Recombinant primases and methods of use

Abstract

The present disclosure relates to recombinant primase polypeptides and compositions thereof, as well as polynucleotides encoding the recombinant primase polypeptides. The present disclosure also provides methods of using the recombinant primase polypeptides or compositions thereof for molecular biological, diagnostic, and other purposes.

Description

RECOMBINANT PRIMASES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional No. 63 / 634,838, filed April 16, 2025, the entire contents of which is incorporated by reference herein.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX10- 265WO2_ST26.xml, created on March 10, 2025, with a file size of 724,479 bytes, is part of the specification and is incorporated by reference herein.BACKGROUND

[0003] Nucleic acid polymerases catalyze the synthesis of nucleic acid molecules from nucleoside triphosphate substrates. Nucleic polymerases can be template-dependent, where the polymerase uses a strand of nucleic acid as a template to generate a copy of the template strand. These templatedependent polymerases may require a primer to initiate the polymerization process while some polymerases can initiate polymerization without the need of a primer, e.g., T7 RNA polymerase.Some polymerases can synthesize nucleic acids in a template-independent manner by adding nucleotides to an initiator nucleic acid, or synthesize a nucleic acid ab initio in the absence of any nucleic acid initiator and template (see, e.g., Zyrina et al., FEMS Microbiol Lett., 2014, 351(1): 1-6).

[0004] Synthesis of modified nucleic acids is typically achieved by chemical synthesis involving phosphoramidite chemistry. As an alternative to chemical synthesis, nucleic acid polymerases have been adapted for synthesis of modified nucleic acids. A primer-template directed synthesis with polymerases are described in WO2023 / 041931 while template directed synthesis in the absence of a primer are described in Meyer et al., Nucleic Acids Res., 2015, 43(15):7480-7488. Templateindependent synthesis of modified oligonucleotides have used terminal deoxynucleotidyl transferases (TdTs), poly(N) polymerases, and certain DNA polymerases, such as polymerase 0 (see, e.g., W02020077227). A limitation of these template-independent polymerases is the need for an initiator nucleic acid with a free 3 ’-OH group for polymerase extension.

[0005] It would be desirable to have polymerases that can be adapted for different modes of synthesis in generating modified oligonucleotides, including template dependent and template -independent synthesis.SUMMARY

[0006] The present disclosure provides recombinant primase polypeptides and compositions thereof, as well as polynucleotides encoding the recombinant primase polypeptides. The present disclosure also provides methods of using the recombinant primase polypeptides and compositions thereof for oligonucleotide synthesis.

[0007] In one aspect, the present disclosure provides a recombinant primase comprising a polypeptide fragment of a primase polypeptide, wherein the polypeptide fragment comprises a primase domain and wherein the recombinant primase displays terminal nucleotidyl transferase activity. In some embodiments, the terminal nucleotidyl transferase activity includes templateindependent terminal nucleotidyl transferase activity.

[0008] In some embodiments, the recombinant primase comprises a polypeptide fragment comprising the primase domain of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, wherein the recombinant primase exhibits template-independent terminal nucleotidyl transferase activity. In some embodiments, the terminal nucleotidyl transferase activity includes template-independent terminal nucleotidyl transferase activity.

[0009] In some embodiments, the recombinant primase comprises a polypeptide fragment comprising the N-terminal fragment containing the primase domain. In some embodiments, the N- terminal fragment comprising the primase domain includes about 250 amino acid residues of the N- terminus comprising the PriS region or domain. In some embodiments, the N-terminal fragment comprising the primase domain includes about 400 amino acid residues of the N-terminus comprising the PriS and PriX regions or domains. In some embodiments, the recombinant primase comprises a primase domain, wherein the primase domain comprises a functionally active PriS region or domain, or functionally active PriS and PriX regions or domains of the primase domain.

[0010] In some embodiments, the recombinant primase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a polypeptide fragment comprising the primase domain of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, wherein the recombinant primase has template independent terminal nucleotidyl transferase activity.

[0011] In some embodiments, the amino acid sequence of the recombinant primase comprises the primase domain that includes the PriS region or domain. In some embodiments, the amino acid sequence of the recombinant primase comprises the primase domain that includes the PriS and PriX regions or domains.

[0012] In some embodiments, the amino acid sequence of the recombinant primase comprises the primase domain of a primase of Methanococcus. In some embodiments, the amino acid sequence of the recombinant primase comprises the primase domain of a primase of Bacillus. In some embodiments, the amino acid sequence of the recombinant primase comprises the primase domain of a primase of Chloroflexota bacterium. In some embodiments, the amino acid sequence of the recombinant primase comprises the primase domain of a primase of Ammonifex.

[0013] In some embodiments, the recombinant primase comprises a chimeric primase domain, wherein the chimeric primase domain comprises a PriS region or domain of a first primase, and thePriX region of domain of a second primase, wherein the first primase and the second primase are different, and wherein the chimeric primase has terminal nucleotidyl transferase activity. In some embodiments, the chimeric primase has template-independent terminal nucleotidyl transferase activity.

[0014] In some embodiments, the chimeric primase domain of the recombinant primase comprises a PriS region or domain of a first primase of Methanococcus, Bacillus, Chloroflexota, Ammonifex, Thermococcus, or Pyrococcus, and a PriX region or domain of a second primase of Methanococcus, Bacillus, Chloroflexota, Ammonifex, Thermococcus, or Pyrococcus, wherein the first primase and second primase are different.

[0015] In some embodiments, the chimeric primase domain of the recombinant primase comprises a PriS region or domain selected from the PriS region or domain of a primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, and a PriX region or domain selected from the PriX domain of a primase of Thermococcus or Pyrococcus.

[0016] In some embodiments, the chimeric primase domain of the recombinant primase comprises a PriS region or domain selected from the PriS region or domain of a primase of Thermococcus or Pyrococcus, and a PriX domain selected from the PriX region or domain of a primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0017] In some embodiments, recombinant primase has nucleotidyl transferase activity at least for a nucleotide donor dATP, ddATP, ddCTP, ddGTP, and / or 2’-F-ATP.

[0018] In some embodiments, the recombinant primase comprising a primase domain comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the residues 12 to the carboxy terminal of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316.

[0019] In some embodiments, the recombinant primase comprising a primase domain comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the residues 12 to the carboxy terminal of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

[0020] In some embodiments, the amino acid sequence of the primase domain of the recombinant primase comprises one or more amino acid differences relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56,58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

[0021] In some embodiments, the amino acid sequence of the primase domain of the recombinant primase comprises at least an amino acid difference at amino acid position 9, 18, 19, 34, 36, 40, 41, 42, 43, 44, 45, 46, 47, 52, 63, 64, 69, 71, 75, 80, 81, 82, 83, 84, 85, 89, 90, 92, 101, 105, 106, 109,111, 113, 119, 124, 128, 136, 137, 142, 157, 159, 171, 184, 187, 191, 192, 195, 197, 200, 201, 203,206, 208, 209, 210, 211, 224, 225, 247, 252, 273, 292, 297, 314, 327, 337, 340, 341, 344, 346, 348,351, 355, 358, 370, 375, 377, 378, 383, 391, 399, 402, 411, 416, 419, 422, and 423, or combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

[0022] In some embodiments, the amino acid sequence of the primase domain of the recombinant primase comprises at least an amino acid difference, or amino acid residue 9A, 18A, I 9G / I / V. 34E, 36T, 40C / L, 41G / L, 42R, 43R / V. 44N, 45S, 46C / G / H / R, 47G, 52K / N, 63G / I / V, 64C / S, 69R / V. 71G / L / R, 75C / M / S, 80L / V, 81A / R, 82G, 83S, 84G / L, 85G / L / P, 89R, 90N / T / V, 92L / T, 101L, 105A / G / R, 106M / S, 109M / R, 111R, 113K, 119E, 124L, 128F, 136D / P, 137G / K, 142D / G / R, 157D, 159E, 171W, 184G / P, 187R, 191V, 192P, 195M, 197A, 200G / Q, 201T, 203L, 206S / T, 208F, 209G, 210G / P / Q, 21 IP, 224N, 225P, 247R, 252E, 273A, 292R, 297D, 314N, 327A, 337L, 340G, 341V, 344H, 346V, 348T, 35 IN, 355D, 358E, 370G, 375H, 377L, 378S, 383G, 391R, 399D, 402P, 41 IL, 416G, 419H, 422G, or 423V, or combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to residues 12 to the carboxy terminus of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

[0023] In some embodiments, the recombinant primase has at least one improved property as compared to a reference primase or a reference recombinant primase having nucleotidyl transferase activity, including template -independent terminal nucleotidyl transferase activity. In some embodiments, the reference primase is the primase of Ammonifex thiophilus (e.g., SEQ ID NO: 24) or the recombinant primase of SEQ ID NO: 60.

[0024] In some embodiments, the improved property of the recombinant primase comprises increased incorporation of ddCTP or ddGTP to an oligonucleotide acceptor. In some embodiments, the improved property is increased incorporation of a nucleotide donor to a modified oligonucleotide acceptor.

[0025] In some embodiments, the recombinant primase comprises a fusion polypeptide. In some embodiments, the fusion is to a peptide affinity tag, e.g., His-tag, or a polyamino acid for conjugation, e.g., a polylysine of 2-12 residues in length.

[0026] In some embodiments, the recombinant primase is a purified preparation. In some embodiments, the recombinant primase is provided in solution, a lyophilizate, or powder form.

[0027] In a further aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding a recombinant primase described herein.

[0028] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3 ’-terminal nucleotide of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 315, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 25, 27, 29, 31, 33, 35, 37 / 38, 39 / 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 315, wherein the recombinant polynucleotide encodes a recombinant primase having terminal nucleotidyl transferase activity. In some embodiments, the encoded recombinant primase has template independent terminal nucleotidyl transferase activity.

[0029] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 3’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, wherein the recombinant polynucleotide encodes a recombinant primase having terminal nucleotidyl transferase activity. In some embodiments, the encoded recombinant primase has template independent terminal nucleotidyl transferase activity.

[0030] In some embodiments, the polynucleotide sequence is codon-optimized for expression of the encoded recombinant primase. In some embodiments, the polynucleotide sequence is codon optimized for expression in bacterial cells, fungal cells, insect cells, or mammalian cells.

[0031] In a further aspect, the recombinant polynucleotide is provided as an expression vector. In some embodiments, the expression vector comprises a recombinant polynucleotide encoding any of the recombinant primases described herein.

[0032] In some embodiments, the recombinant polynucleotide in the expression vector is operably linked to a control sequence. In some embodiments, the control sequence comprises at least a promoter.

[0033] In another aspect, the present disclosure provides a host cell comprising an expression vector for expression of the encoded recombinant primase. In some embodiments, the host cell is an appropriate prokaryotic cell or eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0034] In a further aspect, the host cells are used in a method of producing a recombinant primase, the method comprising culturing the host cell under suitable culture conditions such that the encoded recombinant primase is produced. In some embodiments, the method further comprises recovering the recombinant primase from the culture and / or host cells. In some embodiments, the method comprises purifying the recombinant primase.

[0035] In another aspect, the present disclosure provides a composition comprising a recombinant primase. In some embodiments, the composition comprises a recombinant primase immobilized on a support medium. In some embodiments, the support medium is a solid support, porous support, or membrane support. In some embodiments, the recombinant primase is covalently attached to the support medium.

[0036] In some embodiments, the composition comprising the recombinant primase comprises one or more of a nucleotide acceptor (e.g., polynucleotide acceptor, oligonucleotide acceptor, initiating nucleotide acceptor), and / or a nucleotide donor. In some embodiments, the polynucleotide or oligonucleotide acceptor comprises at least one modified nucleoside, modified intemucleoside linkage, or combinations thereof.

[0037] In some embodiments, the composition comprising the recombinant primase comprises a nucleotide donor dNTP or NTP. In some embodiments, the nucleotide donor comprises a modified nucleoside. In some embodiments, the nucleotide donor comprises a 3 ’-blocking group or a chain terminator nucleotide donor comprising a 3 ’-blocking group. In some embodiments, the 3 ’-blocking group comprises a reversible 3 ’-blocking group.

[0038] In some embodiments, the composition further comprises a second enzyme, such as a pyrophosphatase .

[0039] In another aspect, the present disclosure provides a method of using the recombinant primase for synthesizing polynucleotides and oligonucleotides. In some embodiments, a method of oligonucleotide synthesis comprises reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a recombinant primase under suitable reaction conditions for the extension of the nucleotide acceptor by attachment of the nucleotide donor to the nucleotide acceptor by the primase. In some embodiments, a method of template-independent oligonucleotide synthesis comprises reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a recombinant primase under suitable reaction conditions for the extension of the nucleotide acceptor by attachment of the nucleotide donor to the nucleotide acceptor by the primase.

[0040] In some embodiments of the method, the nucleotide acceptor comprises a polynucleotide acceptor, an oligonucleotide acceptor, or an initiating nucleotide acceptor. In some embodiments, the polynucleotide or oligonucleotide acceptor is DNA, RNA, or a mixture of DNA and RNA. In some embodiments, the polynucleotide or oligonucleotide acceptor has sufficient single stranded region atthe 3 ’-terminal region to act as a substrate for the recombinant primase. In some embodiments, the initiating nucleotide acceptor comprises NTP, NDP, NMP, or a nucleoside, or a modified NTP, NDP, NMP, or a nucleoside, wherein the initiating nucleotide acceptor has a 3 ’-OH. Preferably, the initiating nucleotide acceptor is NDP, NMP, or a nucleoside, or a modified NDP, NMP, or a nucleoside. In some embodiments, the nucleotide acceptor comprises a 5 ’-blocked nucleotide donor.

[0041] In some embodiments of the method, the nucleotide donor comprises an NTP or dNTP. In some embodiments of the method, the nucleotide donor comprises a modified nucleoside. In some embodiments, the nucleotide donor comprises a blocking group, or a terminating nucleotide donor comprising a 3 ’-blocking group, to form a 3 ’-blocked extended polynucleotide or extended oligonucleotide. In some embodiments, the 3 ’-blocking group comprises a reversible 3 ’-blocking group.

[0042] In some embodiments, the method further comprises inactivating the recombinant primase or separating the recombinant primase from the reaction solution containing the 3 ’-blocked extended polynucleotide or extended oligonucleotide. In some embodiments, the method further comprises removing or cleaving the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide.

[0043] In some embodiments, the method further comprises inactivating or separating the deblocking agent from the unblocked extended polynucleotide or oligonucleotide.

[0044] In some embodiments, the method further comprises one or more cycles of: extension with a nucleotide donor; separation of 3 ’-blocked extended polynucleotide or 3’- blocked extended oligonucleotide from the recombinant primase or inactivation of the recombinant primase; removing or cleaving the reversible 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or unblocked extended oligonucleotide; and separating the unblocked extended polynucleotide or unblocked extended oligonucleotide from the deblocking agent, wherein each cycle uses a new nucleotide donor.

[0045] In some embodiments, the nucleotide donor for at least one cycle comprises a mixture of nucleotide different donors.

[0046] In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

[0047] In some embodiments, the method further comprises a pyrophosphatase in the reaction with the recombinant primase.

[0048] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, or initiation nucleotide acceptor is attached to a supported medium, and the nucleotide donor reacted in presenceof a recombinant primase under suitable conditions for attachment of the nucleotide donor to the polynucleotide acceptor, oligonucleotide acceptor, or initiation nucleotide acceptor is attached to a supported medium

[0049] In some embodiments, the recombinant primase is immobilized on a support medium and the polynucleotide acceptor, oligonucleotide acceptor reacted with the nucleotide donor in solution in presence of the recombinant primase immobilized on a support medium under reaction conditions for attachment of the nucleotide donor to the polynucleotide acceptor, oligonucleotide acceptor, or initiation nucleotide acceptor.BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 provides an illustration of the domain structure of a primase in which primase activity is present within a single polypeptide chain.

[0051] FIG. 2 shows an analysis of samples of cell lysates and cell pellets by SDS PAGE, where the cell lysates and pellets were prepared from E. coli cells expressing different recombinant primase polypeptides (see Example 2).

[0052] FIG. 3 shows an analysis of reaction products separated on agarose gels from assays of recombinant primases of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52 (see Example 3).

