phosphate and / or cleavage to the nucleoside and ribose 1,5 -bisphosphate.

[0006] In some embodiments, the enzymatic reaction is carried out in presence of a purine nucleosidase under reaction conditions suitable for cleavage of the NMP to the corresponding nucleoside and ribose-5 -phosphate. In some embodiments, the purine nucleosidase comprises an AMP nucleosidase. In some embodiments, the enzymatic reaction is carried out in the presence of an AMP phosphorylase under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside and ribose 1,5 -bisphosphate. In some aspects, the enzymatic reaction is carried out in the presence of a purine nucleosidase and AMP phosphorylase under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside, ribose-5-phosphate and ribose 1,5-bisphosphate. In some aspects, the reaction conditions for AMP phosphorylase include phosphate. In various aspects, the purine nucleosidase comprise an AMP nucleosidase.

[0007] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, or 10. In some aspects, the amino acid sequence of the purine nucleosidase comprises amino acids 15 to the carboxy terminus of SEQ ID NO: 2, 4, 6, 8, or 10 or comprises SEQ ID NO: 2, 4, 6, 8, or 10.

[0008] In some embodiments, the purine nucleosidase comprises an AMP nucleosidase of Zhizhongheela, Thermoflavifilum, Thermomonas, or Amphiplicatus . In some aspects, the AMP nucleosidase is of Zhizhongheela caldifontis, Thermoflavifilum aggregans, Thermomonas hydrothermalis, or Amphiplicatus xiamenensis.

[0009] In some embodiments, the enzymatic reaction is carried out in presence of a AMP phosphorylase and phosphate under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside and ribose 1,5-bisphosphate.

[0010] In some embodiments, the AMP phosphorylase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminus of SEQ ID NO: 12, 14, or 16, or to a reference sequence corresponding to SEQ ID NO: 12, 14, or 16. In some aspects, the amino acid sequence ofDocket Number CX10-274WO1 the AMP phosphorylase comprises amino acid residues 15 to the carboxy terminus of SEQ ID NO: 12, 14, or 16 or comprises SEQ ID NO: 12, 14, or 16.

[0011] In some embodiments, the AMP phosphorylase is an AMP phosphorylase of Thermococcus, Methanotorris, or Methanocaldococcus . In some aspects, the AMP phosphorylase is an AMP phosphorylase of Thermococcus kodarensis, Methanotorris igneus, or Methanocaldococcus j annas chii.

[0012] In some embodiments, the purine nucleosidase and / or AMP phosphorylase are used in enzymatic reactions that produce NMP as a reaction product, where the enzymatic reaction comprises a nucleic acid ligase, nucleic acid ligase substrate, and an NTP or NAD co-factor.

[0013] In some embodiments, the nucleic acid ligase is an ssDNA ligase or dsDNA ligase and the NTP or NAD co-factor comprises ATP or NAD+. In some aspects, the reaction product AMP results.

[0014] In some embodiments, the nucleic acid ligase comprises dsDNA ligase and the dsDNA ligase substrate(s) comprise cohesive end or blunt ended dsDNA ligase substrate(s).

[0015] In some embodiments, the nucleic acid ligase comprises ssDNA ligase and single stranded DNA ligase substrate(s).

[0016] In some embodiments, the nucleic acid ligase comprises an ssRNA ligase or dsRNA ligase, and the NTP co-factor comprises ATP.

[0017] In some embodiments, the nucleic acid ligase is a dsRNA ligase and a dsRNA ligase substrate comprises cohesive end dsRNA ligase substrate(s). In some embodiments, the dsRNA ligase substrate(s) comprises modified dsRNA ligase substrate(s). In some embodiments, the modified dsRNA ligase substrate(s) comprise modified nucleotide(s) at the ligation junction. In some embodiments, the nucleic acid ligase comprises dsDNA ligase and the dsDNA ligase substrate comprises blunt-ended dsDNA ligase substrates. In some aspects, the nucleic acid ligase comprises ssDNA ligase and the substrates are single stranded nucleic acid substrates.

[0018] In some embodiments, the enzymatic reaction is a ssRNA ligase or dsRNA ligase and the NTP co-factor comprises ATP, thereby resulting in reaction product AMP. In some aspects, the nucleic acid ligase is a dsRNA ligase and the dsRNA ligase substrate comprises cohesive end dsRNA ligase substrate(s). In some aspects, the dsRNA ligase substrate(s) comprise modified dsRNA ligase substrates. In some embodiments, the nucleic acid ligase comprises an ssRNA ligase and ligase substrate comprises ssRNA ligase substrate(s). In some embodiments, the ssRNA ligase substrate(s) comprises modified ssRNA ligase substrate(s). In some embodiments, the modified ssRNA ligase substrate comprises modified nucleotide(s) at least at the 3 ’-terminal nucleotide acceptor and / or 5’- terminal nucleotide donor of the ssRNA ligase substrate(s).Docket Number CX10-274WO1

[0019] In some embodiments, the nucleic acid ligase comprises an RNA splicing ligase. In some embodiments, the RNA splicing ligase is tRNA splicing ligase.

[0020] In some embodiments, the enzymatic reaction of a nucleic acid ligase carried out in presence of a purine nucleosidase and / or AMP phosphorylase further comprises a pyrophosphatase for converting pyrophosphate to phosphate. In some embodiments, the pyrophosphatase is a Type I and / or Type II pyrophosphatase.

[0021] In some embodiments, methods of increasing product formation in ligation of modified single stranded RNA (ssRNA) by a single stranded RNA ligase are provided. The methods comprise ligating the ssRNA ligase substrates with the ssRNA ligase in the presence of a purine nucleosidase, AMP phosphorylase or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for ssRNA ligase mediated ligation of the ssRNA substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

[0022] In some embodiments, methods of increasing product formation in ligation of modified double stranded RNA (dsRNA) by a double stranded RN ligase are provided. The methods comprise ligating the dsRNA ligase substrates with the dsRNA ligase in presence of a purine nucleosidase, AMP phosphorylate or a combination of purine nucleosidase and AMP phosphorylase under reaction conditions suitable for dsRNA ligase mediated ligation of the dsRNA substrates and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

[0023] In some embodiments, methods of performing an enzymatic reaction that produces NMP as a reaction product are provided. The method comprises carrying out the enzymatic reaction in the presence of a purine nucleosidase, AMP phosphorylase or a combination of purine nucleosidase and AMP phosphorylase.

[0024] In some embodiments, methods of performing a nucleic acid ligase reaction with co-factor ATP and nucleic acid ligase substrate are provided. The method comprises carrying out the ligase reaction in the presence of a purine nucleosidase, AMP phosphorylase or a combination of a purine nucleosidase and AMP phosphorylase.DETAILED DESCRIPTION

[0025] The present disclosure provides methods of increasing product formation of enzymatic reactions that produce reaction product nucleotide-5 ’-monophosphate (NMP) by conducting the reaction in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase. In some embodiments, the purine nucleosidase is an AMP nucleosidase. Where AMP phosphorylase is used adjunctively in the enzymatic reaction, phosphate is provided in the reaction for conversion of the NMP to the corresponding nucleobase and ribose-1,5,- bisphosphate.Docket Number CX10-274WO1

[0026] In some embodiments, the enzymatic reactions for use with the purine nucleosidase and / or AMP phosphorylase for increasing product formation include nucleic acid ligases, such as DNA ligases and RNA ligases, as further described herein.Abbreviations and Definitions

[0027] 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.

[0028] 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.

[0029] Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0030] 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.”

[0031] “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.

[0032] ‘ ‘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.

[0033] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0034] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0035] ‘ ‘Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).Docket Number CX10-274WO1

[0036] ‘ ‘Amino acids” and “amino acid” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (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-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0037] ‘ ‘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).

[0038] ‘ ‘Purine nucleosidase” or “purine nucleoside ribohydrolase” refers to an enzyme that converts a purine nucleoside to its constituent purine base and ribose moiety. Where the substrate is a nucleoside-5’ -monophosphate (NMP), the constituent products of the purine nucleosidase is the purine nucleobase and ribose-5’ -monophosphate. In some embodiments, a purine nucleosidase refers to enzymes classified within EC 3.2.2 having the requisite activity. In some embodiments, the purine nucleosidase is capable of acting on different nucleosides, for example adenosine, inosine, xanthosine, guanosine, and deoxyadenosine (see, e.g., Ogawa et al., Appl Environ Microbiol., 2001, 67(4): 1783— 1787). An “AMP nucleosidase” refers to an enzyme capable of converting AMP to its constituent adenine and ribose-5-phosphate. In some embodiments, AMP nucleosidase includes enzymes classified within EC 3.2.2.4.

[0039] “AMP phosphorylase” or “ribose-l,5-bisphosphate isomerase” refers to an enzyme that convers AMP and phosphate into adenine and ribose-1, 5, -bisphosphate. In some embodiments, “AMP phosphorylase” can recognize different nucleoside substrates, for example AMP, CMP, and UMP. In some embodiments, AMP phosphorylase includes enzymes classified within EC 2.4.2.57.Docket Number CX10-274WO1

[0040] “Pyrophosphatase” or “IPP enzyme” or IPPase” refers to an enzyme that coverts pyrophosphate to two phosphates. In particular, pyrophosphatase converts inorganic pyrophosphate to two phosphate molecules, displays specificity towards inorganic pyrophosphate, and has low activity on other phosphorylated substrates. Exemplary pyrophosphatase enzymes include Type I pyrophosphatases, Type II pyrophosphatases,, and Type III pyrophosphatases.

[0041] ‘ ‘DNA ligase” refers to refers to an enzyme that covalently joins the 5’-phosphoryl termini (“donor”) and 3 ’-hydroxyl termini (“acceptor”) of DNA to each other. DNA ligases can be grouped into two families based on cofactor requirements: ATP-dependent ligases and NAD+-dependent ligases. DNA ligases of eukaryl and archael organisms are generally ATP-dependent. DNA ligases of eubacterial origin are generally NAD+ dependent. DNA ligase include enzymes within the general class of EC 6.5.1.

[0042] ‘ ‘RNA ligase” refers to enzymes that covalently joins the 5 ’-phosphoryl termini (donor) of RNA or DNA to the 3 ’-hydroxyl termini (acceptor) of RNA or DNA. Families of known RNA ligases include RNA ligase 1, also referred to as single-stranded RNA ligase or ssRNA ligase, which catalyzes the covalent joining of single-stranded 5 ’-phosphoryl termini of RNA or DNA to singlestranded 3 ’-hydroxyl termini of RNA or DNA. RNA ligase 2, also referred to as double stranded RNA ligase or dsRNA ligase, catalyzes the covalent joining of a 3 ’-hydroxyl terminus of RNA to a 5 ’-phosphorylated RNA or DNA but shows preference for double stranded substrates. In some embodiments, RNA ligases include those enzymes classified in EC 6.5.1.3. It is to be understood that the ligation reaction is not limited to naturally occurring RNA and DNA substrates and includes nucleotide substrates that contain modified nucleotides and / or nucleotide analogs.

[0043] “Polynucleotide,” “nucleic acid,” or “oligonucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2 ’-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’ -deoxyribonucleosides on an individual basis or on an aggregate basis. When a polynucleotide, nucleic acid, or oligonucleotide sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5 ’ to 3’ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. The polynucleotide or nucleic acid may be single -stranded or double-stranded, or may include both singlestranded regions and double-stranded regions.Docket Number CX10-274WO1

[0044] In some embodiments, the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide or nucleic acid or oligonucleotide, which include, among others, nucleosides linked together via other than standard phosphodiester linkages, such as non-standard linkages of phosphoramidates, phosphorothioates, amide linkages, etc.; nucleosides with modified and / or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’- O-alkyl, 2’-halo, 2,3-dideoxy, 2 ’-halo-2 ’-deoxy, p-D-ribo LNA, a-L-ribo-LNA (e.g., locked nucleic acids), etc.; and / or 5 ’-phosphate analogs, including, among others, phosphorothioate, phosphoacetate, phosphoramidate, monomethylphosphate, methylphosphonate, or phosphonocarboxylate.

[0045] “Nucleobase” refers to an unmodified nucleobase or a modified nucleobase. As used herein, in some embodiments, 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.

[0046] “Nucleoside” refers to a compound comprising a nucleobase and a sugar moiety. The nucleobases and sugar moiety are each, independently, unmodified or modified.

[0047] ‘ ‘Intemucleoside linkage” refers to as a linkage that covalently couples two nucleosides together. In some embodiments, intemucleoside linkages covalently couple adjacent nucleosides together, typically forming a bond between the sugar moieties of the adjacent nucleosides. Nonlimiting 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-, as further described herein.

[0048] ‘ ‘Modified oligonucleotide” or “modified polynucleotide” refers to an oligonucleotide or polynucleotide which contains at least one modified intemucleoside linkage and / or a modified nucleoside, or a modified terminal group.

[0049] ‘ ‘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.

[0050] ‘ ‘Modified nucleoside” or “nucleoside modification” refers to a nucleoside modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase. The modified nucleoside comprises a modified nucleobase and / or a modified sugar residue. The term “modified nucleoside” may also be used herein interchangeably with the term “nucleoside analogue.” Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the nucleobase of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson-Crick base pairing.Docket Number CX10-274WO1

[0051] ‘ ‘Modified intemucleoside linkage” refers to as a linkage other than a phosphodiester (PO) linkage that covalently connects two 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-phosphoms containing intemucleoside linkage, including but not limited tomethylenemethylimino (-CH2-N(CH3)- O-CH2), thiodiestcr, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-S1H2-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 thioformacetal (3’-S-CH2-O-5'). In some embodiments, intemucleoside linkages having a chiral atom can be prepared as a mixture of the stereoisomers, or as separate stereoisomers.

[0052] “Phosphorothioate intemucleoside linkage” refers to an intemucleoside 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 intemucleoside 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 intemucleoside linkage. The Sp diastereomer may be referred to as an S-PS intemucleoside 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.

[0053] “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 non-bridging oxygen atom.

[0054] “Non-bridging phosphorodithioate intemucleoside linkage” refers to a modified intemucleoside linkage which is a non-bridging phosphorodithioate intemucleoside linkage. A nonbridging 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.

[0055] “Abasic sugar moiety” refers to a sugar moiety of a nucleoside that is not attached to a nucelobase. In some embodiments, such abasic sugar moieties are referred to as “abasic nucleoside.”Docket Number CX10-274WO1

[0056] ‘ ‘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.

[0057] ‘ ‘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.

[0058] 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 a! ., Bioorganic & Med. Chem. Lett. 2002, 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0059] ‘ ‘Terminal group” as used herein refers to a group located at the first or last nucleoside in a polynucleotide or oligonucleotide. A 5 ’-terminal group refers to the terminal group bonded to 5 '-or 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.

[0060] “5 ’-blocking group” as used herein refers to a moiety or chemical group that prevents or inhibits attachment of another nucleoside, nucleotide or oligonucleotide to the 5’-terminal nucleoside. In context enzymes active on the 5 ’-terminal nucleoside, a 5 ’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide or oligonucleotide to the to the 5’- terminal nucleoside, particularly the 5’-OH of the 5’-terminal nucleoside.

[0061] “3 ’-blocking group” refers to moiety or chemical group that prevents or inhibits attachment off another nucleoside, nucleotide, or oligonucleotide to the 3 ’-terminal nucleoside. In context of single-stranded RNA ligase or other enzymes active on the 3 ’-terminal nucleoside, a 3 ’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide, or oligonucleotide to the 3 ’-terminal nucleoside, particularly the 3 ’-OH of the 3 ’-terminal nucleoside.

[0062] ‘ ‘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.Docket Number CX10-274WO1

[0063] “Enzymatically reversible blocking group” refers to a blocking group that is susceptible to removal or cleaving by an enzyme.

[0064] “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]).

[0065] “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.

[0066] “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.

[0067] “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 doublestranded structure, a triplex structure or a quaternary structure. Annealing or hybridization can occur via Watson-Crick base-pairing interactions, but may be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing. In some embodiments, the nucleobase polymer that anneals or hybridizes to another is a single nucleobase polymer while in other embodiments, the nucleobase polymers are separate nucleobase polymers.

[0068] “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and / or by manipulation using recombinant techniques.

[0069] “Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0070] “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.Docket Number CX10-274WO1

[0071] ‘ ‘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 two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to theDocket Number CX10-274WO1 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.

[0072] ‘ ‘Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0073] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0074] “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 acidDocket Number CX10-274WO1 sequence, such as that of a recombinant primase, 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).

[0075] ‘ ‘Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0076] ‘ ‘Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The amino acid positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. In some instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances, the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in the reference sequence. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a where appropriate. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino acid residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.

[0077] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a The presentDocket Number CX10-274WO1 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.

[0078] “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 primase polypeptides listed in any of the Tables in the Examples. In some embodiments, the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set. In the substitution sets, the individual substitutions are separated by a semicolon or slash “ / ”.

[0079] ‘ ‘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.

[0080] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0081] ‘ ‘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 theDocket Number CX10-274WO1 improved properties of an recombinant primase. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated byand may be present in substitution sets.

[0082] ‘ ‘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.

[0083] ‘ ‘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 primase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0084] ‘ ‘Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant primase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant primase polypeptides provided herein are isolated polypeptides.

[0085] “Substantially pure polypeptide” or “purified” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure primase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant primase polypeptides are substantially pure polypeptide compositions.Docket Number CX10-274WO1

[0086] ‘ ‘Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the primase are codon optimized for optimal production from the host organism selected for expression.

[0087] ‘ ‘Control sequence” refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present disclosure. Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0088] “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.

[0089] ‘ ‘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.

[0090] ‘ ‘Suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzymatic reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, co-factors, etc.) under which the relevant enzyme is capable of carrying out its enzymatic reaction. Exemplary “suitable reaction conditions” are provided herein (see, the Examples).

[0091] ‘ ‘Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the enzyme on a substrate.Docket Number CX10-274WO1

[0092] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0093] “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.

[0094] “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.

[0095] ‘ ‘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.

[0096] “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.

[0097] “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g., a polynucleotide sequences encoding a recombinant primase). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques, and progeny thereof, as known in the art.

[0098] “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 (Ci- C12 or C1-12), and more particularly (Ci-Cs or Cns) 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.

[0099] “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-s)carbon atoms, having at least one double bond. Exemplary “alkenyl” includes, but are not limited to, vinyl ethenyl, allyl, isopropenyl, 1- propenyl, 2 -methyl- 1 -propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-l-butenyl, 3 -methyl -2-Docket Number CX10-274WO1 butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 4-methyl-3 -pentenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl.

[0100] “Alkynyl” refers to a straight or branched chain hydrocarbon 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-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3 -pentynyl, 4- pentynyl, 1 -hexynyl, 2-hexynyl, 3 -hexynyl, 4-hexynyl and 5 -hexynyl.

[0101] “Alkylene”, “alkenylene” and “alkynylene” refers to a straight or branched chain divalent hydrocarbon radical of the corresponding alkyl, alkenyl, and alkynyl, respectively. The “alkylene”, “alkenylene” and “alkynylene” may be optionally substituted, for example with alkyl, alkyloxy, hydroxyl, carbonyl, carboxyl, halo, nitro, and the like.

[0102] ‘ ‘Lower” in reference to substituents refers to a group having between one and six carbon atoms.

[0103] “Heteroalkyl,” heteroalkenyl,” and “heteroalkynyl” refers to the corresponding alkyl, alkenyl, and akynyl in which one or more of the carbon atoms is replaced with a heteroatom, such as O, S and N.

[0100] “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).

[0101] “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.

[0102] “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,Docket Number CX10-274WO1 valency rules permitting. Examples of “aryl” groups include phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, and the like.

[0103] “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.

[0104] “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:

[0105] In some embodiments, a locked nucleoside is a bridged bicyclic compound.Docket Number CX10-274WO1

[0106] ‘ ‘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.

[0107] “Carbonyl” refers to -C(O)-. The carbonyl group may be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones. For example, an -C(O)R’, wherein R’ is an alkyl is referred to as an alkylcarbonyl. In some embodiments, R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0108] “Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

[0109] “Haloalkyl” refers to an alkyl substituted with 1 or more halogen atoms. Preferably, the alkyl is substituted with 1 to 3 halogen atoms.

[0110] “Hydroxy” refers to -OH.

[0111] “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.

[0112] “Acyl” refers to -C(O)R’, where R is hydrogen, or an optionally substituted alkyl, heteroalkyl, cylcoalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl as defined herein. Exemplary acyl groups include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

[0113] “Alkyloxy” or “alkoxy” refers to -OR’, wherein R’ is an optionally substituted alkyl.

[0114] “Aryloxy” refers to -OR’, wherein R’ is an optionally substituted aryl.

[0115] “Carboxy” refers to -COO" or COOM, wherein H or a M+counterion.

[0116] “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.

[0117] “Cyano” refers to -CN.

[0118] ‘ ‘Ester” refers to a group such as -C(=O)OR’, alternatively illustrated as -C(O)OR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocyclolalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.Docket Number CX10-274WO1

[0119] “Silyl” refers to Si, which may have various substituents, for example -SiR’R’R’, where R’ is as defined in the specification. For example, each R’ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. As defined herein, any heterocyloalkyl or heteroaryl group present in a silyl group has from 1 to 3 heteroatoms selected independently from O, N, and S.

[0120] “Thiol” or “sulfhydryl” refers to -SH.

[0121] ‘ ‘Disulfied” refers to -S-S- groups.

[0122] “Sulfanyl” refers to -SR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. For example, -SR, wherein R is an alkyl is an alkylsulfanyl.

[0123] “Sulfonyl” refers to -S(O)2-, which may have various substituents to form different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones. For example, -S(O)2R’, wherein R’ is an alkyl refers to an alkylsulfonyl. In some embodiments of -S(O)2R’, R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0124] “Amino” or “amine” refers to the group -NR’R’ or -NR’R’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, heterocycloalkyl, alkyloxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkyloxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl -oxy-sulfamino, and the like.

[0125] ‘ ‘Amide” refers to a group such as, -C(=0)NR’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0126] “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.

[0127] ‘ ‘Substituted” as used herein means one or more hydrogen atoms of the group is replaced with a substituent atom or group commonly used in pharmaceutical chemistry. Each substituent can be the same or different. Examples of suitable substituents include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, OR (e.g., hydroxyl, alkyloxy (e.g., methoxy, ethoxy, and propoxy), aryloxy, heteroaryloxy, arylalkyloxy, ether, ester, carbamate, etc.),Docket Number CX10-274WO1 hydroxyalkyl, alkyloxycarbonyl, alkyloxyalkyloxy, perhaloalkyl, alkyloxyalkyl, SR (e.g., thiol, alkylthio, arylthio, heteroarylthio, arylalkylthio, etc.), S+R 2, S(O)R, SO2R , NR R (e.g., primary amine (i.e., NH2), secondary amine, tertiary amine, amide, carbamate, urea, etc.), hydrazide, halo, nitrile, nitro, sulfide, sulfoxide, sulfone, sulfonamide, thiol, carboxy, aldehyde, keto, carboxylic acid, ester, amide, imine, and imide, including seleno and thio derivatives thereof, wherein each of the substituents can be optionally further substituted. In embodiments in which a functional group with an aromatic carbon ring is substituted, such substitutions will typically number less than about 10 substitutions, more preferably about 1 to 5, with about 1 or 2 substitutions being preferred.

[0128] ‘ ‘Stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. Thus, “stereoisomer thereof’ with respect to a compound includes any stereoisomer of the compound and mixtures of stereoisomers, and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposable 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.Uses of purine nucleosidase and AMP phosphorylase

[0129] In one aspect, the present disclosure provides a method of increasing product formation in an enzymatic reaction producing NMP as a reaction product by cleaving the NMP produced in the enzymatic reaction. Without being bound by any theory of operation, removal of the NMP decreases reversal of the enzymatic reaction and drives the reaction forward, and also potentially reduces product inhibition. In another mechanism, the AMP can also become adenylated (AppA intermediate), thereby generating an adenylated reaction product.

[0130] In some embodiments, a method of increasing product formation in an enzymatic reaction producing NMP as a reaction product comprises carrying out the enzymatic reaction in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside and ribose-5 -phosphate and / or nucleoside and ribose 1,5-bisphosphate.

[0131] In some embodiments, the enzymatic reaction producing NMP as a reaction product is carried out in presence of a purine nucleosidase under reaction conditions suitable for cleavage of the NMP to the corresponding nucleoside and ribose-5-phosphate.

[0132] In some embodiments, the enzymatic reaction producing NMP as a reaction product is carried out in presence of an AMP phosphorylase under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside and ribose 1,5-bisphosphate.Docket Number CX10-274WO1

[0133] In some embodiments, the enzymatic reaction producing NMP as a reaction product is carried out in presence of a purine nucleosidase and AMP phosphorylase under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside, ribose-5-phosphate, and ribose 1,5- bisphosphate.

[0134] In some embodiments, where the enzymatic reaction that produces NMP as a reaction product is carried out in presence of an AMP phosphorylase, the reaction conditions include phosphate for formation of the ribose 1,5 -bisphosphate.

[0135] In some embodiments, the purine nucleosidase comprises an AMP nucleosidase. In some embodiments, the AMP nucleosidase comprises a bacterial AMP nucleosidase. In some embodiments, the AMP nucleosidase comprises an AMP nucleosidase of Zhizhongheella, Thermoflavifilum, Thermomonas, Thermophagus, Amphiplicatus, Thermococcus, Me thanot orris, or Methanocaldococcus .

[0136] In some embodiments, the purine nucleosidase comprises an AMP nucleosidase of Zhizhongheella caldifontis, Thermoflavifilum aggregans, Thermomonas hydrothermalis, Thermophagus xiamenensis, or Amphiplicatus metriothermophilus .

[0137] In some embodiments, in a method of performing an enzymatic reaction that produces NMP as a reaction product, the improvement comprises carrying out the enzymatic reaction in presence of an purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase.

[0138] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, or 10.

[0139] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0140] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 4, or to the reference sequence corresponding to SEQ ID NO: 4.Docket Number CX10-274WO1

[0141] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6.

[0142] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 8, or to the reference sequence corresponding to SEQ ID NO: 8.

[0143] In some embodiments, the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 10, or to the reference sequence corresponding to SEQ ID NO: 10.

[0144] In some embodiments, the purine nucleosidase comprises an amino acid sequence comprising one or more amino acid differences as compared to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, or 10. In some embodiments, the amino acid differences are based on alignment of the amino acid sequence of the naturally occurring nucleoside phosphorylase, 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 nucleoside phosphorylase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence. In some embodiments, the amino acid difference is a substitution, insertion, or deletion of amino acid residues, for example based on alignment of sequences available for different purine (e.g., AMP) nucleosidases.

[0145] In some embodiments, the purine nucleosidase comprises an amino acid sequence comprising amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or comprises SEQ ID NO: 2, 4, 6, 8, or 10.

[0146] In some embodiments, the AMP phosphorylase is the AMP phosphorylase of Thermococcus, Methanotorris, or Methanocaldococcus . In some embodiments, the AMP phosphorylase is the AMP phosphorylase of Thermococcus kodakarensis, Methanotorris igneus, or Methanocaldococcus j annas chii.

[0147] In some embodiments, the AMP phosphorylase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket Number CX10-274WO194%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, 14, or 16, or to the reference sequence corresponding to SEQ ID NO: 12, 14, or 16.

[0148] In some embodiments, the AMP phosphorylase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0149] In some embodiments, the AMP phosphorylase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: 14.

[0150] In some embodiments, the AMP phosphorylase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16.

[0151] In some embodiments, the AMP phosphorylase comprises an amino acid sequence comprising one or more amino acid differences as compared to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, 14, or 16, or to the reference sequence corresponding to SEQ ID NO: 12, 14, or 16. In some embodiments, the amino acid differences are based on alignment of the amino acid sequence of the naturally occurring AMP phosphorylase, 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 AMP phosphorylase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence. In some embodiments, the amino acid difference is a substitution, insertion, or deletion of amino acid residues, for example based on alignment of sequences available for different AMP phosphorylases.

[0152] In some embodiments, the AMP phosphorylase comprises an amino acid sequence comprising amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, 14, or 16, or comprising SEQ ID NO: 12, 14, or 16.