[0053] FIG. 4 provides an analysis of reaction products separated on agarose gels from assays of recombinant primases of SEQ ID NO: 54, 56, 58, 60, 62, and 316.DETAILED DESCRIPTION

[0054] The present disclosure provides recombinant primase polypeptides and compositions thereof, as well as polynucleotides encoding the recombinant primase polypeptides. The disclosure also provides methods of using of the recombinant primase polypeptides and compositions thereof for synthesis of polynucleotides and oligonucleotides. In some embodiments, the recombinant primase polypeptides display, among others, increased activity, increased stability, increased product yield, and / or increased activity for attachment of a nucleotide donor to a nucleotide or oligonucleotide acceptor.Abbreviations and Definitions

[0055] 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.

[0056] 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.

[0057] 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 (z.e., equivalent to the term “including” and its corresponding cognates).

[0058] 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.”

[0059] “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.

[0060] ‘ ‘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.

[0061] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0062] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0063] ‘ ‘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).

[0064] ‘ ‘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.

[0065] ‘ ‘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).

[0066] ‘ ‘Primase” refers to an enzyme in the class of RNA polymerases that is involved in the replication of DNA by catalyzing the synthesis of short RNA molecules from ribonucleoside triphosphates in the presence of single stranded DNA template. In some embodiments, the “primase” is referred to as a primase-polymerase.

[0067] ‘ ‘PriS region,” “PriS domain,” “PriS-like region,” or “PriS-like domain” as used herein refers to the region or domain with the catalytic activity of the RNA polymerase of a primase.

[0068] ‘ ‘PriX region” or “PriX domain” “Primase X,” “PriX-like region,” or “PriX-like domain” as used herein refers to a domain present in primases and that has homology to C-terminal domain of archael and eukaryotic large primase subunit PriL. Without being bound by theory of operation, the PriX region displays a binding site for the initiating nucleotide triphosphate.

[0069] “Template independent nucleotidyl transferase activity” or “template independent terminal nucleotidyl transferase activity” as used herein refers to the activity of a polymerase in attaching a nucleotide of a nucleotide donor, e.g., NTP, to the 3 ’-OH of a polynucleotide, oligonucleotide, or initiating nucleotide acceptor in the absence of a complementary nucleic acid template.

[0070] “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.

[0071] 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 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.

[0072] “Nucleobase” refers to means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T). cytosine (C). uracil (U). or guanine (G). A “modified nucleobase” refers to a group of bases other than unmodified A, T, C, U. or G capable of pairing with at least one unmodified nucleobase.

[0073] “Nucleoside” refers to a compound comprising a nucleobase and a sugar moiety. The nucleobases and sugar moiety are each, independently, unmodified or modified.

[0074] ‘ ‘Intemucleoside linkage” refers to as a linkage that covalently couples two nucleosides together. In the polynucleotides and oligonucleotides herein, intemucleoside linkages covalently couple adjacent nucleosides together, typically forming a bond between the sugar moieties of the adjacent nucleosides. Non-limiting examples of intemucleoside linkages include phosphodiester -O- P(O)2-O- linkages and modified intemucleoside linkages, such as phosphorothioate -O-P-(O, S)-O- and phosphorodithioate -O-P(S)2-O-.

[0075] ‘ ‘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.

[0076] ‘ ‘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.

[0077] ‘ ‘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 thenucleobase of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson-Crick base pairing.

[0078] ‘ ‘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-SiH2-O-); N,N’- dimethylhydrazine (-CH2-N((CH3)-N((CH3)-); MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)- N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and 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.

[0079] “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.

[0080] “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.

[0081] “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.

[0082] “Abasic sugar moiety” refers to a sugar moiety of a nucleoside that is not attached to a nucleobase. In some embodiments, such abasic sugar moieties are referred to as “abasic nucleoside.”

[0083] ‘ ‘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.

[0084] ‘ ‘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.

[0085] 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.

[0086] ‘ ‘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 ’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.

[0087] “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.

[0088] “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.

[0089] ‘ ‘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.

[0090] “Enzymatically reversible blocking group” refers to a blocking group that is susceptible to removal or cleaving by an enzyme.

[0091] “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]).

[0092] “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.

[0093] “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.

[0094] “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.

[0095] “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.

[0096] ‘ ‘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 (z. 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 numberof 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 the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0097] ‘ ‘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 acidresidues 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.

[0098] “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.

[0099] “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a recombinant primase, 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).

[0100] ‘ ‘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.

[0101] “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. For example, a “residue difference at position X52 as compared to SEQ ID NO: 60” (or a “residue difference at position 52 as compared to SEQ ID NO: 60”) refers to a difference of the amino acid residue at the polypeptide position corresponding to position 52 of SEQ ID NO: 60. Thus, if the reference polypeptide of SEQ ID NO: 60 has a glutamic acid at position 52, then a “residue difference at position X52 as compared to SEQ ID NO: 60” refers to an amino acid substitution of any residue other than glutamic acid at the position of the polypeptide corresponding to position 52 of SEQ ID NO: 60. 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.

[0102] 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(e.g., X52K / X52N, X52K / N, or 52K / N). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either / or both conservative and nonconservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0103] “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 primasepolypeptides 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 semicolone.g., S106G;K251F) or slash

[0104] ‘ ‘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.

[0105] “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.

[0106] ‘ ‘Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity and / or retaining the improved properties of an recombinant primase. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated by and may be present in substitution sets.

[0107] ‘ ‘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.

[0108] ‘ ‘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.

[0109] ‘ ‘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.

[0110] “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.

[0111] “Improved enzyme property” refers to a recombinant primase polypeptide that exhibits an improvement in any enzyme property as compared to a reference primase polypeptide, such as a wildtype primase polypeptide or another recombinant primase polypeptide. Improved properties include but are not limited to such properties as increased enzymatic activity, increased product yield, increased protein expression, increased thermoactivity, increased thermostability, increased stability, increased substrate specificity and / or affinity, increased substrate range, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved solvent stability, increased solubility, and increased inhibitor resistance or tolerance. Exemplary improved properties are provided in the Examples.

[0112] ‘ ‘Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the recombinant primase polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) and / or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of recombinant primase) as compared to the reference primase (e.g., wild-type primase and / or another recombinant primase). Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmaxor kcahchanges of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to about 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring primase or another recombinant primase from which the primase polypeptides were derived.

[0113] ‘ ‘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.

[0114] ‘ ‘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.

[0115] “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.

[0116] ‘ ‘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 ofinterest. 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.

[0117] ‘ ‘Suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a primase polypeptide of the present disclosure is capable of attaching a nucleotide donor to a nucleotide acceptor. Exemplary “suitable reaction conditions” are provided herein (see, the Examples).

[0118] ‘ ‘Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the primase polypeptide on the substrate.

[0119] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0120] “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.

[0121] “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.

[0122] ‘ ‘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.

[0123] “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.

[0124] “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.

[0125] “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 Ci-s) 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.

[0126] “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-l -propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-l-butenyl, 3-methyl-2- butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 4-methyl-3 -pentenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl and 5 -hexenyl.

[0127] “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.

[0128] “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.

[0129] ‘ ‘Lower” in reference to substituents refers to a group having between one and six carbon atoms.

[0130] “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.

[0131] “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).

[0132] “Heterocycloalkyl” or “heterocyclyl” refers to a substituted or unsubstituted 3 to 20 membered, mono- or bicyclic, non-aromatic hydrocarbon, wherein 1 to 3 carbon atoms are 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.

[0133] “Aryl” refers to a six- to fourteen-membered, mono- or bi-carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings in the bicyclic ring is aromatic. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. Examples of “aryl” groups include phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, and the like.

[0134] “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.

[0135] “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:In some embodiments, a locked nucleoside is a bridged bicyclic compound.

[0136] ‘ ‘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.

[0137] “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.

[0138] “Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

[0139] “Haloalkyl” refers to an alkyl substituted with 1 or more halogen atoms. Preferably, the alkyl is substituted with 1 to 3 halogen atoms.

[0140] “Hydroxy” refers to -OH.

[0141] “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.

[0142] “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.

[0143] “Alkyloxy” or “alkoxy” refers to -OR’, wherein R’ is an optionally substituted alkyl.

[0144] “Aryloxy” refers to -OR’, wherein R’ is an optionally substituted aryl.

[0145] “Carboxy” refers to -COO" or COOM, wherein H or a M+counterion.

[0146] “Cyano” refers to -CN.

[0147] ‘ ‘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, heteroarylalkyl, and the like.

[0148] “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.

[0149] “Thiol” or “sulfhydryl” refers to -SH.

[0150] ‘ ‘Disulfied” refers to -S-S- groups.

[0151] “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.

[0152] “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.

[0153] “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.

[0154] “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.

[0155] “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.

[0156] ‘ ‘Substituted” as used herein means one or more hydrogen atoms of the group is replaced with a substituent atom or group commonly used in pharmaceutical chemistry. Each substituent can be the same or different. Examples of suitable substituents include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, OR (e.g., hydroxyl, alkyloxy (e.g., methoxy, ethoxy, and propoxy), aryloxy, heteroaryloxy, arylalkyloxy, ether, ester, carbamate, etc.), hydroxyalkyl, alkyloxy carbonyl, alkyloxyalkyloxy, perhaloalkyl, alkyloxyalkyl, SR (e.g., thiol, alkylthio, arylthio, heteroarylthio, arylalkylthio, etc.), S+R2, S(O)R , SO2R , NRR (e.g., primaryamine (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.

[0157] ‘ ‘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.Recombinant Primase Polypeptides

[0158] In one aspect, the present disclosure provides recombinant primases having nucleotidyl transferase activity, including template independent nucleotidyl transferase activity. Generally, a naturally occurring primase, sometimes referred to as primase-DNA polymerase or primase- polymerase, are template-dependent RNA polymerases that use a DNA template for the synthesis of short RNA fragments, typically of about 8-12 nucleotides in length, which are used for the synthesis of the lagging strand during DNA replication. The short RNA fragments synthesized by the primase are used as primers by a DNA polymerase for synthesis of the lagging strand during DNA replication. The short RNA fragments are subsequently removed by an endonuclease, e.g., Flap endonuclease 1 (FEN 1), and the gap repaired by DNA polymerase and ligase.

[0159] However, polypeptide fragments of primase, wherein the polypeptide fragments contain the primase domain can have nucleotidyl transferase activity. In some instances, the nucleotidyl transferase activity is template-independent terminal nucleotidyl transferase activity, where the template -independent terminal nucleotidyl transferase activity of the primase domain attaches a nucleotide donor to a polynucleotide / oligonucleotide acceptor or a single initiator nucleotide acceptor without the need for a DNA template. The nucleotide acceptor has a competent 3 ’-OH for covalent joining of the nucleotide of the nucleotide donor to the acceptor. In some embodiments, use of polypeptide fragments containing the primase domain can provide a way to extend a polynucleotide / oligonucleotide acceptor or for generating an oligonucleotide from a single initiator nucleotide in a template-independent process.

[0160] In some embodiments, a recombinant primase comprises a polypeptide fragment comprising the primase domain of a primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, whereinthe recombinant primase has nucleotidyl transferase activity. In some embodiments, the nucleotidyl transferase activity is template-independent terminal nucleotidyl transferase activity.

[0161] In some embodiments, a recombinant primase comprises a polypeptide fragment comprising the primase domain of a primase of Thermococcus or Pyrococcus, wherein the recombinant primase has nucleotidyl transferase activity. In some embodiments, the nucleotidyl transferase activity is template -independent terminal nucleotidyl transferase activity.

[0162] In some embodiments, the polypeptide fragment with nucleotidyl transferase activity comprises the N-terminal fragment containing the primase domain, or a polypeptide fragment of the N-terminal region, wherein the polypeptide fragment includes the primase domain. In some embodiments, the primase domain comprises at least the PriS region or domain. In some embodiments, the primase domain consists of the PriS region or domain.

[0163] In some embodiments, the N-terminal fragment containing the primase domain comprises about 200, 210, 220, 230, 240, or 250 amino acid residues of the N-terminus of the full length primase polypeptide, or a polypeptide fragment of about 200, 210, 220, 230, 240, or 250 amino acid residues of the N-terminal region, wherein the polypeptide fragment includes the primase domain. The carboxy terminal end of the polypeptide fragment sufficient to include the PriS domain can be ascertained by activity and / or sequence / structure information.

[0164] In some embodiments, the N-terminal fragment containing the primase domain comprises about 350, 360, 370, 380, 390 or 400 amino acid residues of the N-terminus of the full length primase polypeptide, or a polypeptide fragment of about 350, 360, 370, 380, 390 or 400 amino acid residues of the N-terminal region, wherein the polypeptide fragment includes the primase domain comprising the PriS and PriX regions or domains. In some embodiments, the primase domain consists of the PriS and PriX regions or domains.

[0165] In some embodiments, the primase domain of the recombinant primase comprises functionally active PriS region or domain, or functionally active PriS and PriX regions or domains of the primase domain.

[0166] In some embodiments, the primase domain of the recombinant primase consists of functionally active PriS region or domain, or consists of the functionally active PriS and PriX regions of domains.

[0167] In some embodiments, the primase domain comprises a deletion of the peptide region connecting the PriS region or domain to the PriX region or domain. In some embodiments, the deletion is about 2, 3, 4, 5, or 6 amino acid residues between the PriS region or domain and the PriX region or domain.

[0168] In some embodiments, a recombinant primase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a reference sequence corresponding to the primase domain of a primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, wherein the recombinant primase has template independent nucleotidyl transferase activity. In some embodiments, the recombinant primase comprises a polypeptide fragment comprising the primase domain.

[0169] In some embodiments, the polypeptide fragment comprises a N-terminal fragment comprising the primase domain of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0170] In some embodiments, the primase domain comprises the PriS region or domain, or PriS and PriX regions or domains of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0171] In some embodiments, the primase domain consists of PriS region or domain, or consists of PriS and PriX regions or domains of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0172] In some embodiments, the N-terminal fragment containing the primase domain comprises about 200, 210, 220, 230, 240, or 250 amino acid residues of the N-terminus of the full length primase polypeptide, or a polypeptide fragment of about 200, 210, 220, 230, 240, or 250 amino acid residues of the N-terminal region, wherein the polypeptide fragment includes the primase domain of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex. In some embodiments, the carboxy terminal end of the fragment sufficient to include the PriS region or domain can be ascertained by activity and / or sequence / structure information of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0173] In some embodiments, the N-terminal fragment containing the primase domain comprises about 350, 360, 370, 380, 390 or 400 amino acid residues of the N-terminus of the full length primase polypeptide, or a polypeptide fragment of about 350, 360, 370, 380, 390 or 400 amino acid residues of the N-terminal region, wherein the polypeptide fragment includes the primase domain comprising the PriS and PriX regions or domains of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex. In some embodiments, the primase domain consists of the PriS and PriX regions or domains of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0174] In some embodiments, the primase domain is of the primase of Methanococcus. In some embodiments, the primase domain is of the primase of Methanococcus voltae, Methanococcus halophilus, Methanocaldococcus jannaschii, Methanococcus maripaludis, Methanococcus aeolicus, Methanosarcina mazei Cl 6, Methanococcus vannielii, Methanococcus deltae, Methanothermococcus okinawensis, Methanocaldococcus infernus, Methanothermus sociahilis, Methanomethylovorans hollandica, or Methanosarcina acetivorans.

[0175] In some embodiments, the primase domain is of the primase of Bacillus. In some embodiments, the primase domain is of the primase of Bacillus alhus, Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus australimaris, Bacillus atrophaeus, Bacillus hadius,Bacillus carboniphilus, Bacillus cercus, Bacillus clarus, Bacillus fonticola, Bacillus gaemokensis, Bacillus glycinifermentans, Bacillus halotolerans, Bacillus horti, Bacillus inaquosorum, Bacillus luti, Bacillus methanolicus, Bacillus mobilis, Bacillus mycoides, Bacillus nakamurai, Bacillus norwichensis, Bacillus obstructivus, Bacillus pacificus, Bacillus paralicheniformis, Bacillus paranthracis, Bacillus pinisoli, Bacillus pseudomycoides, Bacillus pumilus, Bacillus safensis, Bacillus salipaludis, Bacillus spizizenii, Bacillus solimangrovi, Bacillus suaedae, Bacillus sub tills, Bacillus spizizenii, Bacillus thuringiensis, Bacillus toyonensis, Bacillus tropicus, Bacillus vallismortis, Bacillus velezensis, Bacillus wiedmannii, Bacillus wudalianchiensis, Bacillus xiamenensis, Bacillus yapensis, or Bacillus zhangzhouensis .

[0176] In some embodiments, the primase domain is of the primase of Chloroflexota bacterium.

[0177] In some embodiments, the primase domain is of the primase of Ammonifex. In some embodiments, the primase domain is of the primase of Ammonifex thiophilus, Ammonifex degensii, Ammonifex sp. Tomsk_66_l 1, or Ammonifex sp. SURF 55.

[0178] In some embodiments, a recombinant primase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the primase domain of the primase of Thermococcus or Pyrococcus, wherein the recombinant primase has nucleotidyl transferase activity. In some embodiments, the recombinant primase has template-independent terminal nucleotidyl transferase activity. In some embodiments, the recombinant primase comprises a polypeptide fragment comprising the primase domain.

[0179] In some embodiments, the polypeptide fragment comprises a N-terminal fragment comprising the primase domain of the primase of Thermococcus or Pyrococcus.

[0180] In some embodiments, the primase domain comprises the PriS region or domain, or PriS and PriX regions or domains of the primase of Thermococcus or Pyrococcus.

[0181] In some embodiments, the primase domain consists of PriS region or domain, or consists of PriS and PriX regions or domains of the primase of Thermococcus or Pyrococcus.

[0182] In some embodiments, the N-terminal fragment containing the primase domain comprises about 200, 210, 220, 230, 240, or 250 amino acid residues of the N-terminus of the full length primase polypeptide, or a polypeptide fragment of about 200, 210, 220, 230, 240, or 250 amino acid residues of the N-terminal region, wherein the polypeptide fragment includes the primase domain of the primase of Thermococcus or Pyrococcus. In some embodiments, the carboxy terminal end of the fragment sufficient to include the PriS domain can be ascertained by activity and / or sequence / structure information of the primase of Thermococcus or Pyrococcus.

[0183] In some embodiments, the N-terminal fragment containing the primase domain comprises about 350, 360, 370, 380, 390 or 400 amino acid residues of the N-terminus of the full length primasepolypeptide, or a polypeptide fragment of about 350, 360, 370, 380, 390 or 400 amino acid residues of the N-terminal region, wherein the polypeptide fragment includes the primase domain comprising the PriS and PriX regions or domains of the primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex. In some embodiments, the primase domain consists of the PriS and PriX regions or domains of the primase of Thermococcus or Pyrococcus.

[0184] In some embodiments, the primase domain is of the primase of Thermococcus. In some embodiments, the primase domain is of the primase of Thermococcus acidaminovorans, Thermococcus ciegaeus, Thermococcus aggregates, Thermococcus alcaliphilus, Thermococcus atlanticus, Thermococcus harophilus, Thermococcus harossii, Thermococcus celer, Thermococcus celericrescens, Thermococcus chitonophagus, Thermococcus cleftensis, Thermococcus coalescens, Thermococcus eurythermalis, Thermococcus fumicolans, Thermococcus gammatolerans, Thermococcus gorgonarius, Thermococcus guaymasensis, Thermococcus hydrothermalis, Thermococcus indicus, Thermococcus kodakarensis, Thermococcus litoralis, Thermococcus marinus, Thermococcus mexicalis, Thermococcus nautili, Thermococcus onnurineus, Thermococcus pacificus, Thermococcus paralvinellae, Thermococcus peptonophilus, Thermococcus piezophilus, Thermococcus prieurii, Thermococcus profundus, Thermococcus radiotolerans, Thermococcus sihiricus, Thermococcus siculi, Thermococcus stetteri, Thermococcus thioreducens, Thermococcus waimanguensis, Thermococcus waiotapuensis, and Thermococcus zilligii. The Thermococcus genus also comprises unclassified strains, including, among others, Thermococcus sp. AEPII la, Thermococcus sp. 101 C5, Thermococcus sp. 11N.A5, Thermococcus sp. 12-4, Thermococcus sp. 13- 2, Thermococcus sp. 13-3, Thermococcus sp. 1519, Thermococcus sp. 175, Thermococcus sp. 17S1, Thermococcus sp. 17S2, Thermococcus sp. 17S3, Thermococcus sp. 17S4, Thermococcus sp. 17S5, Thermococcus sp. 17S6, Thermococcus sp. 17S8, Thermococcus sp. 18S1, Thermococcus sp. 18S2, Thermococcus sp. 18S3, Thermococcus sp. 18S4, Thermococcus sp. 18S5, Thermococcus sp. 21-1, Thermococcus sp. 21S1, Thermococcus sp. 21S2, Thermococcus sp. 21S3, Thermococcus sp. 21S4, Thermococcus sp. 21S5, Thermococcus sp. 21S6, Thermococcus sp. 21S7, Thermococcus sp. 21S8, Thermococcus sp. 21S9, Thermococcus sp. 23-1, Thermococcus sp. 23-2, Thermococcus sp. 2319x1, Thermococcus sp. 26-2, Thermococcus sp. 26-3, Thermococcus sp. 26 / 2, Thermococcus sp. 28-1, Thermococcus sp. 29-1, Thermococcus sp. 300-Tc, Thermococcus sp. 31-1, Thermococcus sp. 31-3, Thermococcus sp. 40 45, Thermococcus sp. 4557, Thermococcus sp. 5-1, Thermococcus sp. 5-4, Thermococcus sp. 70-4-2, Thermococcus sp. 7324, Thermococcus sp. 83-5-2, Thermococcus sp. 9N2, Thermococcus sp. 9N2.20, Thermococcus sp. 9N2.21, Thermococcus sp. 9N3, Thermococcus sp. 9oN- 7, Thermococcus sp. A4, Thermococcus sp. AF1T14.13, Thermococcus sp. AF1T1423, Thermococcus sp. AF1T20.11, Thermococcus sp. AF1T6.10, Thermococcus sp. AF1T6.12, Thermococcus sp.AF1T6.63, Thermococcus sp. AF2T511, Thermococcus sp. Ag85-vw, Thermococcus sp. AM4, Thermococcus sp. AMT11, Thermococcus sp. AMT7, Thermococcus sp. Anhete70478, Thermococcus sp. Anhete70-SCI, Thermococcus sp. Anhete85478, Thermococcus sp. Anhete85-SCI, Thermococcussp. AT1273, Thermococcus sp. AVI, Thermococcus sp. AV2, Thermococcus sp. AV3, Thermococcus sp. AV6, Thermococcus sp. AV7, Thermococcus sp. AV9, Thermococcus sp. AV10, Thermococcus sp. AV11, Thermococcus sp. AVI 3, Thermococcus sp. AVI 4, Thermococcus sp. AVI 5, Thermococcus sp. AVI 6, Thermococcus sp. AV17, Thermococcus sp. AVI 8, Thermococcus sp. AV20, Thermococcus sp. AV21, Thermococcus sp. AV22, Thermococcus sp. Ax00-17, Thermococcus sp. Ax00-27, Thermococcus sp. Ax00-39, Thermococcus sp. Ax00-45, Thermococcus sp. Ax01-2, Thermococcus sp. Ax01-3, Thermococcus sp. Ax01-37, Thermococcus sp. Ax01-39, Thermococcus sp. Ax01-61, Thermococcus sp. Ax01-62, Thermococcus sp. Ax01-65, Thermococcus sp. Ax98-43, Thermococcus sp. Ax98-46, Thermococcus sp. Ax98-48, Thermococcus sp. Ax99-47, Thermococcus sp. Ax99-57, Thermococcus sp. Ax99-67, Thermococcus sp. AXTV6, Thermococcus sp. Bl, Thermococcus sp. B1001, Thermococcus sp. B4, Thermococcus sp. BHI60a21, Thermococcus sp. BHI80a28, Thermococcus sp. BHI80a40, Thermococcus sp. Bubb.Bath, Thermococcus sp. BX13, Thermococcus sp. CARSO, Thermococcus sp. Champagne, Thermococcus sp. CIR10, Thermococcus sp. CKU-1, Thermococcus sp. CKU-199, Thermococcus sp. CL2, Thermococcus sp. CMI, Thermococcus sp. CNRS, Thermococcus sp. CXI, Thermococcus sp. CX2, Thermococcus sp. CX3, Thermococcus sp. CX4, Thermococcus sp. CYA, Thermococcus sp. Dex80a71, Thermococcus sp. Dex80a75, Thermococcus sp. DS-1, Thermococcus sp. DS1, Thermococcus sp. DT4, Thermococcus sp. ENR5, Thermococcus sp. EPl, Thermococcus sp. ES5, Thermococcus sp. ES6, Thermococcus sp. ES7, Thermococcus sp. ES8, Thermococcus sp. ES9, Thermococcus sp. ES10, Thermococcus sp. ESH, Thermococcus sp. ESI 2, Thermococcus sp. ESI 3, Thermococcus sp. EXT 12c, Thermococcus sp. EXT9, Thermococcus sp. Fe85_l_2, Thermococcus sp. GB18, Thermococcus sp. GB20, Thermococcus sp. GE8, Thermococcus sp. Gorda2, Thermococcus sp. Gorda3, Thermococcus sp. Gorda4, Thermococcus sp. Gorda5, Thermococcus sp. Gorda6, Thermococcus sp. GR2, Thermococcus sp. GR4, Thermococcus sp. GR5, Thermococcus sp. GR6, Thermococcus sp. GR7, Thermococcus sp. GT, Thermococcus sp. GU5L5, Thermococcus sp. HJ21, Thermococcus sp. IRI33, Thermococcus sp. IRI35c, Thermococcus sp. IRI48, Thermococcus sp. JCM 11816, Thermococcus sp. JDF-3, Thermococcus sp. JdF3, Thermococcus sp. JdFR-02, Thermococcus sp. KBA1, Thermococcus sp. KI, Thermococcus sp. KS-8, Thermococcus sp. LM0-A1, Thermococcus sp. LM0-A2, Thermococcus sp. LM0-A3, Thermococcus sp. LM0-A4, Thermococcus sp. LM0-A5, Thermococcus sp. LM0-A6, Thermococcus sp. LM0-A7, Thermococcus sp. LM0-A8, Thermococcus sp. EM0-A9, Thermococcus sp. LSI, Thermococcus sp. LS2, Thermococcus sp. M36, Thermococcus sp. M39, Thermococcus sp. MA2.27, Thermococcus sp. MA2.28, Thermococcus sp. MA2.29, Thermococcus sp. MA2.33, Thermococcus sp. MARI, Thermococcus sp. MAR2, Thermococcus sp. MCR132, Thermococcus sp. MCR133, Thermococcus sp. MCR134, Thermococcus sp. MCR135, Thermococcus sp. MCR175, Thermococcus sp. MV1, Thermococcus sp. MV2, Thermococcus sp. MV3, Thermococcus sp. MVS, Thermococcus sp. MV10, Thermococcus sp. MV11, Thermococcus sp. MV12, Thermococcus sp. MV13, Thermococcus sp. MV1031, Thermococcus sp. MV1049, Thermococcus sp.MV1083, Thermococcus sp. MV1092, Thermococcus sp. MV1099, Thermococcus sp. MZ1, Thermococcus sp. MZ2, Thermococcus sp. MZ3, Thermococcus sp. MZ5, Thermococcus sp. MZ6, Thermococcus sp. MZ7, Thermococcus sp. MZ8, Thermococcus sp. MZ9, Thermococcus sp. MZ10, Thermococcus sp. MZ11, Thermococcus sp. MZ12, Thermococcus sp. MZ13, Thermococcus sp. NS85-T, Thermococcus sp. P6, Thermococcus sp. Pd70, Thermococcus sp. Pd85, Thermococcus sp. PK, Thermococcus sp. PK(2011), Thermococcus sp. Rt3, Thermococcus sp. SB611, Thermococcus sp. SN531, Thermococcus sp. SRB55 1, Thermococcus sp. SRB70 1, Thermococcus sp. SRB70 10, Thermococcus sp. SY113, Thermococcus sp. Tc-1-70, Thermococcus sp. Tc-1-85, Thermococcus sp. Tc-1-95, Thermococcus sp. Tc-2-85, Thermococcus sp. Tc-2-95, Thermococcus sp. Tc-365-70, Thermococcus sp. Tc-365-85, Thermococcus sp. Tc-365-95, Thermococcus sp. Tc-4-70, Thermococcus sp. Tc-4-85, Thermococcus sp. Tc-I-70, Thermococcus sp. Tc-I-85, Thermococcus sp. Tc-S-70, Thermococcus sp. Tc-S-85, Thermococcus sp. Tc55_l, Thermococcus sp. Tc55_12, Thermococcus sp. Tc70-4C-I, Thermococcus sp. Tc70-4C-S, Thermococcus sp. Tc70-7C-I, Thermococcus sp. Tc70-7C-S, Thermococcus sp. Tc70-CRC-I, Thermococcus sp. Tc70-CRC-S, Thermococcus sp. Tc70-MC-S, Thermococcus sp. Tc70-SC-I, Thermococcus sp. Tc70-SC-S, Thermococcus sp. Tc70-vw, Thermococcus sp. Tc70_l, Thermococcus sp. Tc70_10, Thermococcus sp. Tc70_ll, Thermococcus sp. Tc70_12, Thermococcus sp. Tc70_20, Thermococcus sp. Tc70_6, Thermococcus sp. Tc70_9, Thermococcus sp. Tc85-0 age SC, Thermococcus sp. Tc85-4C-I, Thermococcus sp. Tc85-4C-S, Thermococcus sp. Tc85-7C-S, Thermococcus sp. Tc85-CRC-I, Thermococcus sp. Tc85-CRC-S, Thermococcus sp. Tc85-MC-I, Thermococcus sp. Tc85-MC-S, Thermococcus sp. Tc85-SC-I, Thermococcus sp. Tc85-SC-ISCS, Thermococcus sp. Tc85-SC-S, Thermococcus sp. Tc85_l, Thermococcus sp. Tc85_10, Thermococcus sp. Tc85_ll, Thermococcus sp. Tc85_12, Thermococcus sp. Tc85_13, Thermococcus sp. Tc85_19, Thermococcus sp. Tc85_2, Thermococcus sp. Tc85_20, Thermococcus sp. Tc85_9, Thermococcus sp. Tc95-CRC-I, Thermococcus sp. Tc95-CRC-S, Thermococcus sp. Tc95-MC-I, Thermococcus sp. Tc95-MC-S, Thermococcus sp. Tc95-SC-S, Thermococcus sp. TK1, Thermococcus sp. TKM 55-W7-A, Thermococcus sp. TM1, Thermococcus sp. TP-33, Thermococcus sp. TP-37, Thermococcus sp. TS3, Thermococcus sp. TVG2, or Thermococcus sp. vpl97. The primase from some Thermococcus species is described in International Patent publication WO2021 / 250265 and U.S. patent publication US2023 / 0235372; incorporated by reference herein.

[0185] In some embodiments, the primase domain is of the primase of Pyrococcus. In some embodiments, the primase domain is of the primase of Pyrococcus ahyssi, Pyrococcus ahyssi GE5, Pyrococcus furiosus, Pyrococcus furiosus C0M1, Pyrococcus furiosus DSM 3638, Pyrococcus kukulkanii, Pyrococcus sp 12-1, Pyrococcus sp. NA2, Pyrococcus sp. ST04, Pyrococcus yayanosii, Pyrococcus yayanosii CHI, or Pyrococcus sp. ST04. In some embodiments, the primase from certain Pyrococcus and variants thereof are described in International Patent publication WO2021 / 250265,U.S. patent publication US2023 / 0235373, and International Patent publication WO2023 / 111180; incorporated by reference herein.

[0186] In some embodiments, the recombinant primase comprises a chimeric primase domain, wherein the chimeric primase domain comprises a PriS region or domain of a first primase and the PriX region or domain of a second primase, wherein the first primase and the second primase are different, and wherein the chimeric primase has terminal nucleotidyl transferase activity. In some embodiments, the chimeric primase has template-independent terminal nucleotidyl transferase activity.

[0187] In some embodiments, the recombinant primase comprises a chimeric primase domain, wherein the chimeric primase domain consists of a PriS region or domain of a first primase, and the PriX region or domain of a second primase, wherein the first primase and the second primase are different, and wherein the chimeric primase has terminal nucleotidyl transferase activity. In some embodiments, the chimeric primase has template-independent terminal nucleotidyl transferase activity.