[0153] In some embodiments, the purine nucleosidase or AMP phosphorylase is provided as a fusion protein. In some embodiments, the purine nucleosidase or AMP phosphorylase described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation,Docket Number CX10-274WO1 polypeptide tags that can be used for detection, purification, immobilization on a support medium, or fusion to another protein. In some embodiments, the fusion protein of the purine nucleosidase or AMP phosphorylase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion protein of the purine nucleosidase or AMP phosphorylase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the purine nucleosidase or AMP phosphorylase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the purine nucleosidase or AMP phosphorylase is fused to a polylysine, for example, for conjugation to a support medium via the amino group of the polylysine. In some embodiments, the polylysine is from 2-10 lysine units in length. In some embodiments, the fusion is to the amino (N-) terminus of purine nucleosidase or AMP phosphorylase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the purine nucleosidase or AMP phosphorylase polypeptide. In some embodiments, the fusion is selected or designed to preserve the activity of the purine nucleosidase or AMP phosphorylase.

[0154] In some embodiments, the purine nucleosidase or AMP phosphorylase described herein is an isolated composition. In some embodiments, the purine nucleosidase or AMP phosphorylase is purified. In some embodiments, the recombinant purine nucleosidase or AMP phosphorylase is provided in solution, as a lyophilizate, or immobilized on a substrate, as further discussed herein.

[0155] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of the purine nucleosidase or AMP phosphorylase. Thus, for each and every embodiment herein of a purine nucleosidase or AMP phosphorylase described herein, a functional fragment or biologically active fragment of the purine nucleosidase or AMP phosphorylase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of a purine nucleosidase or AMP phosphorylase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the purine nucleosidase or AMP phosphorylase polypeptide from which it was derived (i.e., the parent purine nucleosidase or AMP phosphorylase). In some embodiments, functional fragments or biologically active fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the purine nucleosidase or AMP phosphorylase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids, or less than 70 amino acids.

[0156] In some embodiments, a functional fragment of a purine nucleosidase or AMP phosphorylase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the purine nucleosidase or AMP phosphorylase. In some embodiments, the functional fragment will be truncated by less than 5, lessDocket Number CX10-274WO1 than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.Enzymatic reactions generating reaction product NMP

[0157] In some embodiments, the purine nucleosidase and AMP phosphorylase are used for enzymatic reactions that produces NMP as a reaction product. In some embodiments, the enzymatic reaction that produces NMP as a reaction product comprises a nucleic acid ligase, nucleic acid ligase substrate, and an NMP donor co-factor.

[0158] In some embodiments, the nucleic acid ligase is a double stranded (dsDNA) ligase and the NMP donor co-factor comprises ATP or NAD (or dNAD), thereby resulting in reaction product AMP (or dAMP) (see, e.g., U.S. Patent No. 5508179).

[0159] In some embodiments, the nucleic acid ligase comprises a dsDNA ligase and the dsDNA ligase substrate comprises cohesive end dsDNA ligase substrates. In some embodiments, the nucleic acid ligase comprises a dsDNA ligase and the dsDNA substrate comprises blunt ended DNA ligase substrates.

[0160] Various dsDNA ligases that can be used with the purine nucleosidase and / or AMP phosphorylase include, among others, viral, bacterial, fungal, plant, insect, and mammalian dsDNA ligases.

[0161] In some embodiments, the dsDNA ligase uses NAD or dNAD as a cofactor. In some embodiments, dsDNA ligase using NAD as a cofactor include dsDNA ligases of eubacteria, such as E. coli, Thermus filiformis, Thermus aquaticus, Thermus thermophilus, Bacillus stearothermophilus, Haemophilus influenzae, De sulfur olohus ambivalens, Sulfolohus acidocaldarius, Methanothermus fervidus, and Methanococcus vannielii.

[0162] In some embodiments, the dsDNA ligase uses ATP as a cofactor. In some embodiments, dsDNA ligase using ATP as a cofactor include bacteriophage dsDNA ligases (e.g., T3, T4, T6, and T7-DNA ligases), fungal dsDNA ligases (e.g., Saccharomyces pomhe, Schizosaccharomyces pomhe, etc.), human DNA ligase I, vaccinia DNA ligase, and African swine fever virus dsDNA ligase.

[0163] In some embodiments, the dsDNA ligase comprises an engineered or recombinant dsDNA ligase variants or chemically modified dsDNA ligases. In some embodiments, the dsDNA ligase comprises an engineered dsDNA ligase disclosed in U.S. Patent No. 8728725, U.S. Patent No. 10626390, U.S. Patent No. 10837009, U.S. Patent No. 11124789, WO2018208665, WO2024158764, and Wilson et al., Protein Engineering, Design & Selection, 2013, 26(7): 471-478.

[0164] In some embodiments, the nucleic acid ligase that produces NMP as a reaction product is a ssDNA ligase and nucleic acid substrates are single stranded DNA substrates. In some embodiments,Docket Number CX10-274WO1 the ssDNA ligase uses ATP as a cofactor. In some embodiments, the ssDNA ligase is bacteriophage TS2126 RNA ligase (e.g., CircLigase; Epicenter, Biotechnologies), and Methanobacterium thermoautotrophicum RNA ligase 1, and 5' AppDNA / RNA Ligase (New England Biolabs).

[0165] In some embodiments, the nucleic acid ligase that produces reaction product NMP is a single stranded (ssRNA) ligase or double stranded RNA (dsRNA) ligase, and the NMP donor co-factor comprises ATP, thereby resulting in reaction product AMP.

[0166] In some embodiments, the nucleic acid ligase that produces reaction product NMP is a single stranded (ssRNA) ligase and the NMP donor co-factor comprises ATP.

[0167] In some embodiments, in the enzymatic reaction with ssRNA ligase, the substrate comprises at least one single stranded RNA substrate. In some embodiments, the nucleic acid substrates for the ssRNA ligase comprises at least a modified ssRNA ligase substrate, as further described herein. In some embodiments, the modified ssRNA substrate comprises at least one nucleoside comprising a modified sugar moiety, a modified nucleobase, and / or a modified intemucleoside linkage, as further described herein.

[0168] In some embodiments, various ssRNA ligases useful in the enzymatic reactions, include, among others, RNA ligase 1, such as of bacteriophage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Phage TS2126, and Rhodothermus phage RM378.

[0169] In some embodiments, the ssRNA ligase comprises an engineered ssRNA ligase. In some embodiments, the engineered ssRNA ligase is disclosed in U.S. provisional application No. 63 / 634,859, filed April 16, 2024, and U.S. provisional application No. 63 / 646,841, filed May 13, 2024, incorporated by reference herein.

[0170] In some embodiments, the nucleic acid ligase that produces reaction product NMP is a double stranded (dsRNA) ligase and the NMP donor co-factor comprises ATP, thereby resulting in reaction product AMP.

[0171] In some embodiments, the dsRNA ligase comprises an engineered dsRNA ligase disclosed in W02024138200; U.S. provisional application No. 63 / 618,203, filed January 5, 2024; U.S. provisional application No. 63 / 554,938, filed January 16, 2024; U.S. provisional application 63 / 646,753, filed May 13, 2024; and U.S. provisional application No. 63 / 601,699, filed November 21, 2023; all references incorporated herein by reference.

[0172] In some embodiments, in the enzymatic reaction with dsRNA ligases, the nucleic acid substrate comprises at least one double stranded RNA ligase substrate. In some embodiments, the nucleic acid substrates for the dsRNA ligase comprises at least a modified dsRNA ligase substrate, as further described herein. In some embodiments, the modified dsRNA substrate comprises at least oneDocket Number CX10-274WO1 nucleoside comprising a modified sugar moiety, a modified nucleobase, and / or a modified intemucleoside linkage, as further discussed below.

[0173] In some embodiments, the nucleic acid ligase that produces reaction product NMP is an RNA splicing ligase and the NMP donor co-factor comprises ATP, thereby resulting in reaction product AMP. In some embodiments, the RNA splicing ligase is tRNA splicing ligase.

[0174] In some embodiments, the RNA splicing ligase comprises RNA ligase RtcB or rRNA ligase, and homologs thereof, including human and C. elegans.

[0175] In some embodiments, a method of increasing product formation in ligation of single stranded RNAs by a single stranded RNA ligase comprises ligating ssRNA ligase substrates with ssRNA ligase in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for ssRNA ligase-mediated ligation of ssRNA ligase substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

[0176] In some embodiments, a method of increasing product formation in ligation of modified single stranded RNAs by a single stranded RNA ligase comprises ligating modified ssRNA ligase substrates with ssRNA ligase in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for ssRNA ligase-mediated ligation of modified ssRNA ligase substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

[0177] In some embodiments, a method of increasing product formation in ligation of double stranded RNAs by a double stranded RNA ligase comprises ligating dsRNA ligase substrates with dsRNA ligase in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for dsRNA ligase-mediated ligation of dsRNA ligase substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

[0178] In some embodiments, a method of increasing product formation in ligation of modified single stranded RNAs by a double stranded RNA ligase comprises ligating modified dsRNA ligase substrates with dsRNA ligase in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for dsRNA ligase-mediated ligation of modified dsRNA ligase substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

[0179] In some embodiments, in a method of performing an enzymatic reaction that produces NMP as a reaction product, the improvement comprises carrying out the enzymatic reaction in presence ofDocket Number CX10-274WO1 an purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase.

[0180] In some embodiments, in a method of performing a nucleic acid ligase reaction with co-factor ATP and nucleic acid ligase substrate, the improvement comprises carrying out the ligase reaction in presence of a purine nucleosidase, AMP phosphorylase, or a combination of a purine nucleosidase and AMP phosphorylase. In some embodiments, the nucleic acid ligase comprises a double stranded DNA ligase. In some embodiments, the nucleic acid ligase comprises a single stranded DNA ligase. In some embodiments, the nucleic acid ligase comprises a double stranded RNA ligase. In some embodiments, the nucleic acid ligase comprises a single stranded RNA ligase.

[0181] In some embodiments, the enzymatic reaction carried out in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, further comprises a pyrophosphatase, wherein the reaction conditions are suitable for pyrophosphatase mediated hydrolysis of pyrophosphate generated in the enzymatic reaction.

[0182] 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 are disclosed in U.S. provisional application No. 63 / 634,887, fded April 16, 2024, incorporated by reference herein in its entirety. In some embodiments, the pyrophosphatase is a Type 1 group of pyrophosphatases. In some embodiments, the pyrophosphatase is a Type II group of pyrophosphatases.

[0183] In some embodiments, the increase in product yield is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5 folder or greater compared to the enzymatic reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase. In some embodiments, the increase in product yield is at least about 1.1 to 5 fold, about 1.2-4 fold, or about 1.5-3 fold compared to the enzymatic reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase.

[0184] In some embodiments, the increase in product yield of a single stranded DNA ligase reaction is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5 fold or greater compared to the single stranded DNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase. In some embodiments, the increase in product yield in single stranded DNA ligase reaction is at least about 1.1 to 5 fold, about 1.2-4 fold, or about 1.5-3 fold compared to the single stranded DNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase.

[0185] In some embodiments, the increase in product yield of a double stranded DNA ligase reaction is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5 fold or greater compared to the double stranded DNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase. InDocket Number CX10-274WO1 some embodiments, the increase in product yield in double stranded DNA ligase reaction is about 1. 1 to 5 fold, about 1.2-4 fold, or about 1.5-3 fold compared to the double stranded DNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase.

[0186] In some embodiments, the increase in product yield of a single stranded RNA ligase reaction is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5 fold or greater compared to the single stranded RNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase. In some embodiments, the increase in product yield in single stranded RNA ligase reaction is about 1.1 to 5 fold, about 1.2-4 fold, or about 1.5-3 fold compared the single stranded RNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase.

[0187] In some embodiments, the increase in product yield of a double stranded RNA ligase reaction is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5 fold or greater compared to the double stranded RNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase. In some embodiments, the increase in product yield in double stranded RNA ligase reaction is about 1.1 to 5 fold, about 1.2-4 fold, or about 1.5-3 fold or greater compared to the double stranded RNA ligase reaction carried out in absence of a purine nucleosidase and / or AMP phosphorylase.Reaction conditions and process

[0188] In some embodiments, the enzymatic reactions that produce reaction product NMP are carried out under reaction conditions suitable for the enzymatic reaction as well as the purine nucleosidase mediated conversion of NMP to the corresponding nucleobase and ribose-5 -phosphate, or AMP phosphorylase mediated conversion of NMP to the corresponding nucleobase and ribose 1,5- bisphosphate, or the combination of purine nucleosidase and AMP phosphorylase mediated conversion of NMP to the nucleobase and ribose-5 -phosphate and / or nucleobase and ribose 1,5- bisphosphate.

[0189] In some embodiments, the purine nucleosidase and / or AMP phosphorylase is provided at a concentration or amount sufficient to covert the reaction product NMP and improve the product yield of the enzymatic reaction. In some embodiments, the purine nucleosidase and / or AMP phosphorylase 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. In some embodiments, the enzyme / 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.Docket Number CX10-274WO1

[0190] In some embodiments, the substrate or substrates for the enzymatic reaction is present at a concentration of about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 12 mM, 15 mM, 20 mM, or 25 mM or greater. In some embodiments, the substrate or substrates for the enzymatic reaction is present at about a concentration of 0.1-25 mM, 0.2-20 mM, 0.5-15 mM, 1 mM-12 mM, 2 mM-10 mM, or 4-7 mM. In some embodiments, the substrate or substrates for a single stranded nucleic acid ligase comprises a modified single stranded nucleic acid(s). In some embodiments, the substrate or substrates for a double stranded nucleic acid ligase comprises a modified double stranded nucleic acid, as further discussed below.

[0191] In some embodiments, the suitable reaction conditions for the enzymatic reaction includes a NMP donor co-factor. In some embodiments, the NMP donor cofactor is ATP. In some embodiments, the NMP donor cofactor is NAD. In some embodiments, the NMP donor cofactor is at about 0.1-10 mM, 0.5-8 mM, 1-6 mM, or 2-4 mM. In some embodiments, the NMP donor 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 for the enzymatic reaction.

[0192] In some embodiments, the reaction conditions for the enzymatic reaction 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, where the enzymatic reaction is a nucleic acid ligase, the divalent metal is present at a concentration of about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or greater. In some embodiments, the 0.5-100 mM, 1-90 mM, 2-80 mM, 5- 75 mM, 10-70 mM, 15-60 mM, 20-50 mM, or 25-45 mM.

[0193] In some 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.

[0194] 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-I,3-diolDocket Number CX10-274WO1(Tris), and the like. In some embodiments, the buffer is present at a concentration of about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or greater. In some embodiments, the buffer is present at a concentration of about 0.1-100 mM, 0.5-90 mM, 1-80 mM, 2-70 mM, 5-60 mM, 10-50 mM, 15-40 mM, or 20-30 mM.