[0188] In some embodiments, the recombinant primase comprises a chimeric primase domain comprising a PriS region or domain of a first primase of Methanococcus, Bacillus, Chloroflexota, Ammonifex, Thermococcus, or Pyrococcus, and a PriX region or domain of a second primase of Methanococcus, Bacillus, Chloroflexota, Ammonifex, Thermococcus, or Pyrococcus, wherein the first primase and second primase are different.

[0189] In some embodiments, the recombinant primase comprises a chimeric primase domain comprising a PriS region or domain of a first primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, and a PriX region or domain of a second primase of Thermococcus or Pyrococcus.

[0190] In some embodiments, in the recombinant primase comprises a chimeric primase domain comprising a PriS region or domain of a first primase of Thermococcus or Pyrococcus, and a PriX region or domain of a second primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

[0191] In some embodiments, in the recombinant primase comprises a chimeric primase domain comprising a PriS region or domain of a first primase of Thermococcus or Pyrococcus, and a PriX region or domain of a second primase of Thermococcus or Pyrococcus, wherein the first primase and second primase are different.

[0192] In some embodiments, the recombinant primase has nucleotidyl transferase activity at least for nucleotide donor dATP, ddATP, ddCTP, ddGTP, ddUTP, 2’-F-ATP, or any combinations thereof.

[0193] In some embodiments, the polynucleotide or oligonucleotide acceptor for the recombinant primase comprises a modified polynucleotide or modified oligonucleotide acceptor. In some embodiments, the initiating nucleotide acceptor comprises a modified nucleoside.

[0194] In some embodiments, the recombinant primase comprises a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316.

[0195] In some embodiments, the recombinant primase comprises a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

[0196] In some embodiments, the amino acid sequence of the primase domain comprises one or more amino acid differences relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56, 58, 60, 62, or 316, or the reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

[0197] In some embodiments, the amino acid sequence of the primase domain comprises at least an amino acid difference at amino acid position 9, 18, 19, 34, 36, 40, 41, 42, 43, 44, 45, 46, 47, 52, 63, 64, 69, 71, 75, 80, 81, 82, 83, 84, 85, 89, 90, 92, 101, 105, 106, 109, 111, 113, 119, 124, 128, 136, 137, 142, 157, 159, 171, 184, 187, 191, 192, 195, 197, 200, 201, 203, 206, 208, 209, 210, 211, 224, 225, 247, 252, 273, 292, 297, 314, 327, 337, 340, 341, 344, 346, 348, 351, 355, 358, 370, 375, 377, 378, 383, 391, 399, 402, 411, 416, 419, 422, or 423, or combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

[0198] In some embodiments, the amino acid sequence of the primase domain comprises at least an amino acid difference or amino acid residue 9A, 18A, 19G / I / V, 34E, 36T, 40C / L, 41G / L, 42R, 43R / V. 44N, 45S, 46C / G / H / R, 47G, 52K / N, 63G / I / V, 64C / S, 69R / V. 71G / L / R, 75C / M / S, 80L / V, 81A / R, 82G, 83S, 84G / L, 85G / L / P, 89R, 90N / T / V, 92L / T, 101L, 105A / G / R, 106M / S, 109M / R, 1 HR, 113K, 119E, 124L, 128F, 136D / P, 137G / K, 142D / G / R, 157D, 159E, 171W, 184G / P, 187R, 191V, 192P, 195M, 197A, 200G / Q, 201T, 203L, 206S / T, 208F, 209G, 210G / P / Q, 21 IP, 224N, 225P, 247R, 252E, 273A, 292R, 297D, 314N, 327A, 337L, 340G, 341V, 344H, 346V, 348T, 35 IN, 355D, 358E, 370G, 375H, 377L, 378S, 383G, 391R, 399D, 402P, 41 IL, 416G, 419H, 422G, or 423V, or any combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

[0199] In some embodiments, the amino acid sequence of the primase domain comprises at least an amino acid difference or substitution G9A, F18A, F19G / I / V, L34E, L36T, Q40C / L, D41G / L, K42R, L43R / V. T44N, R45S, S46C / G / H / R, F47G, E52K / N, R63G / I / V, L64C / S, K69R / V, V71G / L / R, V75C / M / S, E80L / V, K81A / R, L82G, P83S, P84G / L, E85G / L / P, A89R, A90N / T / V, D92L / T, V101L, C105A / G / R, Q106M / S, I109M / R, KI HR, G113K, G119E, T124L, L128F, V136D / P, D137G / K, L142D / G / R, E157D, D159E, Q171W, E184G / P, N187R, L191V, A192P, L195M, V197A, T200G / Q, R201T, W203L, P206S / T, N208F, P209G, R210G / P / Q, E21 IP, A224N, L225P, L247R, A252E, W273A, K292R, Q297D, S314N, Y327A, T337L, S340G, A341V, Q344H, L346V, E348T, D351N, R355D, D358E, P370G, R375H, F377L, E378S, R383G, E391R, E399D, A402P, P411L, K416G, K419H, R422G, or A423V, or combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

[0200] In some embodiments, the recombinant primase comprises a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314, or to the reference sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314.

[0201] In some embodiments, the recombinant primase comprises a primase domain comprising an amino acid sequence comprising residues 12 to carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314. In some embodiments, the amino acid sequence of the primase domain optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the primase domain optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the recombinant primase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the primase domain optionally includes 1, 2, 3, 4, or 5 substitutions.

[0202] In some of the foregoing embodiments, the amino acid sequence of the primase domain of the recombinant primase has 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence. In some embodiments, the primase domain of the recombinant primase polypeptide has 1, 2, 3, or 4 substitutions in the amino acid sequence. In some embodiments, the substitutions comprises nonconservative or conservative substitutions. In some embodiments, the substitutions comprises conservative substitutions. In some embodiments, the substitutions comprises non-conservative substitutions.

[0203] In some embodiments, the recombinant primase has at least one improved property as compared to a reference primase, or a reference recombinant primase having nucleotidyl transferaseactivity. In some embodiments, the improved property comprises increased template-independent terminal nucleotidyl transferase activity. In some embodiments, the improved property comprises increased incorporation of a nucleotide donor NTP. In some embodiments, the improved property comprises increased incorporation of at least nucleotide donor dATP, dGTP, dCTP, dTTP, 2’-F-ATP, 2’-O-methyl-ATP (mATP), ddATP, ddCTP, ddGTP, or any combinations thereof, to an oligonucleotide acceptor. In some embodiments, the improved property comprises increased incorporation of at least nucleotide donor ddATP and / or mATP to an oligonucleotide acceptor, as provided in the Examples. In some embodiments, the reference primase is the primase of Ammonifex thiophilus (e.g., SEQ ID NO: 24) or the recombinant primase of SEQ ID NO: 60.

[0204] In some embodiments, the improved property is increased incorporation of a nucleotide donor to a modified oligonucleotide acceptor, particularly a modified oligonucleotide acceptor having at least a modification of the 3 ’-terminal nucleoside and / or at least a modified intemucleoside linkage at the 3 ’-terminal nucleoside. In some embodimensts, the increased activity for an oligonucleotide acceptor is selected from FAM-T14ATCfA (SEQ ID NO: 317), 5’-FAM-T17AAA (SEQ ID NO: 318), 5’-FAM-T14ATCfAA (SEQ ID NO: 319), 5’-FAM-T17AAAA (SEQ ID NO: 320), 5’-FAM- T14ATCfAfA (SEQ ID NO: 321), 5’-FAM-T17AAAfA (SEQ ID NO: 322), 5’-FAM-T14ATCfAddA (SEQ ID NO: 323), 5’-FAM-T17AAAddA (SEQ ID NO: 324), 5’-FAM-T14ATCfAddC (SEQ ID NO: 325), 5’-FAM-T17AAAddC (SEQ ID NO: 326), 5’-FAM-T14ATCfAddG (SEQ ID NO: 327), 5’-FAM-T17AAAddG (SEQ ID NO: 328), 5’-FAM-T14ATCfAmA (SEQ ID NO: 329), and 5’-FAM- T17AAAmA (SEQ ID NO: 330).

[0205] In some embodiments, the recombinant primase has at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more fold activity compared to the reference primase or recombinant primase comprising a primase domain. In some embodiments, the increased activity is with modified nucleotide donors and / or modified oligonucleotide acceptor, as discussed herein and in the Examples. In some embodiments, the improved property is increased incorporation of mATP donor of at least 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, or 2 fold or greater as compared to the reference primase or recombinant primase. In some embodiments, the reference primase is the primase of Ammonifex thiophilus (e.g., SEQ ID NO: 24) or the recombinant primase of SEQ ID NO: 60.

[0206] In some embodiments, the recombinant primase is expressed as a fusion protein. In some embodiments, the recombinant primase described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification, nucleic binding domains to enhance activity, and / or for providing a reactive amino acid for immobilization on a support medium. In some embodiments, the fusion polypeptide of the recombinant primase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion polypeptide of the recombinant primase comprises a polylysine, forexample a polylysine of 2-12 amino acids or longer in length. In some embodiments, the fusion protein of the recombinant primase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the recombinant primase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the recombinant primase is fused to protein domains that bind nucleic acids, particularly RNA, for example, zinc knuckle (zk), helix zinc knuckle (hzk), RGG / RG box motifs, RNase III (me), and homeodomains (see, e.g., Tong et al., Nucleic Acids Res., 50(19): 11175-11185, incorporated by reference herein). In some embodiments, the fusion is to the amino (N-) terminus of recombinant primase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the recombinant primase polypeptide.

[0207] In some embodiments, the recombinant primase described herein is an isolated composition. In some embodiments, the recombinant primase is purified. In some embodiments, the recombinant primase is provided in solution, as a lyophilizate, or immobilized on a substrate, as further discussed herein.

[0208] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of recombinant primase polypeptides described herein. Thus, for each and every embodiment herein of a recombinant primase, a functional fragment or biologically active fragment of the recombinant primase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of a recombinant primase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the primase polypeptide from which it was derived (i.e., the parent recombinant primase). 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 recombinant primase. 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.

[0209] In some embodiments, a functional fragment of a recombinant primase 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 recombinant primase. 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, less than 55, less than 60, less than 65, or less than 70 amino acids.

[0210] In some embodiments, the functional fragments or biologically active fragments of the recombinant primase described herein include at least a mutation or mutation set in the amino acid sequence of the recombinant primase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the recombinant primase displays theenhanced or improved property associated with the mutation or mutation set in the parent primase or parent recombinant primase.Polynucleotides Encoding Recombinant Polypeptides, Expression Vectors and Host Cells

[0211] In another aspect, the present disclosure provides recombinant polynucleotides encoding the recombinant primases. 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 recombinant primase.

[0212] 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 primase 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 recombinant primase 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.

[0213] 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 primase 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.

[0214] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure encodes a recombinant primase described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized.

[0215] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an recombinant primase comprising a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316.

[0216] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an recombinant primase comprising a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

[0217] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant primase comprising a primase domain comprising an amino acid sequence having one or more amino acid differences relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

[0218] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an recombinant primase comprising a primase domain comprising an amino acid sequence having at least an amino acid difference at amino acid position 9, 18, 19, 34, 36, 40, 41, 42, 43, 44, 45, 46, 47, 52, 63, 64, 69, 71, 75, 80, 81, 82, 83, 84, 85, 89, 90, 92, 101, 105, 106, 109, 111, 113, 119, 124, 128, 136, 137, 142, 157, 159, 171, 184, 187, 191, 192, 195, 197, 200, 201, 203, 206, 208, 209, 210, 211, 224, 225, 247, 252, 273, 292, 297, 314, 327, 337, 340, 341, 344, 346, 348, 351, 355, 358, 370, 375, 377, 378, 383, 391, 399, 402, 411, 416, 419, 422, and 423, or combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

[0219] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant primase comprising a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12 to carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314, or to the reference sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314.

[0220] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant primase comprising a primase domain comprising an amino acid sequence comprising residues 12 to carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 64- 314, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314.

[0221] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having least 60%, 65%, 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to the sequence from nucleotide residue 34 to the 3 ’-terminal nucleotide of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 315, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 315, wherein the recombinant polynucleotide encodes a recombinant primase having template -independent terminal nucleotidyl transferase activity.

[0222] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 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 the sequence from nucleotide residue 34 to 3 ’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, wherein the recombinant polynucleotide encodes a recombinant primase having template-independent terminal nucleotidyl transferase activity.

[0223] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 3 ’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, or comprising a polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, wherein the recombinant polynucleotide encodes a recombinant primase having nucleotidyl transferase activity. In some embodiments, the recombinant polynucleotide encodes a recombinant primase having template -independent terminal nucleotidyl transferase activity.

[0224] In some embodiments, a recombinant polynucleotide encoding any of the recombinant primases herein is manipulated in a variety of ways to facilitate expression of the recombinant primase. In some embodiments, the recombinant polynucleotide encoding the recombinant primase comprises expression vectors where one or more control sequences is present to regulate the expression of the recombinant primase polynucleotides and / or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0225] 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- 3 -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 P-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.

[0226] 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 primase 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 -phosphatedehydrogenase. 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.

[0227] 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 primase 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 alcohol 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.

[0228] 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.

[0229] In some embodiments, the control sequence is also 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 signalpeptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered 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 betalactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus suhtilis 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 to 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.

[0230] 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 suhtilis alkaline protease (aprE), Bacillus suhtilis 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.

[0231] 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.

[0232] In another aspect, the present disclosure provides a recombinant expression vector comprising a recombinant polynucleotide encoding a recombinant primase 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 recombinant primase polypeptide.

[0233] In some embodiments, 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 primase polynucleotide. The choice of the vector 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.

[0234] 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.

[0235] 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).

[0236] 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 fdamentous 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 (nitratereductase), 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.

[0237] In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a recombinant primase polypeptide described herein, the polynucleotide(s) being operably linked to one or more control sequences for expression of the recombinant primase 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 Spodoptera Sf9 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).

[0238] In another aspect, the present disclosure provides a method of producing the recombinant primase polypeptides, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the recombinant primase polypeptide under conditions suitable for expression of the polypeptide such that the recombinant primase is produced. In some embodiments, the method further comprises isolating the primase polypeptides from the culture and / or host cells. In some embodiments, the method further comprises purifying the expressed primase polypeptide, as described herein.

[0239] In some embodiments, the recombinant primase polypeptide 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.

[0240] Chromatographic techniques for isolation / purification of the recombinant primase 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 recombinant primase 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 primase. For affinity chromatography purification, an antibody that specifically binds the recombinant primase polypeptide may be used. In some embodiments, an affinity tag, e.g., His-tag, can be introduced into the recombinant primase polypeptide for purposes of isolation / purification.

[0241] In some embodiments, recombinant polypeptides (e.g., recombinant primase) can be produced using any suitable methods known the art. For example, there is a wide variety of different mutagenesis techniques known to those skilled in the art. In addition, mutagenesis kits are alsoavailable from many commercial molecular biology suppliers. Methods are available to make specific substitutions at defined amino acids (site-directed), specific or random mutations in a localized region of the gene (region-specific), or random mutagenesis over the entire gene (e.g., saturation mutagenesis). Numerous suitable methods are known to those in the art to generate enzyme variants, including but not limited to site-directed mutagenesis of single -stranded DNA or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: US Pat. No. 6,117,679; US Pat. No. 6,420,175; US Pat. No. 6,376,246; US Pat. No. 6,586,182; US Pat. No. 7,747,391; US Pat. No. 7,747,393; US Pat. No. 7,783,428; and US Pat. No. 8,383,346. Afterthe variants are produced, they can be screened for any desired property (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased stability, increased substrate range, increased inhibitor resistance or tolerance, increased salt tolerance, and / or pH stability, etc.). Exemplary methods are provided in the Examples.

[0242] In some embodiments, the recombinant primase polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or recombinant primase polypeptide to a suitable mutagenesis and / or directed evolution methods known in the art, for example, as described herein. An exemplary directed evolution technique is mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91: 10747- 10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136- S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525- 3529).

[0243] Mutagenesis and directed evolution methods can be applied to recombinant primase-encoding polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods find use in the present disclosure and are known in the art (see, e.g., US Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001,6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related PCT and non-US counterparts; Ling et al., Anal. Biochem., 1997, 254(2): 157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229: 1193-1201; Carter, Biochem. J., 1986, 237: 1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 366:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91: 10747-10751; EP 3 049 973; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; WO 2009 / 152336; and WO 2015 / 048573, all of which are incorporated herein by reference).