[0195] In some embodiments, the enzymatic reaction is carried out at a suitable temperature and reaction time period that is also suitable for the purine nucleosidase and / or AMP phosphorylase. In some embodiments, the reaction temperature is from about 2° C to about 60° C. In some embodiments, the enzymatic 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 enzymatic 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 enzymatic reaction temperature is chosen based on the thermostability and / or the efficiency of the enzymes in the enzymatic reaction, including the purine nucleosidase and / or AMP phosphorylase.

[0196] In some embodiments, the enzymatic reaction time can be a sufficient time for formation of amount of desired product in presence of the purine nucleosidase and / or AMP phosphorylase. In some embodiments, the incubation time for the enzymatic reaction 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.

[0197] In some embodiments, the processes described herein are carried out in a suitable solvent for the enzymatic reaction and the purine nucleosidase and / or AMP phosphorylase. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or co-solvent systems, which generally comprise aqueous solvents, organic solvents and / or polymeric solvents. In some embodiments, the aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the processes using the enzymes can be carried out in an aqueous co-solvent system comprising an organic solvent (e.g., ethanol, isopropanol (IP A), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) ethyl acetate, butyl acetate, 1 -octanol, heptane, octane, methyl t butyl ether (MTBE), toluene, and the like), ionic or polar solvents (e.g., 1-ethyl 4 methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium tetrafluoroborate, 1 -butyl 3 methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, and the like). In some embodiments, the cosolvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. In some embodiments, the non-aqueous co- solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polarDocket Number CX10-274WO1 solvent, and polyol solvent. In general, the co-solvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the enzymes under the reaction conditions.

[0198] In some embodiments, the reaction conditions for the enzymatic reaction also include a reaction enhancing reagent or enzyme protectant, including, among others, DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, dextran (e.g., Dextran 6000), or combinations thereof. In some embodiments, each of the additives, when used is present at an amount sufficient to effectuate the enhancement or protective effects of the additive. In some embodiments, the reagent or protectant is chosen for compatibility with the purine nucleosidase and / or AMP phosphorylase.

[0199] 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-sorbitanmonostearate, hexadecyldimethylamine, etc. In some embodiments, the surfactant is chosen for compatibility with the purine nucleosidase and / or AMP phosphorylase. Any surfactant that may stabilize or enhance the reaction may be employed. In some embodiments, 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.Reactions with nucleic acid ligase substrates

[0200] In some embodiments, the purine nucleosidase and / or AMP phosphorylase are used in conjunction with or as an adjunct to a nucleic acid ligase for ligating nucleic acid substrates. In some embodiments, the purine nucleosidase and / or AMP phosphorylase is used with a nucleic acid ligase where the nucleic acid ligase substrate comprises unmodified nucleotides. In some embodiments, the purine nucleosidase and / or AMP phosphorylase is used with a nucleic acid ligase where the nucleic acid ligase substrate contains modified nucleotides, which can be less efficiently ligated than the corresponding ligase substrates without modified nucleotides.

[0201] In some embodiments, the nucleic acid ligase substrates have at the 3 ’-terminal end of the ligase acceptor substrate a requisite 3 ’-OH, or functional form thereof, to act as the acceptor, and at the 5 ’-terminal end of the ligase donor substrate a requisite 5 ’-phosphate, or functional form thereof, to act as the donor in the ligase reaction. In some embodiments, for nucleic acid ligase substrates having nucleotide modifications, the modification(s) are appropriately selected such that the 3’- terminal end of the ligase acceptor substrate has the requisite 3 ’-OH, or functional form thereof, to actDocket Number CX10-274WO1 as the acceptor, and the 5 ’-terminal end of the ligase donor substrate has the requisite 5 ’-phosphate, or functional form thereof, to act as the donor in the ligase reaction.

[0202] It is to be understood that a polynucleotide acceptor substrate can also act as a polynucleotide donor substrate, e.g., polynucleotide acceptor substrate with a 3’-OH group also having a 5’- phosphate group, and that a polynucleotide donor substrate can also act as a polynucleotide donor substrate, e.g., polynucleotide donor substrate with a 5 ’-phosphate group also having a 3 ’-OH group.

[0203] In some embodiments, the ligation reaction can be controlled by appropriate blocking groups on the polynucleotide acceptor and / or donor substrates, where the blocking group prevents or limits reaction of polynucleotide acceptor and / or donor substrate. In some embodiments, the polynucleotide acceptor substrate comprises a blocked 5 ’-terminus, which prevents or limits the 5 ’-end of the polynucleotide acceptor participating as a substrate for the ligase. In some embodiments, the blocked 5 ’-terminus can be a 5 ’-OH group, or a modification of the 5 ’-OH with a blocking moiety. In some embodiments, the ligase donor substrate comprises a blocked 3 ’-terminus, which prevents or limits the 3 ’-end of the polynucleotide donor participating as a substrate for the ligase. In some embodiments, the 3 ’-terminus of the ligase donor substrate is modified with a reversible blocking group, which can be removed to expose the 3 ’-OH group to participate as a substrate for the ligase. Combinations of 5’-blocked polynucleotide acceptor substrates and 3’-blocked donor substrates can be used to synthesize specific ligated products. Various 5’- and 3’- blocking groups are described herein.

[0204] In some embodiments, where the nucleic acid ligase is a single stranded DNA ligase, the polynucleotide acceptor and / or the polynucleotide donor substrate comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or a combination of one or more modified nucleosides and one or more intemucleoside linkages.

[0205] In some embodiments, the polynucleotide acceptor substrate for the single stranded DNA ligase comprises at least a 5’-terminal modified nucleoside, a 3’-terminal modified nucleoside, or a 5’- and 3 ’-terminal terminal modified nucleosides.

[0206] In some embodiments, the polynucleotide acceptor substrate for the single stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5 ’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor. In some embodiments, the polynucleotide acceptor substrate for the single stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor.Docket Number CX10-274WO1

[0207] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide acceptor substrate are modified. In some embodiments, all of the nucleosides of the polynucleotide acceptor substrate are modified.

[0208] In some embodiments, the polynucleotide acceptor substrate for the single stranded DNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5 ’-terminal region, 3 ’-terminal region, the internal region of the polynucleotide acceptor substrate, or any combinations thereof.

[0209] In some embodiments, the polynucleotide acceptor of the single stranded DNA ligase substrate comprises a 5 ’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0210] In some embodiments, the polynucleotide acceptor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide acceptor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3 ’-terminal nucleoside of the polynucleotide acceptor.

[0211] In some embodiments, the polynucleotide acceptor comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide acceptor comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0212] In some embodiments, the polynucleotide donor substrate for the single stranded DNA ligase comprises at least a 5 ’-terminal modified nucleoside, a 3 ’-terminal modified nucleoside, or a 5’- and 3 ’-terminal terminal modified nucleosides.

[0213] In some embodiments, the polynucleotide donor substrate for the single stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5 ’-terminal nucleoside of the polynucleotide donor, as appropriate for the length of the polynucleotide donor. In some embodiments, the polynucleotide acceptor substrate for the single stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide donor, as appropriate for the length of the polynucleotide donor.

[0214] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide donor substrate are modified. In some embodiments, all of the nucleotides of the polynucleotide donor substrate are modified.Docket Number CX10-274WO1

[0215] In some embodiments, the polynucleotide donor substrate for the single stranded DNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5 ’-terminal region, 3 ’-terminal region, the internal region of the polynucleotide donor substrate, or any combinations thereof.

[0216] In some embodiments, the polynucleotide donor of the single stranded DNA ligase substrate comprises a 5’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0217] In some embodiments, the polynucleotide donor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide donor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3 ’-terminal nucleoside of the polynucleotide donor.

[0218] In some embodiments, the polynucleotide donor comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide donor comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0219] In some embodiments, where the nucleic acid ligase is a single stranded RNA ligase, the substrates for the single stranded RNA ligase comprise a polynucleotide acceptor, and a nucleotide and / or polynucleotide donor. The 3 ’-terminal nucleoside of the polynucleotide acceptor has the requisite 3 ’-OH, or functional form thereof, to act as an acceptor, and a nucleotide or polynucleotide donor has the requisite 5 ’-phosphate, or functional form thereof, to act as a donor for the single stranded RNA ligase.

[0220] In some embodiments, where the nucleic acid ligase is a single stranded RNA ligase, the polynucleotide acceptor and / or the polynucleotide donor substrate comprises one or more modified nucleosides, one or more modified intemucleoside linkages, or a combination of one or more modified nucleosides and one or more intemucleoside linkages.

[0221] In some embodiments, the polynucleotide acceptor substrate for the single stranded RNA ligase comprises at least a 5’-terminal modified nucleoside, a 3’-terminal modified nucleoside, or a 5’- and 3 ’-terminal terminal modified nucleoside.

[0222] In some embodiments, the polynucleotide acceptor substrate for the single stranded RNA ligase comprises a 3 ’-terminal modified nucleoside. In some embodiments, the 3 ’-terminal modified nucleoside comprises a 2’-modified sugar moiety. In some embodiments, the 3’-terminal modified nucleoside comprises 2’-fluoro adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine,Docket Number CX10-274WO12’-fluoro-thymidine, 2’ -O-methyl -adenosine, 2’-O-methyl-guanosine, 2’ -O-methyl -cytidine, 2’-O- methyl-uridine, or 2 ’-O-methyl -thymidine.

[0223] In some embodiments, the polynucleotide acceptor substrate for the single stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5 ’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor. In some embodiments, the polynucleotide acceptor substrate for the single stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor.

[0224] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide acceptor substrate are modified. In some embodiments, all of the nucleotides of the polynucleotide acceptor substrate are modified.

[0225] In some embodiments, the polynucleotide acceptor substrate for the single stranded RNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5’-terminal region, 3’-terminal region, and / or the internal region of the polynucleotide acceptor substrate.

[0226] In some embodiments, the polynucleotide acceptor of the single stranded RNA ligase substrate comprises a 5 ’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0227] In some embodiments, the polynucleotide acceptor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide acceptor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3 ’-terminal nucleoside of the polynucleotide acceptor.

[0228] In some embodiments, the polynucleotide acceptor comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide acceptor comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0229] In some embodiments, the polynucleotide acceptor of the single stranded RNA ligase comprises a combination of one or more modified nucleosides and one or more modified intemucleoside linkages.Docket Number CX10-274WO1

[0230] In some embodiments, where the single stranded RNA ligase reaction uses a polynucleotide donor substrate, the polynucleotide donor comprises one or more modified nucleosides.

[0231] In some embodiments, the polynucleotide donor substrate for the single stranded RNA ligase comprises at least a 5 ’-terminal modified nucleoside, a 3 ’-terminal modified nucleoside, or a 5’- and 3 ’-terminal terminal modified nucleoside.

[0232] In some embodiments, the polynucleotide donor substrate for the single stranded RNA ligase comprises a 5’-terminal modified nucleoside. In some embodiments, the 5’-terminal modified nucleoside comprises a 2’-modified sugar moiety. In some embodiments, the 5’-terminal modified nucleoside comprises 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’-0 methyl-uridine, or 2’-O-methyl thymidine.

[0233] In some embodiments, the polynucleotide donor substrate for the single stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5 ’-terminal nucleoside of the polynucleotide donor, as appropriate for the length of the polynucleotide donor. In some embodiments, the polynucleotide donor substrate for the single stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide donor, as appropriate for the length of the polynucleotide donor.

[0234] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide donor substrate are modified. In some embodiments, all of the nucleotides of the polynucleotide donor substrate are modified.

[0235] In some embodiments, the polynucleotide donor substrate for the single stranded RNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5’-terminal region, 3’-terminal region, and / or the internal region of the polynucleotide donor substrate.

[0236] In some embodiments, the polynucleotide donor of the single stranded RNA ligase substrate comprises a 5 ’-terminal and / or 3’ - terminal modified intemucleoside linkage.

[0237] In some embodiments, the polynucleotide donor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide donor, as appropriate for the length of the polynucleotide donor. In some embodiments, the polynucleotide donor comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3 ’-terminal nucleoside of the polynucleotide donor, as appropriate for the length of the polynucleotide donor.Docket Number CX10-274WO1

[0238] In some embodiments, the polynucleotide donor comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide donor comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0239] In some embodiments, the polynucleotide donor of the single stranded RNA ligase comprises a combination of one or more modified nucleosides and one or more modified intemucleoside linkages.

[0240] In some embodiments, the donor substrate for single stranded RNA ligase comprises a nucleotide donor substrate. In some embodiments, the nucleotide donor substrate for the single stranded RNA ligase comprises the structure pN, where the prefix p represents a 5 ’-phosphate group, and N represents a nucleoside. In some embodiments, the nucleotide donor comprises the stmcture pNp, where prefix p represents a 5 ’-phosphate group, N represents a nucleoside, and the suffix p represents a 3 ’-phosphate group. In some embodiments, the nucleoside N of the nucleotide donor is unmodified. In some embodiments, the nucleoside N of the nucleotide donor is modified. In some embodiments, the modified nucleoside of the nucleotide donor comprises a 2 ’-modified, 3 ’-modified, or 2’- and 3’- modified sugar moiety. In some embodiments, the modified nucleoside comprises a modified nucleobase. In some embodiments, the modified nucleoside comprises a modified sugar moiety and a modified nucleobase. In some embodiments, the nucleoside N for the single stranded RNA ligase comprises a 2’-fluoro adenosine, 2 ’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fhroro-thymidine, 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine, 2’-O-methyl uridine, or 2’-O-methyl thymidine. In some embodiments, the nucleoside N for the single stranded RNA ligase is modified with a conjugate moiety, for example on the 3 ’-position or 3’- phosphate, as described herein. In some embodiments, the conjugate moiety is a cell targeting moiety, such as a GalNAc moiety.

[0241] In some embodiments, the polynucleotide acceptor substrate comprises a 3 ’-terminal modified nucleoside, and the donor substrate, either as a polynucleotide or nucleotide, comprises a 5’- terminal modified nucleoside. In some embodiments, the 3 ’-terminal modified nucleoside of the polynucleotide acceptor substrate and the 5 ’-terminal nucleoside of the donor substrate comprises a modified nucleoside comprise a 2’-modified sugar moiety. In some embodiments, the 3’-terminal modified nucleoside of the polynucleotide acceptor substrate comprises 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, and the 5 ’-terminal nucleoside of the donor substrate comprises 2’ -fluoro adenosine, 2’ -fluoro-guanosine,Docket Number CX10-274WO12’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-0-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine, 2’-O-methyl uridine, or 2’-O-methyl thymidine.

[0242] In some embodiments, where the nucleic acid ligase is a double stranded polynucleotide ligase, the double stranded polynucleotide substrate comprises at least a ligatable nick, wherein the 3’- nucleotide acceptor of the polynucleotide acceptor strand has a requisite 3 ’-OH, or functional form thereof, to act as the acceptor, and the 5 ’-terminal end of the polynucleotide donor strand has a requisite 5 ’-phosphate, or functional form thereof, to act as the donor in the double stranded polynucleotide ligase reaction. In some embodiments, the double stranded polynucleotide substrate comprises unmodified nucleotides. In some embodiments, the double stranded polynucleotide substrate comprises modified nucleotides. In some embodiments, for double stranded polynucleotide substrates having nucleotide modifications, the modification(s) are appropriately selected such that the 3 ’-terminal end of the polynucleotide acceptor strand has the requisite 3 ’-OH, or functional form thereof, to act as the acceptor, and the 5 ’-terminal end of the polynucleotide donor strand has the requisite 5 ’-phosphate group, or functional form thereof, to act as the donor in the double stranded polynucleotide ligase reaction.

[0243] In some embodiments, the double stranded polynucleotide substrate comprises a blunt ended substrate, wherein the double stranded nucleic acid ligase is capable of ligating blunt ended substrates, e.g., T4 DNA ligase or derivative thereof. In some embodiments, the polynucleotide acceptor strand and the polynucleotide donor strand are provided on a single polynucleotide, and the ligatable nick is formed through base pairing of self-complementary regions on the single polynucleotide (e.g., to form a hairpin structure). In some embodiments, the polynucleotide acceptor strand and the polynucleotide donor strand are provided as separate polynucleotides, and form a nick when they base pair to a polynucleotide complementary to the polynucleotide acceptor strand and polynucleotide donor strand. In some embodiments, the double stranded polynucleotide substrate are formed from double stranded polynucleotide fragments that have cohesive ends, which can base pair to form ligatable nicks. In some embodiments, the cohesive end comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more complementary ends. In some embodiments, the double stranded polynucleotide substrate comprises 2, 3, 4, 5, 7, 7, 8, 9, 10, or more ligatable nicks. In some embodiments, the double stranded polynucleotide substrate comprises a plurality of polynucleotide acceptor substrates. In some embodiments, the double stranded polynucleotide substrate comprises a plurality of polynucleotide donor substrates.

[0244] In various embodiments, the modified nucleoside in the polynucleotide acceptor strand, polynucleotide donor strand are selected for compatibility with the double stranded polynucleotide ligase, e.g., double stranded DNA ligase and / or double stranded RNA ligase.

[0245] In some embodiments, wherein the nucleic acid ligase is a double stranded DNA ligase, the polynucleotide acceptor strand comprises a 5 ’-terminal modified nucleoside. In some embodiments,Docket Number CX10-274WO1 the polynucleotide acceptor strand forming the nick in the double stranded polynucleotide substrate comprises a 3’-terminal modified nucleoside. In some embodiments, the polynucleotide acceptor strand comprises a 5 ’-terminal modified and a 3 ’-terminal modified nucleoside.

[0246] In some embodiments, the polynucleotide acceptor strand for the double stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5 ’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor. In some embodiments, the polynucleotide acceptor strand for the double stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor.

[0247] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide acceptor strand are modified. In some embodiments, all of the nucleosides of the polynucleotide acceptor strand are modified.

[0248] In some embodiments, the polynucleotide acceptor strand for the double stranded DNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5’-terminal region, 3’-terminal region, and / or the internal region of the polynucleotide acceptor strand.

[0249] In some embodiments, the polynucleotide acceptor strand of the double stranded DNA ligase substrate comprises a 5 ’-terminal and / or 3’ - terminal modified intemucleoside linkage.

[0250] In some embodiments, the polynucleotide acceptor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide acceptor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3’-terminal nucleoside of the polynucleotide acceptor strand.

[0251] In some embodiments, the polynucleotide acceptor strand comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide acceptor strand comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0252] In some embodiments, the polynucleotide acceptor strand of the double stranded polynucleotide substrate comprises a combination of one or more modified nucleosides and one or more modified intemucleoside linkages.Docket Number CX10-274WO1

[0253] In some embodiments, the polynucleotide donor strand forming the nick in the double stranded polynucleotide substrate comprises a 5’-terminal modified nucleoside. In some embodiments, the polynucleotide donor strand comprises a 3 ’-terminal modified nucleoside. In some embodiments, the polynucleotide donor strand comprises a 5 ’-terminal modified nucleoside and a 3’- terminal modified nucleoside.

[0254] In some embodiments, the polynucleotide donor strand for the double stranded polynucleotide substrate for the double stranded DNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5’-terminal nucleoside of the polynucleotide donor strand, as appropriate for the length of the polynucleotide donor strand. In some embodiments, the polynucleotide donor strand comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide donor strand, as appropriate for the length of the polynucleotide donor strand.

[0255] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide donor strand are modified. In some embodiments, all of the nucleosides of the polynucleotide donor strand are modified.

[0256] In some embodiments, the polynucleotide donor strand for the double stranded DNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5’-terminal region, 3’-terminal region, and / or the internal region of the polynucleotide donor strand.

[0257] In some embodiments, the polynucleotide donor strand of the double stranded DNA ligase substrate comprises a 5 ’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0258] In some embodiments, the polynucleotide donor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide donor strand. In some embodiments, the polynucleotide acceptor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3’-terminal nucleoside of the polynucleotide donor strand.

[0259] In some embodiments, the polynucleotide donor strand comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide donor strand comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0260] In some embodiments, the polynucleotide donor strand of the double stranded polynucleotide substrate comprises a combination of modified nucleoside and modified intemucleoside linkage.Docket Number CX10-274WO1

[0261] In some embodiments, the polynucleotide strand or strands, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand forming the ligatable nick comprise one or more modified nucleosides.

[0262] In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide acceptor strand comprises a modified nucleoside at the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide acceptor strand comprises a modified nucleoside at the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide acceptor strand.

[0263] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand comprises one or more modified nucleosides at nucleoside position 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 5’-terminal nucleoside of the polynucleotide acceptor strand, to the nucleosides 3’ of the nucleoside complementary to the 5’- terminal nucleoside of the polynucleotide acceptor strand.

[0264] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand comprises one or more modified nucleosides at nucleoside position 2,3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 3’-terminal nucleoside of the polynucleotide acceptor strand, to the nucleosides 5’ of the nucleoside complementary to the 3’- terminal nucleoside of the polynucleotide acceptor strand.

[0265] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand are modified.

[0266] In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide donor strand comprises a modified nucleoside at the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide donor strand. In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide donor strand comprises a modified nucleoside at the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide donor strand.

[0267] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide donor strand comprises one or more modified nucleosides at nucleoside position 2, 3,4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide donor strand, to the nucleosides 3’ of the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide donor strand.Docket Number CX10-274WO1

[0268] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide donor strand comprises one or more modified nucleosides at nucleoside position 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide donor strand, to the nucleosides 5’ of the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide acceptor strand.

[0269] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide, or segment thereof, complementary to the polynucleotide donor strand are modified.

[0270] In some embodiments, the polynucleotide complementary to the polynucleotide acceptor strand and the polynucleotide donor strand of the double stranded DNA ligase substrate comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5 ’-terminal region, 3 ’-terminal region, and / or the internal region of the complementary polynucleotide.

[0271] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises a 5’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0272] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the complementary polynucleotide. In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3 ’-terminal nucleoside of the complementary polynucleotide.

[0273] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide donor strand comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0274] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand of the double stranded DNA ligase substrate comprises a combination of modified nucleoside and modified intemucleoside linkage.Docket Number CX10-274WO1

[0275] In some embodiments, wherein the nucleic acid ligase is a double stranded RNA ligase, the polynucleotide acceptor strand comprises a 5 ’-terminal modified nucleoside. In some embodiments, the polynucleotide acceptor strand forming the nick in the double stranded polynucleotide substrate comprises a 3 ’-terminal modified nucleoside. In some embodiments, the polynucleotide acceptor strand comprises a 5’-terminal modified and a 3’-terminal modified nucleoside.

[0276] In some embodiments, the polynucleotide acceptor strand of the double stranded RNA ligase substrate comprises a 3 ’-terminal modified nucleoside. In some embodiments, the 3 ’-terminal modified nucleoside comprises a 2’-modified sugar moiety. In some embodiments, the 3’-terminal modified nucleoside of the polynucleotide acceptor strand comprises 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.

[0277] In some embodiments, the polynucleotide acceptor strand for the double stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5 ’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor. In some embodiments, the polynucleotide acceptor strand for the double stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide acceptor, as appropriate for the length of the polynucleotide acceptor.

[0278] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide acceptor strand are modified. In some embodiments, all of the nucleosides of the polynucleotide acceptor strand are modified.

[0279] In some embodiments, the polynucleotide acceptor strand for the double stranded RNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5’-terminal region, 3’-terminal region, and / or the internal region of the polynucleotide acceptor strand.

[0280] In some embodiments, the polynucleotide acceptor strand of the double stranded RNA ligase substrate comprises a 5 ’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0281] In some embodiments, the polynucleotide acceptor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide acceptor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3’-terminal nucleoside of the polynucleotide acceptor strand.Docket Number CX10-274WO1

[0282] In some embodiments, the polynucleotide acceptor strand comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide acceptor strand comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0283] In some embodiments, the polynucleotide acceptor strand of the double stranded polynucleotide substrate comprises a combination of one or more modified nucleosides and one or more modified intemucleoside linkages.

[0284] In some embodiments, the polynucleotide donor strand forming the nick in the double stranded polynucleotide substrate comprises a 5’-terminal modified nucleoside. In some embodiments, the polynucleotide donor strand comprises a 3 ’-terminal modified nucleoside. In some embodiments, the polynucleotide donor strand comprises a 5 ’-terminal modified nucleoside and a 3’- terminal modified nucleoside.

[0285] In some embodiments, the polynucleotide acceptor strand of the double stranded RNA ligase substrate comprises a 5 ’-terminal modified nucleoside. In some embodiments, the 5 ’-terminal modified nucleoside comprises a 2’-modified sugar moiety. In some embodiments, the 5 ’-terminal modified nucleoside of the polynucleotide acceptor strand comprises 2’-fluoro adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro thymidine, 2’-O-methyl adenosine, 2’-O- methyl guanosine, 2’-O-methyl cytidine, 2’-O-methyl uridine, or 2’-O-methyl thymidine.

[0286] In some embodiments, the polynucleotide donor strand for the double stranded polynucleotide substrate for the double stranded RNA ligase comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 5’-terminal nucleoside of the polynucleotide donor strand, as appropriate for the length of the polynucleotide donor strand. In some embodiments, the polynucleotide donor strand comprises a modified nucleoside at one or more nucleoside residue positions 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the 3’-terminal nucleoside of the polynucleotide donor strand, as appropriate for the length of the polynucleotide donors strand.

[0287] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide donor strand are modified. In some embodiments, all of the nucleosides of the polynucleotide donor strand are modified.

[0288] In some embodiments, the polynucleotide donor strand for the double stranded RNA ligase reaction comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5’-terminal region, 3’-terminal region, and / or the internal region of the polynucleotide donor strand.Docket Number CX10-274WO1

[0289] In some embodiments, the polynucleotide donor strand of the double stranded RNA ligase substrate comprises a 5 ’-terminal and / or 3’ - terminal modified intemucleoside linkage.

[0290] In some embodiments, the polynucleotide donor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the polynucleotide donor strand. In some embodiments, the polynucleotide acceptor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3’-terminal nucleoside of the polynucleotide donor strand.

[0291] In some embodiments, the polynucleotide donor strand comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the polynucleotide donor strand comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0292] In some embodiments, the polynucleotide donor strand of the double stranded polynucleotide substrate comprises a combination of one or more modified nucleosides and one or more modified intemucleoside linkages.

[0293] In some embodiments, the polynucleotide strand (or strands), or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand forming the ligatable nick comprise one or more modified nucleosides.

[0294] In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide acceptor strand comprises a modified nucleoside at the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide acceptor strand. In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide acceptor strand comprises a modified nucleoside at the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide acceptor strand.

[0295] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand comprises one or more modified nucleosides at nucleoside position 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 5’-terminal nucleoside of the polynucleotide acceptor strand, to the nucleosides 3’ of the nucleoside complementary to the 5’- terminal nucleoside of the polynucleotide acceptor strand.

[0296] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand comprises one or more modified nucleosides at nucleoside position 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 3’-terminal nucleoside of the polynucleotide acceptor strand, to the nucleosides 5’ of the nucleoside complementary to the 3’- terminal nucleoside of the polynucleotide acceptor strand.Docket Number CX10-274WO1

[0297] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand are modified.

[0298] In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide donor strand comprises a modified nucleoside at the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide donor strand. In some embodiments, the polynucleotide, or a segment thereof, complementary to the polynucleotide donor strand comprises a modified nucleoside at the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide donor strand.

[0299] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide donor strand comprises one or more modified nucleosides at nucleoside position 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide donor strand, to the nucleosides 3’ of the nucleoside complementary to the 5 ’-terminal nucleoside of the polynucleotide donor strand.

[0300] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide donor strand comprises one or more modified nucleosides at nucleoside position 2, 3, 4, 5, 6, 7, 8, 9, or 10 from the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide donor strand, to the nucleosides 5’ of the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide acceptor strand.

[0301] In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleosides of the polynucleotide, or segment thereof, complementary to the polynucleotide donor strand are modified.

[0302] In some embodiments, the polynucleotide complementary to the polynucleotide acceptor strand and the polynucleotide donor strand of the double stranded RNA ligase substrate comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage is present at the 5 ’-terminal region, 3 ’-terminal region, and / or the internal region of the complementary polynucleotide.

[0303] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises a 5’-terminal and / or 3’- terminal modified intemucleoside linkage.

[0304] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises one or more modifiedDocket Number CX10-274WO1 intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 5 ’-terminal nucleoside of the complementary polynucleotide strand. In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises one or more modified intemucleoside linkages at the intemucleoside linkage position 1, 2, 3, 4, or 5 from the 3 ’-terminal nucleoside of the complementary polynucleotide.