[0244] In some embodiments, the clones obtained following mutagenesis treatment are screened by subjecting the enzyme preparations to a defined treatment conditions or assay conditions (e.g., temperature, pH, type of primase substrate, input substrate concentration, nucleotide cofactors, etc.) and measuring enzyme activity after the treatments or other suitable assay conditions. Clones containing a polynucleotide encoding the polypeptide of interest are then isolated from the gene, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art and as described in the Examples.

[0245] In some embodiments, for engineered polypeptides of known sequence, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated methods) to form any desired continuous sequence (see, e.g., Hughes et al., Cold Spring Harb Perspect Biol. 2017 Jan; 9(1) :a023812) . For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tet. Lett., 1981, 22: 1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors.

[0246] In some embodiments, a method for preparing the recombinant primase polypeptide can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant as described herein, and (b) expressing the primase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1- 22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions are conservative or non-conservative substitutions.Composition of Primases

[0247] In a further aspect, the present disclosure provides compositions of the recombinant primases disclosed herein. In some embodiments, the recombinant primase polypeptide in the compositions is isolated or purified. In some embodiments, the recombinant primase is combined with other components and compounds to provide compositions and formulations comprising the recombinant primase polypeptide as appropriate for different applications and uses.

[0248] In some embodiments, a recombinant primase described herein is provided in solution, as a lyophilizate, or is immobilized on a substrate. In some embodiments, the composition comprises an additive, such as a preservative, a buffer, 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).

[0249] In some embodiments, the composition comprises a recombinant primase described herein immobilized on a support medium. In some embodiments, the recombinant primase is immobilized on a support medium by adsorption, covalent attachment, entrapment, or affinity interactions.

[0250] In some embodiments, the substrate 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.

[0251] In some embodiments, the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed on the support medium. In some embodiments, the enzyme is immobilized on the support medium 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, polyvinyl chloride, polyacrylamide, polyacrylate, polymethacrylate, polystyrene, polyethylene glycol, dextran, agarose (e.g., cross-linked agarose, e.g., Sepharose) and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.

[0252] In some embodiments, in the composition comprising the recombinant primase immobilized on a support medium, the immobilized recombinant primase retains at least 90%, 91%, 92%, 93%„ 94%, 95%, 96%, 96%, 97%, 98%, or greater activity following attachment to the support medium.

[0253] In some embodiments, the composition further comprises one or more of a nucleotide acceptor and / or a nucleotide donor. In some embodiments, the composition further comprises a polynucleotide acceptor, oligonucleotide acceptor, or an initiating nucleotide acceptor.

[0254] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, or the initiating nucleotide acceptor in the composition comprises at least one modified nucleoside, modified intemucleoside linkage, or combinations thereof.

[0255] In some embodiments, the nucleotide donor in the composition comprises a dNTP (e.g., dUTP, dCTP, dTTP, dATP, dGTP). In some embodiments, the nucleotide donor comprises a modified dNTP. In some embodiments, the nucleotide donor comprises a ddNTP (e.g., ddUTP, ddCTP, ddTTP, ddATP, ddGTP). In some embodiments, the nucleotide donor comprises a 2’- modification of furanosyl sugar moiety. In some embodiments, the nucleotide donor comprises a 2’- modification of the furanosyl sugar moiety (e.g., 2-fluoro, 2’-O-methyl, etc.). Exemplary 2’-modified nucleotide donors, include among others, 2’-fluoro-ATP and 2’-O-methyl-ATP. In some embodiments, the nucleotide donor comprises a modified nucleobase. In some embodiments, the nucleotide donor comprises a modification of the phosphate group (e.g., with a phosphorothioate). Various modifications to the nucleotide donor are described herein.

[0256] In some embodiments, the nucleotide donor comprises a 3 ’-blocking group or a chain terminator nucleotide donor (e.g., ddNTP), or a chain terminating nucleotide donor comprising a 3’- blocking group. In some embodiments, the 3 ’-blocking group comprises a reversible 3 ’-blocking group or chain terminating nucleotide donor comprising a reversible 3 ’-blocking group, as described herein.

[0257] In some embodiments, the composition comprises a recombinant primase and another (e.g., second) enzyme for use with the recombinant primase. In some embodiments, the composition further comprises a pyrophosphatase. In some embodiments, the pyrophosphatase comprises apyrophosphatase of Type 1 pyrophosphatase, a Type II pyrophosphatase, or a combinations thereof (see Holmes, et al., Front. Mol. Biosci., 2019, Volume 6, Article 132., incorporated by reference herein).

[0258] 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)ethane sulfonic acid (MES), 3-(N-morpholino)propanesulfonic 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.

[0259] In some embodiments, the composition further comprises a cofactor. In some embodiments, the cofactor is a divalent metal ion, such as Ni2+, Mg2+, Mn2+, or Co2+. In some embodiments, the cofactor is present in the form of a salt, such MgCE. MnCE. or CoCE. In some embodiments, the cofactor is a nonnatural or non-preferred cofactor for the recombinant primase and / or the second enzyme. In some embodiments, the cofactor is a natural or preferred cofactor for the recombinant primase and / or the second enzyme. In some embodiments, the cofactor is a preferred or natural cofactor for a second enzyme and a nonpreferred or nonnatural cofactor for the recombinant primase. In some embodiments, the cofactor is present at a higher concentration compared to an in vivo concentration. In some embodiments, the cofactor is present at a concentration of 0.1 mM to 5 mM.Modified Nucleotides

[0260] In some embodiments, in the compositions or methods of using recombinant primase for polynucleotide or oligonucleotide synthesis, the polynucleotide acceptor, oligonucleotide acceptor, initiating nucleotide acceptor, or nucleotide donor, or any combinations thereof, can have various modifications. In some embodiments, the modifications occur on the nucleobase, the sugar moiety, or terminal groups. The various modifications are described below.Intemucleoside linkages

[0261] In some embodiments, the polynucleotide acceptor or oligonucleotide acceptor comprises at least one modified, non-naturally occurring intemucleoside linkage. In some embodiments, the modified polynucleotide or oligonucleotide comprises at least one modified, non-naturally occurring intemucleoside linkage. In some embodiments, the polynucleotide acceptor or oligonucleotide acceptor 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.

[0262] In some embodiments, the modified intemucleoside linkage is a phosphorous containing modified intemucleoside linkage. Exemplary phosphorous-containing intemucleoside linkagesincluding, without limitation, phosphotriester, alkylphosphonate (e.g., methyl phosphonate, ethyl phosphonate, etc.), phosphoramidate, phosphorothioate, and phosphorodithioate.

[0263] 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 (-0- C(=0)(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 intemucleside 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).

[0264] 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 oligonucleotide acceptor and / or oligonucleotide donor have a mixture of stereoisomers in the intemucleoside linkage. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have greater than 50% of the intemucleoside linkages as Rp or Sp configuration. In some embodiments, the oligonucleotide acceptor and / or oligonucleotide donor have at least 60%, 70%, 80%, 90%, or greater of Rp or Sp stereomeric configuration.

[0265] In some embodiments, the modified intemucleoside linkage(s) is / are present in the 5’- terminal region of the polynucleotide acceptor oligonucleotide acceptor. In some embodiments, at least 1, 2, 3, 4, or 5 modified intemucleoside linkages are present at the 5 ’-terminal region of the polynucleotide or oligonucleotide acceptor. In some embodiments at least 1 or 2 phosphorothioate intemucleoside linkages are present at the 5 ’-terminal region of the polynucleotide or oligonucleotide acceptor. In some embodiments, the phosphorothioate linkage is a non-bridging phosphorothioate intemucleoside linkage.

[0266] In some embodiments, the modified intemucleoside linkage(s) is / are present in the 3’- terminal region of a polynucleotide acceptor or oligonucleotide acceptor. In some embodiments, at least 1, 2, 3, 4, or 5 modified intemucleoside linkages are present at the 3 ’-terminal region of the polynucleotide or oligonucleotide acceptor. In some embodiments, at least 1 or 2 phosphorothioate intemucleoside linkages are present at the 3 ’-terminal region of a polynucleotide acceptor or oligonucleotide acceptor.

[0267] In some embodiments, the modified intemucleoside linkages are present in the internal portions of the polynucleotide acceptor or oligonucleotide acceptor.

[0268] In some embodiments, the polynucleotide acceptor or oligonucleotide acceptor 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 configuration in the population of polynucleotide acceptor or oligonucleotide acceptor.

[0269] In some embodiments, the initiating nucleotide acceptor comprises a 5’-alpha-thiophosphate. In some embodiments, the initiating nucleotide acceptor comprises an NTPaS, preferably NDPaS or NMPaS.2’- and 3 ’-modifications

[0270] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, initiating nucleotide acceptor 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 fiiranosyl sugar moiety, for example ribose or deoxyribose. In some embodiments, the furanosyl sugar moiety is modified or substituted at the 2’, 3’, or a combination of 2’ and 3’ positions. Modifications at the 4’, and / or 5 ’-positions are described below for “terminal group.”

[0271] In some embodiments, the modification is at the 2’-position of the sugar moiety. In some embodiments, substitutions at the 2’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O- alkyl or 2’-alkoxy (e.g., O-methyl, O-ethyl, etc.). In some embodiments, other modifications at the 2’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCH3, SO2CH ,. ONO2, NO2, N3, 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 unsubsiituted C1-C10 alkyl or C1-C10 alkenyl and alkynyl. In some embodiments, substituent groups at the 2 ’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl. In some embodiments, the substitution at the 2 ’-position is a phosphate (see, e.g., Current Protocols in Nucleic Acid Chemistry, 13.1.1-13.1.31, John Wiley & Sons (2003).

[0272] 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 -CFFCFfi or -CFFCFFOCFb. In some embodiments, the modified 2’-position is 2’-O-(2-methoxyethyl), 2’-O-allyl, 2’-O-propargyl, 2’-O- ethylamine, 2’-O-cyanoethyl, -2’-0-amine, or 2’-O-acetate ester.

[0273] 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.

[0274] In some embodiments, other locked nucleosides include, among others, 5’ -methyl -LNA, 2’- amino-LNA, alpha-L-LNA, and thio-LNA. Structures of certain locked nucleosides are shown below:where R in the above is alkyl or acyl.

[0275] In some embodiments, a modification at the 2 ’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker, or a linker, as described herein.

[0276] 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 primase 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, CFs, OCFs, SCH3, SOCH3, SO2CH3, ONO2, NO2, N3, 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 unsubsiituted 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.

[0277] 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 -CH2CH3or -C H2CH2OCH ,. In some 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.

[0278] 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 the recombinant primase or another terminal nucleotidyl transferase. In some embodiments, exemplary reversible or cleavable 3 ’-blocking groups include, among others, 3’-O-azidomethyl, 3’-O-(2- methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O-ethylamine, 3’-O-cyanoethyl, -3’-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, as is known in the art. Other reversible or cleavable 3 ’-blocking groups are described in International patent publication WO2023183569, incorporated by reference herein.

[0279] In some embodiments, a modification at the 3 ’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker, or a linker, as described herein.

[0280] In some embodiments, the modified sugar moiety comprises an unlocked nucleoside. In some embodiments, in the unlocked nucleoside, the fiiranosyl 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.Modified nucleobase s

[0281] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor comprises at least one modified nucleobase. In some embodiments, the nucleotide donor comprises a modified nucleobase.

[0282] In some embodiments, the nucleobase comprise an inosine nucleoside (i.e., nucleosides comprising a hypoxanthine 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).

[0283] 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.

[0284] 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 a polynucleotide or oligonucleotide acceptor. In some embodiments, an abasic nucleoside is attached to the 3’- or 5 ’-terminal end, which is in certain embodiments denoted herein as a terminal group.

[0285] In some embodiments, the modified nucleobase is present on the 5 ’-terminal nucleoside of the polynucleotide acceptor or oligonucleotide acceptor, 3 ’-terminal nucleoside of the polynucleotide or oligonucleotide acceptor, and / or present on the internal nucleosides of the polynucleotide or oligonucleotide acceptor. In some embodiments, blocks or contiguous stretches of nucleosides in the polynucleotide or oligonucleotide acceptor have modified nucleobases. In some embodiments, the initiating nucleotide acceptor comprises a modified nucleobase.

[0286] In some embodiments, the nucleotide donor comprises a modified nucleobase. In some embodiments, the modified nucleobase on the nucleotide donor is selected for compatibility with the recombinant primase activity and that does not inhibit activity of the recombinant primase.Terminal groups

[0287] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, initiating nucleotide acceptor, and / or nucleotide donor comprises a terminal group. In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor comprises a terminal group at the 5 ’-terminal nucleoside. In some embodiments, the terminal group is attached to the 5 ’-OH or 4 ’-carbon atom of the terminal nucleoside.

[0288] 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).

[0289] 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’-(Eor Z)-vinylphosphate, or 5’-methylenephosphonate.

[0290] 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.

[0291] In some embodiments, the nucleotide donor comprises a 3 ’-terminal group. In some embodiments, the 3 ’-terminal group comprises a 3 ’-phosphate, which can also function as a reversible blocking group. In some embodiments, the 3’-phosphate is modified, such as with 3’-(E or Z)- vinylphosphate, 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

[0292] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor comprises a conjugate moiety. In some embodiments, the nucleotide donor comprises a conjugate moiety that is compatible with recombinant primase activity and that does not interfere with the nucleotide donor acting as a substrate for the recombinant primase.

[0293] In some embodiments, the conjugate moiety (i.e., non-nucleotide moiety) includes, among others, carbohydrates (e.g. GalNAc), lipids, sterols, drug substances, hormones, polymers (e.g., polyethylene glycol, etc.), proteins, peptides, toxins (e.g. bacterial toxins, etc.), vitamins (e.g., folate, tocopherol, retinoic acid, etc.), or combinations thereof. In some embodiments, the conjugate moiety is used to affect the pharmacokinetics of the oligonucleotide and / or oligonucleotide cell targeting.

[0294] In some embodiments, the conjugate moiety can be attached to the 5 ’-terminal nucleotide, the 3 ’-terminal nucleotide, or in a polynucleotide or oligonucleotide an internal nucleotide. In some embodiments, the conjugate moiety is attached the 2 ’-position of the sugar moiety of a nucleoside, for example, to the 2’-OH. In some embodiments, the conjugate moiety is attached to the 3 ’-position of the sugar moiety of the nucleoside, for example 3 ’-OH. In some embodiments, the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res. 2019 Feb 20; 47(3): 1082-1096). In some embodiments, the conjugate moiety is attached directly or attached using a linker.

[0295] In some embodiments, the conjugate moiety comprises a C6-C22 alkyl, Ce-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, Ci2-alkyl, Cis-alkyl, Ci4-alkyl, Cis-alkyl, Cie-alkyl, Cn-alkyl, Cis-alkyl, Cig-alkyl, C2o-alkyl, C21 -alkyl, or C22-alkyl. In some embodiments, the conjugate moiety comprises a Ce alkenyl, C7 alkenyl, Cs alkenyl C9 alkenyl, C10 alkenyl, Cn-alkenyl, Ci2-alkenyl, Cis-alkenyl, C14- alkenyl, Cis-alkenyl, Cie-alkenyl, Ci7-alkenyl, Cis-alkenyl, Cig-alkenyl, C2o-alkenyl, C2i-alkenyl, or C22-alkenyl. In some embodiments, the conjugate moiety comprises a Ce alkynyl, C7 alkynyl, Cs alkynyl, C9 alkynyl, C10 alkynyl, Cn-alkynyl, Ci2-alkynyl, Cis-alkynyl, Ci4-alkynyl, Cie-alkynyl, Cie- alkynyl, Ci7-alkynyl, Cis-alkynyl, Cig-alkynyl, C2o-alkynyl, C2i-alkynyl, or C22-alkynyl.

[0296] 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.

[0297] In some embodiments, the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group. In some embodiments, the cycloalkyl includes, by way of example and not limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3 -cyclohexenyl, and cycloheptyl. In some embodiments, the heterocycloalkyl includes, among others, l-(l,2,5,6-tetrahydropyridine, 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl.

[0298] In some embodiments, the conjugate moiety comprises an aryl or heteroaryl moiety. In some embodiments, the aryl group includes, by way of example and not limitation, 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.

[0299] 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- groups.

[0300] 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, W02024 / 040041; incorporated herein by reference).

[0301] 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.