[0305] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand comprises 1, 2, 3, 4, 5, or more of internal modified intemucleoside linkages. In some embodiments, the complementary polynucleotide strand comprises at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of intemucleoside linkages as modified intemucleoside linkages.

[0306] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand of the double stranded RNA ligase substrate comprises a combination of one or more modified nucleosides and one or more modified intemucleoside linkages.

[0307] In some embodiments, the polynucleotide acceptor strand, the polynucleotide donor strand, and / or the polynucleotide strand complementary to the acceptor strand and the donor strand comprises modified nucleosides, modified intemucleoside linkages, or a combination of modified nucleosides and modified intemucleoside linkages of the double stranded polynucleotide substrate(s), particularly at the ligation junction. As used herein, the “ligation junction” in context of a double stranded ligase substrate includes at least the 3 ’-terminal nucleotide of the polynucleotide acceptor strand and the 5’- terminal nucleotide of the polynucleotide donor strand forming the nick when the polynucleotide acceptor strand and the polynucleotide donor strand are hybridized to a complementary polynucleotide strand. In some embodiments, the polynucleotide acceptor strand and polynucleotide donor strand has perfect complementarity to the complementary polynucleotide strand at the ligation junction. In some embodiments, the polynucleotide acceptor strand and the polynucleotide donor strand has imperfect complementarity at the ligation junction. In some embodiments, one or more nucleosides 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases from the ligation junction on the polynucleotide acceptor strand are modified. In some embodiments, one or more nucleosides 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases from the ligation junction on the polynucleotide donor strand are modified.

[0308] In some embodiments, the ligation junction comprises one or more modified intemucleoside linkages. In some embodiments, the polynucleotide acceptor strand forming the ligation comprises a modified intemucleoside linkage. In some embodiments, the polynucleotide donor strand forming the ligation junction comprises a modified intemucleoside linkage. In some embodiments, the polynucleotide complementary to the polynucleotide acceptor stand and polynucleotide donor strandDocket Number CX10-274WO1 comprises a modified intemucleoside linkage. In some embodiments, the modified intemucleoside linkage

[0309] In some embodiments, the ligation junction of a double stranded RNA ligase substrate comprises a polynucleotide acceptor strand comprising a 3 ’-terminal modified nucleoside, and a polynucleotide donor strand comprising a 5 ’-terminal modified nucleoside. In some embodiments, the 3 ’-terminal modified nucleoside of the polynucleotide acceptor strand and the 5 ’-terminal modified nucleoside of the polynucleotide donor strand comprises a 2’ -modified sugar moiety. In some embodiments, the 3 ’-terminal modified nucleoside of the polynucleotide acceptor strand comprises 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, and the 5’-terminal modified nucleoside of the polynucleotide donor strand 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.

[0310] In some embodiments, the polynucleotide, or segment thereof, complementary to the polynucleotide acceptor strand and polynucleotide donor strand at the ligation junction comprises a modified nucleoside at the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide acceptor strand, and a modified nucleoside at the nucleoside complementary to the 5’- terminal nucleoside of the polynucleotide donor strand. In some embodiments, the modified nucleoside at the nucleoside complementary to the 3 ’-terminal nucleoside of the polynucleotide acceptor strand comprises 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, and the modified nucleoside at the nucleoside complementary to the 5’-terminal nucleoside of the polynucleotide donor strand comprises 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.

[0311] In some embodiments, the modification or modifications in the ligase substrate, such as modified nucleosides and / or modified intemucleoside linkages, can comprise modifications described herein and below.2’- and 3 ’-sugar modifications

[0312] In some embodiments, the modified nucleotide 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 someDocket Number CX10-274WO1 embodiments, the furanosyl sugar moiety is modified or substituted at the 2’, 3’, or a combination of 2’ and 3’ positions, as appropriate. 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, SOCHS, SO2CH3, ONO2, NO2, Ns, and NH2. In some embodiments, substituent groups at the 2 ’-position include, among others, 0-(Ci-Cio)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O-alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or Ci- 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).

[0313] In some embodiments, the modified 2’ -position of the sugar moiety is halo, 2’-O-R’, or 2’-O- COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a Ci-C4alkyl. In some embodiments, the modified 2’-position is a 2’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, the 2 ’-position of the sugar moiety of the nucleoside substrate is -O-R’, wherein R’ is -CH3or -C tLCHs or -CFLCFLOCHs. In some embodiments, the modified 2’-position is 2’-O-(2 -methoxy ethyl), 2’-O-allyl, 2’-O-propargyl, 2’-O- ethylamine, 2’-O-cyanoethyl, -2’-0-amine, or 2’-O-acetate ester.

[0314] In some embodiments, a modification at the 2 ’-position comprises a locked nucleoside. In some embodiments, locked nucleosides comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a “2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring (see, e.g., Obika et al., Tetrahedron Letters, 1997, 38(50):8735-8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11): 1138-1142). In some embodiments, the ribose moiety of the locked nucleotide is in the C3’-endo (beta-D) or C2’-endo (alpha-L) conformation. In some embodiments, the bridge is a methylene bridge. In some embodiments, the bridge is an ethylene bridge, also referred to as ENA (see, e.g., Morita et al., Bioorg Med Chem Lett., 2002, 12( 1): 73-6). Other locked nucleoside are described in International patent publication WO 2121249993, incorporated by reference herein.

[0315] 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:Docket Number CX10-274WO1where R in the above is alkyl or acyl, and B refers to a nucleobase.

[0316] In some embodiments, a modification at the 2’ -position comprises a reactive moiety or a conjugate moiety, including a conjugate moiety attached via a linker or a linker, as described herein.

[0317] 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 some embodiments, the modification at the 3 ’-position are similar to those at the 2’- position. In some embodiments, substitutions at the 3’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 3’-alkoxy (e.g., O-methyl, O-ethyl, etc.). In some embodiments, other modifications at the 3 ’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCHS, SO2CH3, ONO2, NO2, Ns, and NH2. In some embodiments, substituent groups at the 3 ’-position include, among others, 0-(Ci-Cio)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O-alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C1-C10 alkenyl and alkynyl. In some embodiments, substituent groups at the 3 ’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl. In some embodiments, In some embodiments, the substitution at the 3 ’-position is a phosphate.

[0318] In some embodiments, the modified 3’-position of the sugar moiety is halo, 3’-O-R’, or 3’-O- COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a Ci-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 -CH3or -CHsCH, or -C HsCHsOCHs. In someDocket Number CX10-274WO1 embodiments, the modified 3’-position is 3 ’-O-(2 -methoxy ethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O- ethylamine, 3’-O-cyanoethyl, -3’-0-amine, or 3’-O-acetate ester.

[0319] 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 singlestranded 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- methoxy ethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O-ethylamine, 3’-O-cyanoethyl, -3’-0-amine, 3’-O- acetate ester, 3’-phosphate, 3 ’-diphosphate, or 3 ’-triphosphate. In some embodiments, the 3’- blocking group is paired with the corresponding deblocking agent used in the deblocking or cleavage of the 3 ’-blocking group. Other reversible or cleavable 3 ’-blocking groups are described in International patent publication WO2023183569, incorporated by reference herein.

[0320] In some embodiments, a modification at the 3 ’-position comprises a reactive moiety or a conjugate moiety, including a conjugate moiety attached via a linker’ or a linker, as described herein.

[0321] In some embodiments, the modified sugar moiety comprises an unlocked nucleoside. In some embodiments, in the unlocked nucleoside, the furanosyl ring is opened to result in the structure below:where B represents the nucleobase. Unlocked nucleosides are described in, among others, International patent publication W02022 / 098990 and Snead et al., Molecular Therapy-Nucleic Acids, 2013, 2, el03.

[0322] As described herein, in some embodiments, where the modification is to the 3 ’-terminal nucleotide of the polynucleotide acceptor, the 3 ’-OH group of the nucleoside, or equivalent position thereof, is maintained to act as an acceptor for the ligase reaction. In some embodiments, where the modification is to the 5 ’-terminal nucleotide of the polynucleotide donor, the 5 ’-phosphate group of the nucleoside, or equivalent position thereof, is maintained to act as a donor for the ligase reaction. In some embodiments, the 5 ’-phosphate group of the polynucleotide donor strand comprises a 5’- phosphorothioate (see, e.g., U.S. Patent No. 6811986, incorporated by reference herein).Modified nucleobasesDocket Number CX10-274WO1

[0323] In some embodiments, the modified nucleotide comprises a modified nucleobase. In some embodiments, modified nucleobase that is capable of hydrogen bonding to form Watson and Crick type base pairing is selected.

[0324] In some embodiments, the nucleobase comprise an inosine nucleoside (i.e., nucleosides comprising a hypoxantine nucleobase). In 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 modified nucleobases include tricyclic pyrimidines, e.g., l,3-diazaphenoxazine-2-one. 1,3- diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1.3-diazaphenoxazine-2-one (G-clamp).

[0325] In some embodiments, the modified nucleobase includes, among others, nucleobases based on 2,4-dihalotolene and benzimidazole groups. In some embodiments, the modified nucleobase is 4- methylbenzimidazole, 2,4-difluorotoluene, 9-methylimidazo[(4,5)-b]pyridine, 2,4-dibromotoluene, benzimidazole, 5 -nitrobenzimidazole, 6-nitrobenzimidazole, and 5 -nitroindole. In some embodiments, the modified nucleobase is 7-azaindole, and isocarbostyril (see, e.g., Berdis et al., Front. Chem. 10: 1051525). Other modified nucleobases are described in, among others, patent publication WO2021249993.

[0326] 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.

[0327] In some embodiments, the modified nucleobase is present on the 5 ’-terminal nucleoside of the polynucleotide acceptor or polynucleotide donor, 3 ’-terminal nucleoside of the polynucleotide acceptor or polynucleotide donor, and / or present on the internal nucleosides of the polynucleotide acceptor or polynucleotide donor, as described herein. In some embodiments, the blocks or contiguous stretches of nucleosides in the polynucleotide acceptor or polynucleotide donor have modified nucleobases.Docket Number CX10-274WO1

[0328] In some embodiments, where the nucleic acid ligase substrate is a double stranded nucleic acid ligase substrate, the polynucleotide complementary to the polynucleotide acceptor strand and / or the polynucleotide donor strand comprises one or more modified nucleobases.Modified intemucleoside linkages

[0329] In some embodiments, the modified nucleotide comprises at least one modified, non-naturally occurring intemucleoside linkage. In some embodiments, the modified polynucleotide 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.

[0330] 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.

[0331] 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-SiFE-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 p-amino acids (see, e.g., Banerjee et al., Bioconjugate Chem., 2015, 26, 8, 1737-1742).

[0332] 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 stereomeric configuration. In some embodiments, the polynucleotide acceptor and / or polynucleotide donor have a mixture of stereoisomers in the intemucleoside linkages. In some embodiments, the polynucleotide acceptor and / or polynucleotide donor have greater than 50% of the intemucleoside linkages as Rp or Sp configuration. In some embodiments, the polynucleotide acceptor and / or polynucleotide donor have at least 60%, 70%, 80%, 90%, or greater of Rp or Sp stereomeric configuration.

[0333] In some embodiments, the modified intemucleoside linkages are present in the 5 ’-terminal region of the polynucleotide acceptor and / or polynucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified intemucleoside linkages are present at the 5 ’-terminal region of the polynucleotide acceptor and / or polynucleotide donor. In some embodiments at least 1 or 2 phosphorothioate intemucleoside linkages are present at the 5 ’-terminal region of the polynucleotideDocket Number CX10-274WO1 acceptor and / or polynucleotide donor. In some embodiments, the phosphorothioate linkage is a nonbridging phosphorothioate intemucleoside linkage.

[0334] In some embodiments, the modified intemucleoside linkages are present in the 3 ’-terminal region of the polynucleotide acceptor or polynucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified intemucleoside linkages are present at the 3 ’-terminal region of the polynucleotide acceptor and / or polynucleotide donor. In some embodiments, at least 1 or 2 phosphorothioate intemucleoside linkages are present at the 3 ’-terminal region of an polynucleotide acceptor or polynucleotide donor. In some embodiments, the modified intemucleoside linkages are present in the internal portions of the polynucleotide acceptor or polynucleotide donor.

[0335] In some embodiments, the polynucleotide acceptor and / or polynucleotide 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 nucleotides of the polynucleotide acceptor and / or polynucleotide donor strand.

[0336] In some embodiments, where the nucleic acid ligase substrate is a double stranded nucleic acid ligase substrate, the polynucleotide complementary to the polynucleotide acceptor strand and / or the polynucleotide donor strand comprises one or more modified intemucleoside linkages.Terminal groups

[0337] In some embodiments, the polynucleotide acceptor and / or polynucleotide donor comprises a terminal group. In some embodiments, where the nucleic acid ligase substrate is a double stranded nucleic acid ligase substrate, the polynucleotide complementary to the polynucleotide acceptor strand and / or the polynucleotide donor strand comprises a terminal group.

[0338] In some embodiments, the terminal group is attached to the 5 ’-OH or 4 ’-carbon atom of the terminal nucleoside. 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)

[0339] 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.

[0340] 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.Docket Number CX10-274WO1

[0341] In some embodiments, the terminal group 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 ’).Conjugate moiety

[0342] In some embodiments, the modified nucleotide comprises a conjugate moiety. In some embodiments, the purine nucleosidase and / or AMP phosphorylase are used with nucleic acid ligases, wherein the nucleic acid substrate includes a conjugate moiety. In some embodiments, the polynucleotide acceptor and / or the polynucleotide donor comprises a conjugate moiety. In some embodiments, where the nucleic acid ligase substrate is a double stranded nucleic acid ligase substrate, the polynucleotide complementary to the polynucleotide acceptor strand and / or the polynucleotide donor strand comprises a conjugate moiety.

[0343] 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 an oligonucleotide, including for cellular targeting of an oligonucleotide.

[0344] In some embodiments, the conjugate moiety can be attached to the 5’-terminal nucleotide, the 3 ’-terminal nucleotide, or an internal nucleotide of a ligase substrate. In some embodiments, the conjugate moiety is attached the 2’-position of the sugar moiety of a nucleoside, for example, to the 2 ’-OH. In some embodiments, the conjugate moiety is attached to the 3 ’-position of the sugar moiety of the nucleoside, for example 3 ’-OH. In some embodiments, the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res. 2019 Feb 20; 47(3): 1082-1096). In some embodiments, the conjugate moiety is attached directly or attached using a linker.