[0302] In some embodiments, the conjugate moiety comprises a vitamin or vitamin derivative, including, by way of example and not limitation, folate, tocopherol, retinoic acid, vitamin D, and the like (see, e.g., US Patent No. 9789197).

[0303] In some embodiments, the conjugate moiety comprises a phospholipid. In some embodiments, the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.

[0304] 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, carbohydrate moiety includes the following:

[0305] 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 monovalent, divalent, trivalent or tetravalent, GalNAc.

[0306] In some embodiments, the GalNAc moiety has the following structure,where L is a linker, and W is a heteroatom, such as O or S. 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 a polynucleotide or oligonucleotide (see, e.g., W02024 / 040041).

[0307] In some embodiments, the conjugate moiety is a trivalent GalNAc. Tri-valent N- acetylgalactosamine conjugate moieties are described in, for example, International patent publication WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620. “Trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker. In some embodiments, the trivalent GalNac is L96. Exemplary trivalent GalNAc conjugate moieties are depicted below:

[0308] In some embodiments, the conjugate moiety comprises a reporter molecule. Example of reporter molecules include, among others, fluorescent moieties, such as fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxy- fluorescein, 6-carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy- 3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), IRDyes (e.g., IRD40, IRD 700, IRD 800). (See, e.g., “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., R.P. Haugland, 2002, Molecular Probes, Inc., Eugene, Oregon).

[0309] 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.

[0310] 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).

[0311] In some embodiments, the conjugate moiety comprises a peptide. In some embodiments, the peptide comprises a cellular targeting peptide and / or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate modified oligonucleotide. In some embodiments, the cell penetrating peptide is attached via a linker, including a cleavable linker. Cell penetrating peptides, include among others, TAT, penetratin, MAP, transportan / 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. Sequences of some penetrating peptides are described in Copolovici et al., 2014, 8(3): 1972-1994 and some are provided below:

[0312] 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.Reactive Moiety

[0313] In some embodiments, the modification comprises a reactive group that is conjugated to a nucleoside. In some embodiments, the reactive group is attached to the nucleoside via a linker. In some embodiments, the reactive group is a cyano, azido, alkynyl, amino, carboxyl, sulfhydryl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine. In some embodiments, the reactive group is those used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below:

[0314] Other reactive groups used in click chemistry, particularly for nucleic acids, is described in Fantoni et al., Chem. Rev. 2021, 121, 7122-7154, incorporated by reference herein.Linker

[0315] In some embodiments, as described above, the conjugate moiety or reactive moiety is attached to the nucleoside or the terminal group through a linker. Various linkers are known in the art for conjugating chemical groups to nucleosides and phosphate groups.

[0316] In some embodiments, mixtures of linkers are used. In some embodiments, different linker types are connected to form a longer linker or linkers with branched or dendritic structure. For example, an alkylene linker is connected to a polyethylene linker through a functional group, e.g., an amide; and an arylene linker is attached to an alkylene linker. As such, different combinations of linker types can be connected to provide for longer linkers and / or branched linkers, for example for attaching multile conjugate moieties.

[0317] In some embodiments, linkers include, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene, heteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroarylheteroalkylene, and heteroarylalkenylene. In some embodiments, the linker comprises substituted or unsubstituted C2-C22 alkylene, heteroalkylene, or polyethylene glycol. In some embodiments, the linkers have functional groups for conjugation.

[0318] In some embodiments, the linker comprises a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms, 1 to 20 carbon atoms, or 1 to 14 carbon atoms, wherein one or more of the carbon atoms in the hydrocarbon chain is optionally replaced by -O-, -NR1-, -NR'-C^O)-, -C(=O)-NR1, or -S-, and wherein R1is hydrogen or (Ci- Cejalkyl. wherein the hydrocarbon chain, is optionally substituted with one or more (e.g. 1, 2, 3, or 4) substituents selected from (Ci-Ce)alkoxy, (Cs-Cejcycloalkyl. (Ci-Ce)alkanoyl, (Ci-Ce)alkanoyloxy, (Ci-Ce)alkoxycarbonyl, (Ci-Ce)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0319] In some embodiments, the L is attached to the nucleoside and / or conjugate through -NH-, -O-, -S-, -(C=O)-, -(C=0)-NH-, -NH-(C=0)-, -(C=O)-O-, -NH-(C=0)-NH-, or -NH-(SO2)-.

[0320] In some embodiments, the linker L has the structure below:

[0321] In some embodiments, the linker comprises a substituted or unsubstituted polyethylene glycol linker. In some embodiments, the polyethylene glycol linker has the formula:

[0322] In some embodiments, n is 2-24. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0323] In some embodiments, the polyethylene glycol linker has the structure below:

[0324] In some embodiments, exemplary polyethylene glycol linkers have the structure below:

[0325] In some embodiments, the linker is a cleavable linker in which the linker can be cleaved, for example to detach a conjugate moiety. Example of a cleavable linker includes, by way of example and not limitation, a disulfide linkage, enzymatically cleavable linkers (e.g., peptide linkers), and photocleavable linkers (see, e.g., Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2ndEd., S.S. Mark ed., 2011, Humana Press).

[0326] In some embodiments, bifunctional linkers can be used to attach a conjugate moiety to the linker and attach the linker-conjugate to the nucleoside or vice versa (see, e.g., Hermanson, G., supra; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, supra). In some embodiments, an activating group can be attached to an atom to activate the atom to form a covalent bond with another reactive group. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When an activating group is attached to an oxygen atom of a carboxylic acid, the activating group can be a group that is derivable from a known coupling reagent. Examples of such coupling reagents include, but are not limited to, N,N'-dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3 -dimethylaminopropyl)-N'-ethylcarbonate (EDC), (denzotriazol- 1 - yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1 -yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-l-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU).Uses of Recombinant Primase Polypeptides

[0327] In another aspect, the present disclosure provides uses of the recombinant primases for polynucleotide or oligonucleotide synthesis. In some embodiments, the recombinant primases are use for template-independent polynucleotide or oligonucleotide synthesis.

[0328] In some embodiments, a method of oligonucleotide synthesis comprises reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a recombinant primase described herein under suitable reaction conditions for the extension of the nucleotide acceptor by attachment of the nucleotide donor to the nucleotide acceptor by the recombinant primase. In some embodiments, the reaction is in the absence of the template (i.e., template-independent reaction).

[0329] In some embodiments, the nucleotide acceptor comprises a polynucleotide acceptor, an oligonucleotide acceptor, or an initiating nucleotide acceptor. As used herein, an initiating nucleotide acceptor refers to an acceptor having a single nucleoside.

[0330] In some embodiments, the oligonucleotide acceptor is at least 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length.

[0331] In some embodiments, the polynucleotide or oligonucleotide acceptor is DNA, RNA, or a mixture of DNA and RNA.

[0332] In some embodiments, the 3 ’-terminal nucleotide of the polynucleotide or oligonucleotide acceptor is a ribonucleotide.

[0333] In some embodiments, the 3 ’-terminal nucleotide of the polynucleotide or oligonucleotide acceptor is a deoxyribonucleotide.

[0334] In some embodiments, the initiating nucleotide acceptor comprises NTP, NDP, NMP, or a nucleoside. Preferably, the initiating nucleotide acceptor is NDP, NMP, or a nucleoside to limit polymerization of the initiating nucleotide acceptor by the recombinant primase. In some embodiments, the initiating nucleotide acceptor comprises a modification to limit unwanted polymerization by the recombinant primase. In some embodiments, the initiating nucleotide acceptor is conjugated at the 5’-OH or 5’-phosphate, or has other 5’-blocking groups to inhibit polymerization of the initiating nucleotide acceptor. In some embodiments, the initiating nucleotide acceptor is conjugated to a support medium, preferably via a linker. In some embodiments, when the initiating nucleotide acceptor comprises an NTP, the nucleotide donor comprises a 5 ’-blocking group for controlling the reaction with the recombinant primase and limiting the extension reaction to the nucleotide donor.

[0335] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, and initiating nucleotide acceptor comprises a competent 3 ’-end for reaction with the nucleotide donor. In some embodiments, the 3 ’-end of the polynucleotide, oligonucleotide acceptor, or initiating nucleotide acceptor comprises at least a 3 ’-OH. In some embodiments, the 3 ’-end of the polynucleotide acceptor or oligonucleotide acceptor has sufficient single stranded region for recognition by the recombinant primase. In some embodiments, the 3 ’-end of the polynucleotide or oligonucleotide acceptor comprises at least a single stranded region of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 nucleotides in length. In some embodiments, the polynucleotide acceptor or oligonucleotide acceptoris single stranded, which may have some internal regions of complementarity to another portion of the singled stranded polynucleotide or oligonucleotide acceptor. In some embodiments, the 3 ’-end of the polynucleotide or oligonucleotide acceptor comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous ribonucleotide residues, wherein the contiguous region is single stranded and recognized by the recombinant primase.

[0336] In some embodiments of the method, the nucleotide donor comprises a blocking group, or a chain terminating nucleotide donor comprising a 3 ’-blocking group, to form a 3 ’-blocked extended polynucleotide or extended oligonucleotide.

[0337] In some embodiments, the 3 ’-blocking group comprises a reversible 3 ’-blocking group to form a reversible 3 ’-blocked extended polynucleotide or extended oligonucleotide.

[0338] In some embodiments, the method further comprises inactivating the recombinant primase or separating the recombinant primase from the reaction solution containing the 3 ’-blocked extended polynucleotide or extended oligonucleotide.

[0339] In some embodiments, where the 3 ’-blocking group on the nucleotide donor comprises a reversible 3 ’-blocking group, the method further comprises removing or cleaving the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide.

[0340] In some embodiments, the method further comprises inactivating the deblocking agent or separating the deblocking agent from the unblocked extended polynucleotide or oligonucleotide.

[0341] In some embodiments, the method further comprises one or more cycles of: extension with a nucleotide donor; separation of a 3 ’-blocked extended polynucleotide or oligonucleotide from the recombinant primase or inactivation of the recombinant primase; removing or cleaving the reversible 3 ’-blocking group using a deblocking agent; and separating or removing the unblocked extended polynucleotide or oligonucleotide from the deblocking agent, wherein each cycle uses a new nucleotide donor.

[0342] In some embodiments, the nucleotide donor for at least one cycle comprises a mixture of different nucleotide donors. For example, and mixture of dATP and dGTP with a 3 ’-blocking group forms a mixture of polynucleotides or oligonucleotides with dA or dG in the reaction with the recombinant primase; and mixture of ATP and GTP with a 3 ’-blocking group forms a mixture of polynucleotides or oligonucleotides with A or G in the reaction with the recombinant primase.

[0343] In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

[0344] In some embodiments of the method, the reaction further comprises a pyrophosphatase in the reaction with the recombinant primase. In some embodiments, the pyrophosphatase comprises apyrophosphate belonging to the Type 1 pyrophosphatases. In some embodiments, the pyrophosphatase comprises a pyrophosphatase belonging to the Type II pyrophosphatases.

[0345] In some embodiments, the suitable reaction conditions for the reaction with recombinant primase comprises a temperature of about 30 °C to about 95 °C. In some embodiments, the reaction temperature is about 35 °C to about 90 °C, about 40 °C to about 85 °C, about 45 °C to about 80 °C, about 50 °C to about 75 °C, or about 55 °C to about 70 °C. In some embodiments, the suitable reaction conditions for the reaction with the recombinant primase comprises a temperature of about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.

[0346] In some embodiments, the suitable reactions for the reaction with the recombinant primase comprises a pH suitable for activity of the recombinant primase. In some embodiments, the suitable reaction conditions comprise a pH of about 6-9, pH of about 6.5-8.5, or pH of about 7-8. In some embodiments, the suitable reactions comprises a pH of about 6, 6.5, 7, 7.5, 8, 8.5, or 9.

[0347] In some embodiments, the suitable reaction conditions includes, among others, one or more of a buffer for maintaining or adjusting reaction pH, a divalent metal cofactor, and additives to stabilize the recombinant primase.

[0348] In some embodiments, the nucleotide acceptor is provided at a concentration of about 0.05 to about 5 mM. In some embodiments, the nucleotide acceptor is provided at a concentration of about 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient attachement of the nucleotide donor to the nucleotide acceptor.

[0349] In some embodiments, the nucleotide donor is provided at a concentration of about 0.05 to about 5 mM. In some embodiments, the nucleotide donor is provided at a concentration of about 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient extension of the nucleotide donor. In some embodiments, the ratio of nucleotide donor to nucleotide acceptor is 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or higher.Methods Using Acceptor Attached to a Support Medium

[0350] In some embodiments, the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor is attached to a support medium, and the recombinant primase and nucleotide donor are reacted with the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor attached to a support medium. In some embodiments, the recombinant primase and the nucleotide donor are in solution.

[0351] In some embodiments, the nucleotide donor comprises a 3 ’-blocking group to form a 3’- blocked extended polynucleotide or extended oligonucleotide attached to the support medium. In some embodiments, the 3 ’-blocking group comprises a reversible 3 ’-blocking group.

[0352] In some embodiments, method further comprises separating the recombinant primase and nucleotide donor from the 3 ’-blocked extended polynucleotide or 3 ’-extended oligonucleotide attached to the support medium.

[0353] In some embodiments, where the 3 ’-blocking group is a reversible or cleavable blocking group, the method further comprises removing or cleaving the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide attached to the support medium.

[0354] In some embodiments, the method further comprises inactivating or separating the deblocking agent from the unblocked extended polynucleotide or oligonucleotide attached to the support medium.

[0355] In some embodiments, the method further comprises one or more cycles of: extension with a nucleotide donor; separation of the recombinant primase from the 3 ’-blocked extended polynucleotide or oligonucleotide attached to the support medium; removing or cleaving the reversible 3 ’-blocking group with a deblocking agent to form the unblocked extended polynucleotide or extended oligonucleotide attached to the support medium; and separating the unblocked extended polynucleotide or oligonucleotide attached to the support medium from the deblocking agent, wherein each cycle uses a new nucleotide donor.

[0356] In some embodiments, the nucleotide donor in at least one cycle comprises a mixture of different nucleotide donors. In some embodiments, the mixture of nucleotide donors comprises 2, 3, 4, 5, or 6 different nucleotide donors. In some embodiments, the nucleotide donor can have different nucleobases, sugar moiety, or different modifications to the sugar moiety. By way of example and not limitation, use of a mixture of dATP and dGTP having a 3 ’-blocking group for a single cycle results in a mixture of 3 ’-blocked extended polynucleotide or oligonucleotide having dA or dG at the 3’- terminal nucleotide.

[0357] In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

[0358] In some embodiments, the method further comprises a pyrophosphatase in the reaction with the recombinant primase. In some embodiments, the pyrophosphatase comprises a pyrophosphatase of Type 1 pyrophosphatases. In some embodiments, the pyrophosphatase comprises a pyrophosphatase of Type II pyrophosphatases. In some embodiments, the recombinant primase and pyrophosphatase comprise a fusion protein.

[0359] In some embodiments, the polynucleotide or oligonucleotide acceptor attached to a support medium is housed in a reaction chamber. Generally, the support material is insoluble in aqueous solution. In some embodiments, the reaction chamber is a column or vessel, where the column or vessel retains the polynucleotide or oligonucleotide acceptor attached to the support medium.

[0360] In some embodiments, a method of template -independent oligonucleotide synthesis comprises reacting a nucleotide acceptor having a 3 ’-OH group, wherein the nucleotide acceptor is attached to a support medium in a chamber, with a nucleotide donor in presence of a recombinant primase described herein under suitable reaction conditions for the extension of the nucleotide acceptor to form an extended nucleotide acceptor attached to the support medium. In some embodiments, the reaction solution comprising the recombinant primase and the nucleotide donor is fed into the chamber for reaction with the nucleotide acceptor attached to the support medium in the chamber.

[0361] In some embodiments, the nucleotide acceptor comprises a polynucleotide acceptor, oligonucleotide acceptor, or an initiation nucleotide acceptor attached to a support medium to form an extended polynucleotide or extended oligonucleotide.

[0362] In some embodiments, the nucleotide donor comprises a 3 ’-blocking group to form a 3’- blocked extended polynucleotide or extended oligonucleotide attached to the support medium.

[0363] In some embodiments, a pyrophosphatase is used concurrently in the reaction with the recombinant primase.