[0345] 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 Ce-alkyl, Cy-alkyl. Cs-alkyl, C9- alkyl, Cio-alkyl, Cn-alkyl, Cn-alkyl, Cis-alkyl, Ci4-alkyl, Cis-alkyl, Ci6-alkyl, Cn-alkyl, Cis-alkyl, Cis-alkyl, C2o-alkyl, C2i-alkyl, or C22-alkyl. In some embodiments, the conjugate moiety comprises a C, alkenyl, C7 alkenyl, Cs alkenyl C9 alkenyl, C10 alkenyl, Cn-alkenyl, Ci2-alkenyl, Cis-alkenyl, C14- alkenyl, Cis-alkenyl, Ci6-alkenyl, Cn-alkenyl, Cis-alkenyl, Ci9-alkenyl, C2o-alkenyl, C2i-alkenyl, orDocket Number CX10-274WO1C22-alkenyl. In some embodiments, the conjugate moiety comprises a C„ alkynyl, C7 alkynyl, Cs alkynyl, C9 alkynyl, C10 alkynyl, Cn-alkynyl, Ci2-alkynyl, Cis-alkynyl, Ci4-alkynyl, Cis-alkynyl, Ci6- alkynyl, Ci7-alkynyl, Cis-alkynyl, C19 -alkynyl, C2o-alkynyl, C2i-alkynyl, or C22-alkynyl.

[0346] 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.

[0347] 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-(l,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.

[0348] 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.

[0349] In some embodiments, the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl- heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, heteroarylalkenyl-, cycloalkylheteroalkenyl- heterocycloalkylheteroalkenyl-, arylheteroalkenyl-, or heteroarylheteroalkenyl-.

[0350] In some embodiments, the conjugate moiety comprises a lipid or lipophilic moiety, for example a fatty acid. In some embodiments, the fatty acid comprises a saturated fatty acid, unsaturated fatty acid, or a polyunsaturated fatty acid. In some embodiments, the fatty acid comprises caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans-vaccenic acid, linoleic acid, alpha-linoleic acid, gammalinoleic 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, W02024040041; incorporated herein by reference).

[0351] 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.Docket Number CX10-274WO1

[0352] In some embodiments, the conjugate moiety comprises a phospholipid. In some embodiments, the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.

[0353] 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.

[0354] 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. In some embodiments, the GalNAc targeting moiety is L96.

[0355] In some embodiments, the GalNAc moiety has the following structure,Docket Number CX10-274WO1where 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. An exemplary monovalent GalNAc moiety iswherein the monovalent GalNAc is attached via the linker to the 2’ -position of a nucleoside, such as adenine or guanine. These conjugate moieties can be present in contiguous nucleotides in an oligonucleotide (see, e.g., W02024040041).

[0356] In some embodiments, the conjugate moiety is a trivalent GalNAc. Tri-valent N- acetylgalactosamine conjugate moieties are described in, for example, WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620. The term “trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker. Exemplary trivalent GalNAc conjugate moiety is depicted below:Docket Number CX10-274WO1

[0357] 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 FluorDocket Number CX10-274WO1680), 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)

[0358] 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.

[0359] 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).

[0360] In some embodiments, the conjugate moiety comprises a peptide. In some embodiments, the peptide comprises a cellular targeting peptide and / or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate-modified polynucleotide. 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, incorporated by reference herein.

[0361] 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.Polynucleotides, expression vectors, and host cells

[0362] In another aspect, the present disclosure provides recombinant polynucleotides encoding the purine nucleosidase or AMP phosphorylase described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the purine nucleosidase or AMP phosphorylase.Docket Number CX10-274WO1

[0363] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode a recombinant purine nucleosidase or AMP phosphorylase of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that could be made that encode the purine nucleosidase or AMP phosphorylase described herein by selecting combinations based on the possible codon choices, and all such variations of polynucleotides are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples and in the accompanying Sequence Listing.

[0364] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. In some embodiments, preferred codons in bacterial cells are used for expression in bacterial cells. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, codon optimized polynucleotides encoding a recombinant purine nucleosidase or AMP phosphorylase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.

[0365] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure encodes a purine nucleosidase or AMP phosphorylase described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized.

[0366] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant purine nucleosidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, or 10.

[0367] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant purine nucleosidase comprising an AMP nucleosidase. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant AMP nucleosidase comprising a bacterial AMP nucleosidase. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant AMPDocket Number CX10-274WO1 nucleosidase comprising an AMP nucleosidase of Zhizhongheella, Thermoflavifilum, Thermomonas, Thermophagus, Amphiplicatus, Thermococcus, Methanotorris, or Methanocaldococcus .

[0368] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a purine nucleosidase comprising an AMP nucleosidase of Zhizhongheella caldifontis, Thermoflavifilum aggregans, Thermomonas hydrothermalis, Thermophagus xiamenensis, or Amphiplicatus me trio thermophilus.

[0369] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a purine nucleosidase comprising an amino acid sequence comprising amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or comprising SEQ ID NO: 2, 4, 6, 8, or 10.

[0370] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 45 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, or 9, wherein the polynucleotide sequence encodes a purine nucleosidase, or to the reference polynucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, or 9.

[0371] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 45 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, or 9, or comprising SEQ ID NO: 1, 3, 5, 7, or 9.

[0372] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an AMP phosphorylase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, 14, or 16, or corresponding to SEQ ID NO: 12, 14, or 16.

[0373] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an AMP phosphorylase of Thermococcus, Methanotorris, or Methanocaldococcus . In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an AMP phosphorylase of Thermococcus kodakarensis, Methanotorris igneus, or Methanocaldococcus j annas chii.

[0374] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 45 to the 3’-terminus of SEQ ID NO: 11, 13, or 15, or to the referenceDocket Number CX10-274WO1 polynucleotide sequence comprising SEQ ID NO: 11, 13, or 15, wherein the polynucleotide sequence encodes an AMP phosphorylase.

[0375] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 45 to the 3’-terminus of SEQ ID NO: 11, 13, or 15, or comprising SEQ ID NO: 11, 13, or 15.

[0376] As noted above, in some embodiments, the recombinant polynucleotide is codon-optimized for expression of the encoded recombinant purine nucleosidase or AMP phosphorylase. In some, the polynucleotide sequence is codon optimized for expression in a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0377] In some embodiments, a recombinant polynucleotide encoding a purine nucleosidase or AMP phosphorylase is manipulated in a variety of ways to facilitate expression of the encoded polypeptide. In some embodiments, the recombinant polynucleotide encoding the purine nucleosidase or AMP phosphorylase comprises expression vectors where one or more control sequences is present to regulate the expression polynucleotides and / or encoded polypeptides. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0378] In some embodiments, suitable promoters are selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-Docket Number CX10-274WO13 -phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in insect cells include, but are not limited to, polyhedrin, plO, ELT, OpIE2, and hr5 / iel promoters. Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken [3-actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor-la or glyceraldehyde -3 -phosphate dehydrogenase, and from Gallus P-actin.

[0379] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the purine nucleosidase or AMP phosphorylase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho- independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3 -phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8(6):423-88). Exemplary terminators for insect cells and mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.

[0380] In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is used for translation by the host cell. In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the [urine nucleosidase or AMP phosphorylase polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholDocket Number CX10-274WO1 dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include but are not limited to the 5 -UTR element present in orthopoxvirus mRNA.

[0381] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3 ’ UTR sequences for insect and mammalian host cells include, but are not limited to, OpIE2 polyA sequence, D. melanogaster metallothionein (Mt) polyA signal sequence, D. melanogaster alcohol dehydrogenase (adh), SV40 polyA signal sequence, and the 3'-UTRs of a- and [3-globin mRNAs harboring sequence elements that increase the stability and translation of mRNA.

[0382] In some embodiments, the control sequence, where appropriate, comprises a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway). In some embodiments, the 5’ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5’ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the recombinant polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57: 109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited toDocket Number CX10-274WO1 those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for insect and mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.

[0383] In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

[0384] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0385] In another aspect, the present disclosure provides a recombinant expression vector comprising a recombinant polynucleotide encoding a purine nucleosidase or AMP phosphorylase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce recombinant expression vectors capable of expressing the encoded purine nucleosidase or AMP phosphorylase.

[0386] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the polynucleotide encoding the purine nucleosidase or AMP phosphorylase. The choice of the vectorDocket Number CX10-274WO1 typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0387] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon is utilized.

[0388] In some embodiment, the recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0389] In some embodiments, the expression vector contains one or more selectable markers, which permit selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5 '-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

[0390] In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a purine nucleosidase or AMP phosphorylase polypeptide described herein, the polynucleotide(s) being operably linked to one or more control sequences for expression of the recombinant phosphatase enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are known in the art and include but are not limited to, bacterial cells, such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and SpodopteraDocket Number CX10-274WO1Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21).

[0391] In another aspect, the present disclosure provides a method of producing the purine nucleosidase or AMP phosphorylase, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the purine nucleosidase or AMP phosphorylase under conditions suitable for expression of the polypeptide such that the purine nucleosidase or AMP phosphorylase is produced. In some embodiments, the method further comprises isolating the purine nucleosidase or AMP phosphorylase polypeptides from the culture and / or host cells. In some embodiments, the method further comprises purifying the expressed purine nucleosidase or AMP phosphorylase polypeptide, as described herein.

[0392] In some embodiments, the purine nucleosidase or AMP phosphorylase is expressed in a host cell is recovered from the cells and / or the culture medium using any one or more of the known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography, such as described herein.

[0393] Chromatographic techniques for isolation / purification of the single-stranded RNA ligase polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography, sizeexclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the single-stranded RNA ligase depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the phosphatase. For affinity chromatography purification, an antibody that specifically binds the purine nucleosidase or AMP phosphorylase may be used. In some embodiments, an affinity tag, e.g., His-tag, can be introduced into the purine nucleosidase or AMP phosphorylase for purposes of isolation / purification.Compositions of purine nucleosidase and / or AMP phosphorylase

[0394] In a further aspect, the present disclosure provides compositions of the purine nucleosidase or AMP phosphorylase disclosed herein. In some embodiments, the composition comprises a recombinant purine nucleosidase or AMP phosphorylase polypeptide described herein. In some embodiments, the recombinant purine nucleosidase or AMP phosphorylase polypeptide in the compositions is isolated or purified.

[0395] In some embodiments, the recombinant purine nucleosidase or AMP phosphorylase is combined with other components and compounds, including other enzymes and reagents of those other enzymes used in conjunction with purine nucleosidase and / or AMP phosphorylase, to provideDocket Number CX10-274WO1 compositions and formulations comprising the recombinant purine nucleosidase or AMP phosphorylase polypeptide, as appropriate for different applications and uses.

[0396] In some embodiments, the composition further comprises a buffer. Suitable reaction buffers are well known in the art and include but are not limited to, borate, phosphate, 2-(N- morpholino)ethanesulfonic acid (MES), 3 -(N-morpholino)propane sulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris), and the like. Any suitable buffer may be used. In some embodiments, the buffer is present at a concentration of 10-100 mM, 50-500 mM, or 300 mM- 1 M.

[0397] In some embodiments, the composition further comprises a nucleoside-5 ’-monophosphate (NMP) substrate. In some embodiments, the composition comprises NMP concentration of about 0.01-10 mM. In some embodiments, the NMP concentration is about 0.01, 0.05, 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.

[0398] In some embodiments, the composition further comprises a divalent metal ion. In some embodiments, the divalent metal ion, such as Ni2+, Mg2+, Mn2+, or Co2+. In some embodiments, the divalent is present in the form of a salt, such MgCE, MnCE, or C0CI2. In some embodiments, the divalent metal is present in the composition at about 0.1 mM-25 mM, 0.2 mM-20 mM, 0.5 mM-15 mM, 1 mM-10 mM, or 2 mM-5 mM. In some embodiments, the divalent metal is present at about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, or greater. In some embodiments, the divalent metal ion is compatible with the enzyme used in conjunction with the purine nucleosidase and / or AMP phosphorylase.

[0399] In some embodiments, the composition comprises one or more of EDTA, DTT, and glycerol, such as for storage of the recombinant purine nucleosidase or AMP phosphorylase. In some embodiments, the composition further comprises a preservative or cryoprotectant. In some embodiments, the additive comprises a preservative / cryoprotectant, for example, dextran, polyethylene glycol, or glycerol. In some embodiments, the additive comprises a surfactant, for example, polysorbate (e.g., polysorbate 20 or 80).

[0400] In some embodiments, the composition further comprises another enzyme used in conjunction with the purine nucleosidase and / or AMP phosphorylase. In some embodiments, the other enzyme includes a nucleic acid ligase, for example a dsDNA ligase, ssDNA ligase, dsRNA ligase, ssRNA ligase, or an RNA splice ligase. In some embodiments, the composition further comprises a substrate for the nucleic acid ligase.

[0401] In some embodiments, the composition further comprises another accessory enzyme, particularly a pyrophosphatase. In some embodiments, the pyrophosphatase enzyme is a Type I pyrophosphatase, which are generally homo-hexameric in structure; a Type II pyrophosphatase, whichDocket Number CX10-274WO1 are generally from bacterial and archaeal sources; or a Type III pyrophosphatase, which are characterized by a haloalkanoate dehalogenase superfamily (HADSF) protein fold.

[0402] In some embodiments, the composition further comprises an enzyme additive, including, among others, one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000). In some embodiments, the composition comprises 1% to 40% v / v of DMSO. In some embodiments, the composition comprises 0.1 M to 3 M betaine. In some embodiments, the composition comprises 0.5% to 20% w / v of PEG (e.g., PEG6000 or PEG8000).

[0403] In some embodiments, a recombinant purine nucleosidase and / or AMP phosphorylase described herein is provided in solution, as a lyophilizate, or is immobilized on a support medium. In some embodiments, the support medium is a solid substrate, porous substrate, membrane, or particles. The enzyme can be entrapped in matrixes or membranes. In some embodiments, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, agarose or derivatives thereof (e.g., cross-linked agarose), and collagen, or solid matrices, such as activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0404] In some embodiments, the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed on the support material. In some embodiments, the enzyme is immobilized on the support material by covalent attachment. Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polyacrylate, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.EXAMPLES

[0405] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

[0406] In the experimental disclosure below, the following abbreviations where relevant apply: ppm (parts per million); M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mb (milliliter); ul, uL, ml, and mb (microliter); cm (centimeters); mm (millimeters); um and pm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); OD (optical density); MW (molecular weight); rpm (rotations per minute); ref (relative centrifugal force); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); NGS (next-Docket Number CX10-274WO1 generation sequencing); ds (double stranded); ss (single-stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FPLC (fast protein liquid chromatography); ddH2O (double distilled water); PBS (phosphate buffered saline); BSA (bovine serum albumin); DTT (dithiothreitol); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); IPTG (isopropyl P-D-l -thiogalactopyranoside); FIOPC or FIOP (fold improvements over positive control or parent); LB (Luria-Bertani); TB (Terrific-Broth).Abbreviations for modified nucleotidesDocket Number CX10-274WO1List of oligonucleotide acceptors, donors, and productsExample 1Shake Flask Expression and Purification of NucleosidasesShake Flask ExpressionDocket Number CX10-274WO1

[0407] Selected E. coli strains expressing genes of interest were plated onto LB agar plates with 1% glucose and 30 pg / mL chloramphenicol and grown overnight at 37 °C. A single colony from each culture was transferred to 5 mL of LB broth with 1% glucose and 30 pg / mL chloramphenicol. The cultures were grown for 20 h at 30 °C, 250 rpm, and subcultured at a dilution of approximately 1:50 into 250 mL of Terrific Broth with 30 pg / mL of chloramphenicol, to a final OD600 of about 0.05. The cultures were incubated for approximately 195 min at 30 °C, 250 rpm, to an OD600 of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM. The induced cultures were incubated for 20 h at 30 °C, 250 rpm. Pollowing this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 mL of 20 mM triethanolamine, pH 7.5. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-l 10L). The crude lysate was pelleted by centrifugation (11,000 rpm for 60 min at 4 °C), and the supernatant was then filtered through a 0.2 pm PES membrane to further clarify the lysate.Purification of nucleosidases from Shake Flask Lysates

[0408] Nucleosidase lysates were supplemented with 1 / 1 Oth volume of SF elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.02% v / v Triton X-100 reagent) per well. Lysates were then purified using an AKTA Start purification system and a 5 mL HisTrap FF column (GE Healthcare) using the AC Step HiF setting (the run parameters are provided below). The SF wash buffer comprised 50mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v / v Triton X-100 reagent.

[0409] Elution fractions containing protein were identified by UV absorption (A280) and pooled, then dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0. 1 mM EDTA, and 50% glycerol) in a 3.5K Slide-A-Lyzer™ dialysis cassette (ThermoFisher™) for buffer exchange. Nucleosidase concentrations in the preparations were measured by absorption at 280 nm.Example 2Docket Number CX10-274WO1Capillary Electrophoresis (CE) Analysis of OligonucleotidesSample preparation for reaction analysis using CE

[0410] For analysis of the reaction samples, capillary electrophoresis was performed using an ABI 3500x1 Genetic Analyzer (ThermoFisher). Reactions (1 pL) were quenched by the addition of 99 pL of 1 mM aqueous EDTA. Quenched reactions were diluted in water to 1.25 nM FAM-labeled oligonucleotide, and a 2-pL aliquot of this solution was transferred to a new 96-well Micro Amp Optical PCR plate or 384-well Micro Amp Optical PCR plate containing 18 pL Hi -Di™ Formamide (ThermoFisher) containing an appropriate size standard (LIZ or Alexa633). The ABI3500xl was configured with POP6 polymer, 50 cm capillaries, and a 55 °C oven temperature. Pre-run settings were 18KV for 50 sec. Injection was 10KV for 2 sec, and the run settings were 19KV for 620 sec. FAM-labeled oligo substrates and products were identified by their mobility relative to the sizing ladder.Example 3 Gene Acquisition and Expression of Wild-type Nucleosidases

[0411] Synthetic genes encoding an N-terminal 6-histidine tagged version of multiple wild-type (WT) nucleosidase enzymes were cloned into the pCKl 10900 vector system (See e.g., US Pat. No. 9,714,437, which is hereby incorporated by reference in its entirety) and subsequently expressed in an E. coli strain derived from W3110.

[0412] Cells transformed with the nucleosidase expression constructs were grown at shake-flask scale as described in Example 1. Cells were then lysed, purified, and dialyzed into storage buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0. 1 mM EDTA, and 50% glycerol). After overnight dialysis, protein samples were removed, and enzyme concentrations were measured by absorption at 280 nm using a NanoDrop™ 1000 spectrophotometer. Soluble protein concentrations are summarized in Table 3.1 below, showing a fold improvement in soluble protein production following shake-flask purification relative to the enzyme isolated from Amphiplicatus metriothermophilus (SEQ ID NO: 10).Docket Number CX10-274WO1Example 4Activity of SEQ ID NO: 2-16 in a ssRNA ligation reaction with a modified RNA acceptor substratesActivity of shake-flask purified nucleosidases

[0413] Nucleosidase variants SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16 were produced in shake flask and purified as described in Example 1.

[0414] Reactions were performed in 96-well format 200 pL BioRad PCR plates. Reactions included 200 pM oligonucleotide acceptor, 500 pM oligonucleotide donor, 1 mM ATP, 25 vol % nucleosidase solution (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16), 10 uM ssRNA ligase (SEQ ID NO: 18), 1 pM IPP (SEQ ID NO: 20), 100 mM triethanolamine (pH 7.5), 1 mM CoC’E and 10 mM MgCE. Control reactions omitted the nucleosidase. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. ssRNA ligases are known in the art and include, but are not limited to, those described in PCT Application No. PCT / US25 / 25017 filed on April 16, 2025. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.

[0415] Percent conversion to product (% product) was defined as the sum of the area of the ligation product divided by the sum of the total peak area times one hundred. The % byproduct was calculated as the sum of peak areas attributed to by-products divided by the sum of the total peak area times one hundred. The ratio %product / %by-product was calculated for each enzyme. This ratio was then divided by the ratio observed in the control experiment with no nucleoside present (n=8) to obtain the fold improvement. The results are shown in Table 4.2.Docket Number CX10-274WO1Example 5Activity of SEQ ID NO: 4-16 in a ssRNA ligation reaction with an RNA acceptor substrateActivity of shake-flask purified nucleosidases

[0416] Nucleosidase variants SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16 were produced in shake flask and purified as described in Example 1.

[0417] Reactions were performed in 96-well format 200 pL BioRad PCR plates. Reactions included 200 pM oligonucleotide acceptor, 500 pM oligonucleotide donor, 1 mM ATP, 25 vol % nucleosidase solution (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16), 10 uM ssRNA ligase (SEQ ID NO: 18), 1 pM IPP (SEQ ID NO: 20), 100 mM triethanolamine (pH 7.5), 1 mM C0CI2 and 10 mM MgCE. Control reactions omitted the nucleosidase. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.Docket Number CX10-274WO1

[0418] Percent conversion to product (% product) was calculated as the percent ligation product, defined as the sum of the area of product divided by the sum of the total peak area. The % byproduct was calculated as the sum of peak areas attributed to by-products divided by the sum of the total peak area. The ratio %product / %by-product was calculated for each enzyme. This ratio was then divided by the ratio observed in the control experiment with no nucleoside present (n=8) to obtain the foldEXAMPLE 6Activity of SEQ ID NO: 24, 26 in a ssRNA ligation reaction with an RNA acceptor substrate

[0419] Activity of shake-flask purified nucleosidases

[0420] Nucleosidase variants SEQ ID NO: 24 and 26 were produced in shake flask and purified as described in Example 1.

[0421] Reactions were performed in 384-well format BioRad PCR plates. Reactions included 1900 pM oligonucleotide acceptor unlabelled, 100 pM oligonucleotide acceptor labelled, 3000 pM oligonucleotide donor, 10 mM ATP, 0.1 pM nucleosidase (SEQ ID NO: 24 or 26), 20 uM ssRNA ligase (SEQ ID NO: 28) (PCT / US25 / 025017 filed May 2, 2025), 100 mM 3-(N- morpholino)propanesulfonic acid (MOPS, pH 8.0), 1 mM C0CI2 and 10 mM MgCT. Control reactions omitted the nucleosidase. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 384-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicatedDocket Number CX10-274WO1 temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.

[0423] Percent conversion to product (% product) was calculated as the percent ligation product, defined as the sum of the area of product divided by the sum of the total peak area. The % byproduct was calculated as the sum of peak areas attributed to by-products divided by the sum of the total peak area. The ratio %product / %by-product was calculated for each enzyme. This ratio was then divided by the ratio observed in the control experiment with no nucleosidase to obtain fold improvement. The results are shown in Table 6.2.

[0424] While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material,Docket Number CX10-274WO1 composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

[0425] For all purposes, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

Claims

Docket Number CX10-274WO1CLAIMSWHAT IS CLAIMED IS:

1. A method of increasing product formation in an enzymatic reaction producing NMP as a reaction product, comprising carrying out the enzymatic reaction in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for cleavage of the NMP to the corresponding nucleoside and ribose-5-phosphate and / or nucleoside and ribose 1,5-bisphosphate.

2. The method of claim 1, wherein the enzymatic reaction is carried out in presence of a purine nucleosidase under reaction conditions suitable for cleavage of the NMP to the corresponding nucleoside and ribose-5 -phosphate.

3. The method of claim 1, wherein the enzymatic reaction is carried out in presence of a AMP phosphorylase under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside and ribose 1,5-bisphosphate.

4. The method of claim 1, wherein the enzymatic reaction is carried out in presence of a purine nucleosidase and AMP phosphorylase under reaction conditions suitable for cleavage of NMP to the corresponding nucleoside, ribose-5-phosphate, and ribose 1,5-bisphosphate.

5. The method of any one of claims 1, 3, and 4, wherein the reaction conditions for AMP phosphorylase includes phosphate.

6. The method of any one of claims 1, 2, and 4, wherein the purine nucleosidase comprises an AMP nucleosidase.

7. The method of any one of claims 1, 2, 4, and 6, wherein the purine nucleosidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, or 10.

8. The method of claim 6, wherein the AMP nucleosidase comprises an AMP nucleosidase of Zhizhongheela, Thermoflavifilum, Thermomonas, or Amphiplicatus .

9. The method of claim 7 or 8, wherein the AMP nucleosidase is of Zhizhongheela caldifontis, Thermoflavifilum aggregans, Thermomonas hydrothermalis, or Amphiplicatus xiamenensis .Docket Number CX10-274WO110. The method of any one of claims 7-9, wherein the amino acid sequence of the purine nucleosidase comprises amino acid residues 15 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, or 10, or comprises SEQ ID NO: 2, 4, 6, 8, or 10.

11. The method of any one of claims 1, 3, 4, and 5, wherein the AMP phosphorylase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, 14, or 16, or to a reference sequence corresponding to SEQ ID NO: 12, 14, or 16.

12. The method of claim 11, wherein the AMP phosphorylase is AMP phosphorylase of Thermococcus, Me thanot orris, or Methanocaldococcus .

13. The method of claim 11 or 12, wherein the AMP phosphorylase is AMP phosphorylase of Thermococcus kodakarensis, Methanotorris igneus, or Methanocaldococcus jannaschii.

14. The method of any one of claims 11-13, wherein the amino acid sequence of the AMP phosphorylase comprises amino acid residues 15 to the carboxy terminal of SEQ ID NO: 12, 14, or 16, or comprises SEQ ID NO: 12, 14, or 16.

15. The method of any one of claims 1-14, wherein the enzymatic reaction that produces NMP as a reaction product comprises a nucleic acid ligase, nucleic acid ligase substrate, and an NTP or NAD+ co-factor.

16. The method of claim 15, wherein the nucleic acid ligase is an ssDNA ligase or dsDNA ligase and the NTP or NAD co-factor comprises ATP or NAD+, thereby resulting in reaction product AMP.

17. The method of claim 16, wherein the nucleic acid ligase comprises dsDNA ligase and the dsDNA ligase substrate comprises cohesive end dsDNA ligase substrates.

18. The method of claim 16, wherein the nucleic acid ligase comprises dsDNA ligase and the dsDNA ligase substrate comprises blunt-ended dsDNA ligase substrates.

19. The method of claim 16, wherein the nucleic acid ligase comprises ssDNA ligase and single stranded nucleic acid substrates.

20. The method of claim 15, wherein the enzymatic reaction is a ssRNA ligase or dsRNA ligase, and the NTP co-factor comprises ATP, thereby resulting in reaction product AMP.Docket Number CX10-274WO121. The method of claim 20, wherein the nucleic acid ligase is a dsRNA ligase and a dsRNA ligase substrate comprises cohesive end dsRNA ligase substrate(s).

22. The method of claim 21, wherein the dsRNA ligase substrate(s) comprise modified dsRNA ligase substrate(s).

23. The method of claim 20, wherein the nucleic acid ligase comprises an ssRNA ligase and ligase substrate comprises ssRNA ligase substrate(s).

24. The method of claim 23, wherein the ssRNA ligase substrate(s) comprise modified ssRNA ligase substrate(s).

25. The method of claim 15, wherein the nucleic acid ligase is an RNA splicing ligase.

26. The method of claim 25, wherein the RNA splicing ligase is tRNA splicing ligase.

27. The method of any one of claims 1-26, further comprising carrying out the reaction in presence of a pyrophosphatase.

28. The method of claim 27, wherein the pyrophosphatase is a Type 1 or Type 2 pyrophosphatase .

29. A method of increasing production formation in ligation of modified single stranded RNA (ssRNA) by a single stranded RNA ligase, comprising ligating the ssRNA ligase substrates with the ssRNA ligase in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for ssRNA ligase mediated ligation of the ssRNA substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

30. A method of increasing production formation in ligation of modified double stranded RNA (dsRNA) by a double stranded RNA ligase, comprising ligating the dsRNA ligase substrates with the dsRNA ligase in presence of a purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase, under reaction conditions suitable for dsRNA ligase mediated ligation of the dsRNA substrates, and conditions suitable for purine nucleosidase and / or AMP phosphorylase activity.

31. In a method of performing an enzymatic reaction that produces NMP as a reaction product, the improvement comprising carrying out the enzymatic reaction in presence of an purine nucleosidase, AMP phosphorylase, or a combination of purine nucleosidase and AMP phosphorylase.Docket Number CX10-274WO132. In a method of performing a nucleic acid ligase reaction with co-factor ATP and nucleic acid ligase substrate, the improvement comprising carrying out the ligase reaction in presence of a purine nucleosidase, AMP phosphorylase, or a combination of a purine nucleosidase and AMP phosphorylase.

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