[0364] In some embodiments, the recombinant primase, and if present the pyrophosphatase, and byproducts of the reaction are removed from the chamber, for example by washing the chamber (e.g., column).

[0365] In some embodiments, the method further comprises feeding a deblocking agent into the chamber to remove or cleave the 3 ’-blocking agent to form an unblocked extended polynucleotide or unblocked extended oligonucleotide attached to the support medium.

[0366] In some embodiments, the deblocking agent and any by-products of the deblocking reaction are removed or separated from the unblocked extended polynucleotide or unblocked extended oligonucleotide attached to the support medium in the chamber, for example by washing.

[0367] In some embodiments, the method further comprises cleaving or detaching the unblocked extended polynucleotide or unblocked extended oligonucleotide from the support medium. In some embodiments, the cleaving or detaching is carried out using a cleaving or detaching agent, for example a chemical detaching agent or a nuclease.

[0368] In some embodiments, the 3 ’-blocked extended polynucleotide or oligonucleotide attached to the support medium is cleaved or detached from the support medium using a cleaving or detaching agent, and 3 ’-blocked extended polynucleotide or oligonucleotide reacted with the deblocking agent to form an unblocked extended polynucleotide or oligonucleotide.

[0369] In some embodiments, the method further comprises one or more cycles of: extending a polynucleotide, oligonucleotide, or initiating nucleotide acceptor with a nucleotide donor comprising a 3 ’-blocking group in presence of a recombinant primase, and optionally a pyrophosphatase to form a 3 ’-blocked extended polynucleotide or oligonucleotide, wherein the polynucleotide, oligonucleotide,or initiating nucleotide acceptor is attached to a support medium in a chamber; removing or separating the recombinant primase, and if present the pyrophosphatase, and by-products of the reaction from the chamber by washing the chamber, wherein the 3 ’-blocked extended polynucleotide or oligonucleotide is retained in the chamber; reacting the 3 ’-blocked extended polynucleotide or oligonucleotide with a deblocking agent by feeding a deblocking agent into the chamber to form an unblocked extended polynucleotide or unblocked extended oligonucleotide attached to the support medium; and removing or separating the deblocking agent and any by-products of the deblocking reaction from the unblocked extended polynucleotide or unblocked extended oligonucleotide attached to the support medium in the chamber by washing the chamber.

[0370] In some embodiments, following the synthesis of the desired extended polynucleotide or oligonucleotide the method further comprises cleaving or detaching the unblocked extended polynucleotide or unblocked extended oligonucleotide from the support medium to form an unblocked extended polynucleotide or oligonucleotide in solution. In some embodiments, the cleaving or detaching is carried out using a cleaving or detaching agent. In some embodiments, the cleaving or detaching can be done in the chamber, e.g., by adding the cleaving or detaching agent into the chamber. In some embodiments, the unblocked extended polynucleotide or oligonucleotide attached to the support medium is removed from the chamber, and then reacted with the cleaving or detaching agent. In some embodiments, the released unblocked extended polynucleotide or oligonucleotide is separated from the support medium, and optionally further purified, e.g., by chromatography or filtration.

[0371] In some embodiments, a cycle comprises cleaving or detaching of the 3 ’-blocked polynucleotide or oligonucleotide from the support medium by use of a cleaving or detaching agent, and optionally removing or separating the cleaving or detaching agent from the 3 ’-blocked extended polynucleotide or oligonucleotide; and reacting the 3'-blocked extended polynucleotide or oligonucleotide with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide in solution. Generally, this cycle is the final cycle in the synthesis of the desired unblocked extended polynucleotide or oligonucleotide.

[0372] In some embodiments, the nucleotide donor in at least one cycle comprises a mixture of different nucleotide donors. In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

[0373] In some embodiments, the 3 ’-blocked extended polynucleotide or oligonucleotide, or the unblocked extended polynucleotide or oligonucleotide is purified, for example using chromatography and / or filtration.Methods Using Immobilized Enzymes

[0374] In some embodiments, a method of nucleic acid synthesis uses the recombinant primase immobilized on a support medium. In some embodiments, a method of template-independent nucleic acid synthesis uses the recombinant primase immobilized on a support medium. In some embodiments, a method of nucleic acid synthesis comprises reacting a polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor with a nucleotide donor in presence of a recombinant primase immobilized on a support medium in a first chamber, such as a column of immobilized recombinant primase, to from an extended polynucleotide or extended oligonucleotide. In some embodiments, the acceptor, e.g., the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor, and nucleotide donor are provided in solution.

[0375] In some embodiments, the nucleotide donor comprises a 3 ’-blocking group to form a 3’- blocked extended polynucleotide or extended oligonucleotide in solution. In some embodiments, the 3 ’-blocking group comprises a reversible 3 ’-blocking group.

[0376] In some embodiments, the method further comprises separating or removing the 3 ’-blocked extended polynucleotide or extended oligonucleotide in solution from the recombinant primase immobilized on the support medium in the first chamber.

[0377] In some embodiments, where the 3 ’-blocked extended polynucleotide or oligonucleotide comprises a reversible or removable 3 ’-blocking group, the method further comprises cleaving or removing the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide in solution. In some embodiments, the solution of the 3 ’-blocked extended polynucleotide or oligonucleotide having a reversible or removable 3 ’-blocking group is separated into a second chamber, wherein the second chamber provides a deblocking agent or a deblocking agent is fed into the second chamber.

[0378] In some embodiments, the method further comprises inactivating or separating the deblocking agent, and optionally deblocking reaction by-products, from the unblocked extended polynucleotide or oligonucleotide in solution. In some embodiments, an inactivating agent is added to the second chamber for inactivation of the deblocking agent. In some embodiments, a deblocking agent, e.g., an enzymatic deblocking agent, immobilized on a support medium is provided in the second chamber. In some embodiments, the unblocked extended polynucleotide or oligonucleotide in solution is removed from the second chamber to yield a solution of unblocked extended polynucleotide or oligonucleotide.

[0379] In some embodiments, the method further comprises one or more cycles of: extension of a polynucleotide acceptor, oligonucleotide acceptor, or an initiating nucleotide acceptor with a nucleotide donor in presence of a recombinant primase immobilized on a support medium in a first chamber, wherein the nucleotide donor comprises a 3 ’-blocking group; separation or removal of the 3 ’-blocked extended polynucleotide or oligonucleotide from the recombinant primase immobilized on a support medium in the first chamber to a second chamber; removing or cleaving the reversible 3’- blocking group with a deblocking agent to form the unblocked extended polynucleotide or extendoligonucleotide in solution in the second chamber; and separating the unblocked extended polynucleotide or oligonucleotide from the deblocking agent in the second chamber, wherein each cycle uses a new nucleotide donor. In some embodiments, one or more reservoirs can be used for storing the reaction solution from the first chamber prior to flowing the reaction solution to the second chamber.

[0380] In some embodiments, the unblocked extended polynucleotide or unblocked extended oligonucleotide in solution is returned or fed into the first chamber for reaction with the new nucleotide donor and immobilized recombinant primase. In some embodiments, the unblocked extended polynucleotide or unblocked extended oligonucleotide in solution is fed into a third chamber comprising a recombinant primase different than the recombinant primase in the first chamber and reaction with a new nucleotide donor.

[0381] In some embodiments, the nucleotide donor in at least one cycle comprises a mixture of different nucleotide donors. In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

[0382] In some embodiments, the method further comprises a pyrophosphatase in the reaction with the recombinant primase in the first chamber or the third chamber. In some embodiments, the pyrophosphatase is immobilized on a support medium. In some embodiments, the recombinant primase and pyrophosphatase are co-immobilized on a support medium. In some embodiments, the recombinant primase and pyrophosphatase are provided as a fusion protein in the first or third chamber, where the fusion protein is immobilized on a support medium.

[0383] In some embodiments, the unblocked extended polynucleotide or unblocked extended oligonucleotide removed or separated from the second chamber is further purified. In some embodiments, the unblocked extended polynucleotide or unblocked extended oligonucleotide is purified by chromatography or filtration.EXAMPLES

[0384] 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.

[0385] 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 m (milliliter); ul, uL, ml, and m (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 andPSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); 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); HPLC (high pressure liquid chromatography); FPLC (fast protein liquid chromatography); PBS (phosphate buffered saline); BSA (bovine serum albumin); DTT (dithiothreitol); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); IPTG (isopropyl [3-D-l -thiogalactopyranoside); FIOPC or FIOP (fold improvements over positive control or parent); LB (Luria-Bertani); TB (Terrific-Broth).Example 1 Primase Gene Acquisition and Construction of Expression Vectors

[0386] Genes encoding an N-terminal 6-histidine tagged version of the truncated primases (SEQ ID NO: 25-62 and 315-316) were designed with codon optimization for E. coli expression, synthesized, and subcloned into the E. coli expression vector pCK100900i (See e.g., U.S. Pat. No. 7,629,157 and US Pat. Appln. Publn. 2016 / 0244787). The plasmid construct was transformed into an E. coli strain derived from W3110.Example 2 Growth and Purification of Primase Variants at Shakeflask Scale2,1: Shakeflask scale growth of Primase Variants

[0387] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose and 30 pg / ml chloramphenicol. After overnight incubation 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 OD6oo of about 0.05. The cultures were incubated for approximately 195 min at 30 °C., 250 rpm, to an OD6oo 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 4000 rpmx 10 min. The culture supernatant was discarded, and the pellets were resuspended in 30 ml of 50 mM Tris, pH 7.5, 150 mM NaCl. 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 (10,000 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. Samples from the clarified lysate and resuspended pellet were analyzed by SDS-PAGE (see FIG. 2).2,2: Purification of Primase Variants

[0388] Lysates were then purified using an AKTA Pure purification system and a 5 ml HisTrap FF column (GE Healthcare). The run parameters are provided below. The wash buffer comprised 50 mM Tris-HCl, pH 7.5 150 mM NaCl, 20 mM imidazole, and the elution buffer contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 500 mM imidazole.

[0389] Purification Parameters:

[0390] Fractions containing the desired protein, identified by UV absorption (A280), were pooled and dialyzed overnight in dialysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 50% glycerol) overnight in a 3.5K Slide-A-Lyzer™ dialysis cassette (Thermo Fisher). Purified primases were used for activity characterization.Example 3 Activity Characterization of Purified Recombinant Primases3,1: Ab initio activity of Recombinant Primases

[0391] Methods for ab initio activity characterization of SEQ IDS NO: 26 - 52 are described here. Reactions were performed in 96-well BioRad PCR plates. Reactions included 100 pM pooled deoxynucleotide triphosphates, 1 X Thermopol buffer (20 mM Tris-HCl, 10 mM (NEL^SO^ 10 mM KC1, 5 mM MgSO4, pH 8.8 @ 25 °C). The reactions were set up as follows: (i) all reaction components, except for primase were pre-mixed in a single solution, and 15 pL of this solution was aliquoted into each well of the 96-well plates (ii) 5 pL of primase solution was then added into the wells to initiate the reaction. The mixture was then sealed and incubated at the indicated temperature. The reactions were mixed with a DNA loading dye and run on a 6-8% agarose gel. Gels were stained with Sybr Green II (ThermoFisher) and imaged under UV transillumination (see FIG. 3)

[0392] Methods for ab initio activity characterization of SEQ ID NO: 54 - 62 and 316 are described here. Reactions were performed in 96-well BioRad PCR plates. Reactions included 200 pM nucleotide triphosphates (dATP, dGTP, dCTP, dTTP, 2-fluoro-ATP, or 2-fluoro-GTP), lOOmM Tris- HCl pH 7.5, and 1 mM MgCF. The reactions were set up as follows: (i) all reaction components, except for primase were pre-mixed in a single solution, and 15 pL of this solution was aliquoted into each well of the 96-well plates (ii) 5 pL of primase solution was then added into the wells to initiate the reaction. The mixture was then sealed and incubated for one hour at 70 °C. The reactions were mixed with a DNA loading dye and run on a 6-8% agarose gel. Gels were stained with Sybr Green II (ThermoFisher) and imaged under UV transillumination (see FIG. 4)3,2: Extension of Oligonucleotide Acceptor Molecules with Nucleotide Donors by Primase

[0393] Methods for oligonucleotide extension activity characterization of SEQ IDS NO: 26 - 52 are described here. Reactions were performed in 96-well BioRad PCR plates. Reactions included 1-4 pMoligonucleotide (FAM-T14ATCfA), 25-200 pM nucleotide triphosphate (dATP, 2’-F-ATP, or ddATP), 1 pM inorganic pyrophosphatase, 1 X Thermopol buffer (20 mM Tris-HCl, 10 mM (NFU^SCh, 10 mM KC1, 5 mM MgSCh, pH 8.8 at 25 °C). It is possible that the poly-T section of the substrate oligonucleotide could act as a template for addition of dATP, 2’-F-ATP, and ddATP. The reactions were set up as follows: (i) all reaction components, except for primase were pre-mixed in a single solution, and 15 pL of this solution was aliquoted into each well of the 96-well plates (ii) 5 pL of primase 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 70 °C overnight. Following reaction, samples were analyzed by capillary electrophoresis as described in example 5. Substrate (FAM-T14ATCfA) and products (FAM-T14ATCfAA and further polymerization products, FAM- T14ATCfAfA and further polymerization products, and FAM-T14ATCfAddA) were quantified. See Appendix for details on oligonucleotide substrates and products.

[0394] Methods for oligonucleotide extension activity characterization of SEQ ID NOs: 54-62 and 316 is described here. Reactions were performed in 96-well BioRad PCR plates. Reactions included 2 pM oligonucleotide, 200 pM nucleotide triphosphate, 1 pM inorganic pyrophosphatase, 50 mM Tris- HCl pH 8.0, 1 mM MgCT. It is possible that the poly-T section of the substrate oligonucleotide could act as a template for addition of dATP, 2’-F-ATP, and ddATP. The reactions were set up as follows: (i) all reaction components, except for primase were pre-mixed in a single solution, and 15 pL of this solution was aliquoted into each well of the 96-well plates, and (ii) 5 pL of primase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelablealuminum seal and incubated in a thermocycler at 70°C for 1 hour. Following reaction, samples were analyzed by capillary electrophoresis as described in example 5. Substrates (FAM-T14ATCfA and FAM-T17AAA) and products (FAM-T14ATCfAA and further polymerization products, FAM- T14ATCfAfA and further polymerization products, FAM-T14ATCfAddA, FAM-T17AAAA and further polymerization products, FAM-T17AAAfA and further polymerization products, and FAM- T17AAAddA) were quantified. See Appendix for details on oligonucleotide substrates and products.Example 4 High-Throughput (HTP) Growth of Primase Variants and Screening Conditions4, 1: HTP Growth of Primase and Variants

[0395] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose and 30 pg / ml chloramphenicol. After overnight incubation at 37 °C, colonies were inoculated into 96- well shallow flat bottom plates (NUNC™, Thermo-Scientific) filled with 180 pl / well LBsupplemented with 1% glucose and 30 pg / ml chloramphenicol. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kuhner).

[0396] Overnight growth samples (20 pL) were transferred into Costar 96-well deep plates fdled with 380 pL of Terrific Broth supplemented with 30 pg / mL chloramphenicol. The plates were incubated for 130 minutes in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner). The cells were then induced with 40 pL of 10 mM IPTG in sterile water and incubated overnight for 20-24 hours in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner). The cells were pelleted (4000 rpm x 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to analysis.4,2: Lysis of HTP Cell Pellets

[0397] Lysis was carried out by addition of 200 pL of lysis buffer (100 mM triethanolamine (TEoA) pH 8.0 containing 0.1 mg / ml lysozyme) into each well of cell pellets. Plates were sealed, agitated until pellets were resuspended, and then transferred to a 96-well BioRad PCR plate. Lysates were incubated at 55 °C for one hour. Following incubation, plates were centrifuged (4000 rpm x 15 min) and clarified lysates were transferred to new 96-well PCR plates. In some cases, clarified lysates were stored at 4 °C or were further diluted in lOOmM TEoA pH 8.0 prior to use.4,3: Primase activity assay with clarified lysate: addition of ddATP to an unmodified oligo

[0398] Reactions were performed in 384-well format BioRad PCR plates. Reactions included 2 pM of acceptor oligonucleotide FAM-T17AAA, 200 pM dideoxy adenosine triphosphate (ddATP), 1 pM IPP inorganic pyrophosphatase, 100 mM TEoA pH 8.0, and 1 mM CoCL. It is possible that the poly- T section of the substrate oligonucleotide could act as a template for addition of dATP, 2’-F-ATP, and ddATP. The reactions were set up as follows: (i) all reaction components, except for primase were pre-mixed in a single solution, and 0.9 pL of this solution was aliquoted into each well of the 384- well plates (ii) 0.1 pL of 30-fold diluted clarified lysate was then added into the wells to initiate the reaction. Low volume reagent transfers were performed using the I.DOT Liquid Handler (Dispendix) and the Echo Acoustic Liquid Handler (Beckman Coulter). The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at 60 °C for 1 hour followed by a 5 minute heat kill step at 95 °C. Samples were saved for later analysis by capillary electrophoresis. Substrate (FAM-T17AAA) and product (FAM-T17AAAddA) were quantified. See Appendix for details on oligonucleotide substrates and products.4,4: Primase activity assay with clarified lysate: addition of mATP to an unmodified oligo

[0399] Reactions were performed in 384-well format BioRad PCR plates. Reactions included 2 pM of acceptor oligonucleotide FAM-T17AAA, 1 pM IPP inorganic pyrophosphatase, 100 mM TEoA pH 8.0, and 1 mM CoCL. Reactions also contained 200 pM of 2 ’-methoxy adenosine triphosphate (mATP). It is possible that the poly-T section of the substrate oligonucleotide could act as a template for addition of mATP. The reactions were set up as follows: (i) all reaction components, except forprimase were pre-mixed in a single solution, and 0.2 pL of this solution was aliquoted into each well of the 384-well plates (ii) 0.8 uL of undiluted clarified lysate was then added into the wells to initiate the reaction. Low volume reagent transfers were performed using the I. DOT Liquid Handler (Dispendix) and the Echo Acoustic Liquid Handler (Beckman Coulter). The reaction plate was heat- sealed with a peelable aluminum seal and incubated in a thermocycler at 60 °C for 1 hour followed by a 5 minute heat kill step at 95 °C. Following reaction, samples were analyzed by capillary electrophoresis as described in example 5. Substrate (FAM-T17AAA) and product (FAM- T17AAAmA) were quantified. See Appendix for details on oligonucleotide substrates and products.Example 5 Capillary electrophoresis (CE) analysis of oligonucleotidesSample preparation and reaction analysis using CE

[0400] For analysis of the reaction samples, capillary electrophoresis was performed using an ABI 3500x1 Genetic Analyzer (ThermoFisher). Reactions were quenched by the addition of water and EDTA such that the final concentration of EDTA in the quenched reaction was 1 mM. Quenched reactions were diluted in water to approximately 0. 1 nM 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 oligonucleotide substrates and products were identified by their sizes relative to the sizing ladder.Example 6 Screening Results of Primase Variants derived from SEQ ID NO: 60

[0401] The polynucleotide sequence of SEQ ID NO: 59 encoding the polypeptide sequence of SEQ ID NO: 60 was used as the backbone for the construction of new variants. The variants generated from homolog diversity and saturation mutagenesis were grown and screened according to the methods outlined in Example 4. Addition of ddATP and 2’-O-methyl-ATP (mATP) to an oligonucleotide substrate was investigated. Analysis of the data relative to SEQ ID NO: 60 is listed in Table 6.1.

[0402] 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, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

[0403] 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.APPENDIX

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A recombinant primase comprising a polypeptide fragment comprising the primase domain of a primase of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, wherein the recombinant primase exhibits terminal nucleotidyl transferase activity.

2. The recombinant primase of claim 1, wherein the polypeptide fragment comprises the N- terminal fragment containing the primase domain.

3. The recombinant primase of claim 1, wherein the N-terminal fragment containing the primase domain comprises (a) about 250 amino acid residues of the N-terminus comprising the PriS domain, or (b) about 400 amino acid residues of the N-terminus comprising the PriS and PriX domains.

4. The recombinant primase of any one of claims 1-3, wherein the primase domain consists of functionally active PriS domain, or consists of the functionally active PriS and PriX domains.

5. A recombinant primase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a polypeptide fragment comprising the primase domain of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, wherein the recombinant primase has template independent terminal nucleotidyl transferase activity.

6. The recombinant primase of claim 6, wherein the polypeptide fragment comprises the N- terminal fragment comprising the primase domain.

7. The recombinant primase of claim 5 or 6, wherein the primase domain includes PriS, or PriS and PriX domains.

8. The recombinant primase of any one of claims 1-7, wherein the primase domain is of Methanococcus.

9. The recombinant primase claim 8, wherein the primase domain is of a primase of Methanococcus voltae, Methanococcus halophilus, Methanocaldococcus jannaschii, Methanococcus maripaludis, Methanococcus aeolicus, Methanosarcina mazei Cl 6, Methanococcus vannielii, Methanococcus deltae, Methanothermococcus okinawensis, Methanocaldococcus infemus, Methanothermus sociabilis, Methanomethylovorans hollandica, or Methanosarcina acetivorans.

10. The recombinant primase of any one of claims 1-9, wherein the primase domain is of Bacillus.

11. The recombinant primase of claim 12, wherein the primase domain is of a primase of Bacillus albus, Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus australimaris, Bacillus atrophaeus, Bacillus badius, Bacillus carboniphilus, Bacillus cereus, Bacillus clarus, Bacillus fonticola, Bacillus gaemokensis, Bacillus glycinifermentans, Bacillus halotolerans, Bacillus horti,Bacillus inaquosorum, Bacillus luti, Bacillus methanolicus, Bacillus mobilis, Bacillus mycoides, Bacillus nakamurai, Bacillus norwichensis, Bacillus obstructivus, Bacillus pacificus, Bacillus paralicheniformis, Bacillus paranthracis, Bacillus pinisoli, Bacillus pseudomycoides, Bacillus pumilus, Bacillus safensis, Bacillus salipaludis, Bacillus spizizenii, Bacillus solimangrovi, Bacillus suaedae, Bacillus subtilis, Bacillus spizizenii, Bacillus thuringiensis, Bacillus toyonensis, Bacillus tropicus, Bacillus vallismortis, Bacillus velezensis, Bacillus wiedmannii, Bacillus wudalianchiensis, Bacillus xiamenensis, Bacillus yapensis, Bacillus zhangzhouensis.

12. The recombinant primase of any one of claims 1-9, wherein the primase domain is of Chloroflexota bacterium or Ammonifex.

13. The recombinant primase of claim 12, wherein the primase domain is of a primase of Ammonifex thiophilus, Ammonifex degensii, Ammonifex sp. Tomsk_66_l 1, or Ammonifex sp. SURF 55.

14. A recombinant primase comprising a chimeric primase domain, wherein the chimeric primase domain comprises of a PriS domain of a first primase and the PriX domain of a second primase, wherein the first primase and the second primase are different, and wherein the chimeric primase exhibits terminal nucleotidyl transferase activity.

15. The recombinant primase of claim 14, wherein the chimeric primase domain comprises a PriS domain of a first primase of Methanococcus, Bacillus, Chloroflexota, Ammonifex, Thermococcus, or Pyrococcus, and a PriX domain of a second primase of Methanococcus, Bacillus, Chloroflexota, Ammonifex, Thermococcus, or Pyrococcus, wherein the first primase and second primase are different.

16. The recombinant primase of claim 14, wherein the PriS domain is selected from the PriS domain of Methanococcus, Bacillus, Chloroflexota, or Ammonifex, and the PriX domain is selected from the PriX domain of Thermococcus or Pyrococcus.

17. The recombinant primase of claim 14, wherein the PriS domain is selected from the PriS domain of Thermococcus or Pyrococcus, and the PriX domain is selected from the PriX domain of Methanococcus, Bacillus, Chloroflexota, or Ammonifex.

18. The recombinant primase of claim 15-17, wherein the Thermococcus comprises Thermococcus acidaminovorans, Thermococcus aegaeus, Thermococcus aggregans, Thermococcus alcaliphilus, Thermococcus atlanticus, Thermococcus barophilus, Thermococcus barossii, Thermococcus celer, Thermococcus celericrescens, Thermococcus chitonophagus, Thermococcus cleftensis, Thermococcus coalescens, Thermococcus eurythermalis, Thermococcus fumicolans, Thermococcus gammatolerans, Thermococcus gorgonarius, Thermococcus guaymasensis, Thermococcus hydrothermalis, Thermococcus indicus, Thermococcus kodakarensis, Thermococcus litoralis, Thermococcus marinus, Thermococcus mexicalis, Thermococcus nautili, Thermococcus onnurineus, Thermococcus pacificus, Thermococcus paralvinellae, Thermococcus peptonophilus, Thermococcuspiezophilus, Thermococcus prieurii, Thermococcus profundus, Thermococcus radiotolerans, Thermococcus sibiricus, Thermococcus siculi, Thermococcus stetteri, Thermococcus thioreducens, Thermococcus waimanguensis, Thermococcus waiotapuensis, Thermococcus zilligii, Thermococcus sp. AEPII la, Thermococcus sp. 101 C5, Thermococcus sp. 11N.A5, Thermococcus sp. 12-4, Thermococcus sp. 13-2, Thermococcus sp. 13-3, Thermococcus sp. 1519, Thermococcus sp. 175, Thermococcus sp. 17S1, Thermococcus sp. 17S2, Thermococcus sp. 17S3, Thermococcus sp. 17S4, Thermococcus sp.17S5, Thermococcus sp. 17S6, Thermococcus sp. 17S8, Thermococcus sp. 18S1, Thermococcus sp.18S2, Thermococcus sp. 18S3, Thermococcus sp. 18S4, Thermococcus sp. 18S5, Thermococcus sp. 21-1, Thermococcus sp. 21 S 1, Thermococcus sp. 21S2, Thermococcus sp. 21S3, Thermococcus sp. 21S4,Thermococcus sp. 21S5, Thermococcus sp. 21S6, Thermococcus sp. 21S7, Thermococcus sp. 21S8, Thermococcus sp. 21S9, Thermococcus sp. 23-1, Thermococcus sp. 23-2, Thermococcus sp. 2319x1, Thermococcus sp. 26-2, Thermococcus sp. 26-3, Thermococcus sp. 26 / 2, Thermococcus sp. 28-1, Thermococcus sp. 29-1, Thermococcus sp. 300-Tc, Thermococcus sp. 31-1, Thermococcus sp. 31-3, Thermococcus sp. 40_45, Thermococcus sp. 4557, Thermococcus sp. 5-1, Thermococcus sp. 5-4, Thermococcus sp. 70-4-2, Thermococcus sp. 7324, Thermococcus sp. 83-5-2, Thermococcus sp. 9N2, Thermococcus sp. 9N2.20, Thermococcus sp. 9N2.21, Thermococcus sp. 9N3, Thermococcus sp. 9oN-7, Thermococcus sp. A4, Thermococcus sp. AF1T14.13, Thermococcus sp. AF1T1423, Thermococcus sp. AF1T20.11, Thermococcus sp. AF1T6.10, Thermococcus sp. AF1T6.12, Thermococcus sp. AF1T6.63, Thermococcus sp. AF2T511, Thermococcus sp. Ag85-vw, Thermococcus sp. AM4, Thermococcus sp. AMT11, Thermococcus sp. AMT7, Thermococcus sp. Anhete70478, Thermococcus sp. Anhete70-SCI, Thermococcus sp. Anhete85478, Thermococcus sp. Anhete85-SCI, Thermococcus sp. AT1273, Thermococcus sp. AVI, Thermococcus sp. AV2, Thermococcus sp. AV3, Thermococcus sp. AV6, Thermococcus sp. AV7, Thermococcus sp. AV9, Thermococcus sp. AV10, Thermococcus sp. AVI 1, Thermococcus sp. AV 13, Thermococcus sp. AV 14, Thermococcus sp. AV 15, Thermococcus sp. AV 16, Thermococcus sp. AV17, Thermococcus sp. AV18, Thermococcus sp. AV20, Thermococcus sp. AV21, Thermococcus sp. AV22, Thermococcus sp. Ax00-17, Thermococcus sp. Ax00-27, Thermococcus sp. Ax00-39, Thermococcus sp. Ax00-45, Thermococcus sp. Ax01-2, Thermococcus sp. Ax01-3, Thermococcus sp. Ax01-37, Thermococcus sp. Ax01-39, Thermococcus sp. Ax01-61, Thermococcus sp. Ax01-62, Thermococcus sp. Ax01-65, Thermococcus sp. Ax98-43, Thermococcus sp. Ax98-46, Thermococcus sp. Ax98-48, Thermococcus sp. Ax99-47, Thermococcus sp. Ax99-57, Thermococcus sp. Ax99-67, Thermococcus sp. AXTV6, Thermococcus sp. Bl, Thermococcus sp. B1001, Thermococcus sp. B4, Thermococcus sp. BHI60a21, Thermococcus sp. BHI80a28, Thermococcus sp. BHI80a40, Thermococcus sp. Bubb.Bath, Thermococcus sp. BX13, Thermococcus sp. CAR-80, Thermococcus sp. Champagne, Thermococcus sp. CIR10, Thermococcus sp. CKU-1, Thermococcus sp. CKU-199, Thermococcus sp. CL2, Thermococcus sp. CMI, Thermococcus sp. CNR-5, Thermococcus sp. CXI, Thermococcus sp. CX2, Thermococcus sp. CX3, Thermococcus sp. CX4, Thermococcus sp. CYA, Thermococcus sp. Dex80a71, Thermococcus sp. Dex80a75, Thermococcus sp. DS-1, Thermococcus sp.DS1, Thermococcus sp. DT4, Thermococcus sp. ENR5, Thermococcus sp. EPl, Thermococcus sp. ES5, Thermococcus sp. ES6, Thermococcus sp. ES7, Thermococcus sp. ES8, Thermococcus sp. ES9, Thermococcus sp. ES10, Thermococcus sp. ES11, Thermococcus sp. ES12, Thermococcus sp. ES13, Thermococcus sp. EXT12c, Thermococcus sp. EXT9, Thermococcus sp. Fe85_l_2, Thermococcus sp. GB18, Thermococcus sp. GB20, Thermococcus sp. GE8, Thermococcus sp. Gorda2, Thermococcus sp. Gorda3, Thermococcus sp. Gorda4, Thermococcus sp. Gorda5, Thermococcus sp. Gorda6, Thermococcus sp. GR2, Thermococcus sp. GR4, Thermococcus sp. GR5, Thermococcus sp. GR6, Thermococcus sp. GR7, Thermococcus sp. GT, Thermococcus sp. GU5L5, Thermococcus sp. HJ21, Thermococcus sp. IRI33, Thermococcus sp. IRI35c, Thermococcus sp. IRI48, Thermococcus sp. JCM 11816, Thermococcus sp. JDF-3, Thermococcus sp. JdF3, Thermococcus sp. JdFR-02, Thermococcus sp. KBA1, Thermococcus sp. KI, Thermococcus sp. KS-8, Thermococcus sp. LM0-A1, Thermococcus sp. LM0-A2, Thermococcus sp. LM0-A3, Thermococcus sp. LM0-A4, Thermococcus sp. LM0-A5, Thermococcus sp. LM0-A6, Thermococcus sp. LM0-A7, Thermococcus sp. LM0-A8, Thermococcus sp. LM0-A9, Thermococcus sp. LSI, Thermococcus sp. LS2, Thermococcus sp. M36, Thermococcus sp. M39, Thermococcus sp. MA2.27, Thermococcus sp. MA2.28, Thermococcus sp. MA2.29, Thermococcus sp. MA2.33, Thermococcus sp. MARI, Thermococcus sp. MAR2, Thermococcus sp. MCR132, Thermococcus sp. MCR133, Thermococcus sp. MCR134, Thermococcus sp. MCR135, Thermococcus sp. MCR175, Thermococcus sp. MV1, Thermococcus sp. MV2, Thermococcus sp. MV3, Thermococcus sp. MVS, Thermococcus sp. MV 10, Thermococcus sp. MV11, Thermococcus sp. MV 12, Thermococcus sp. MV 13, Thermococcus sp. MV 1031, Thermococcus sp. MV 1049, Thermococcus sp. MV1083, Thermococcus sp. MV1092, Thermococcus sp. MV1099, Thermococcus sp. MZ1, Thermococcus sp. MZ2, Thermococcus sp. MZ3, Thermococcus sp. MZ5, Thermococcus sp. MZ6, Thermococcus sp. MZ7, Thermococcus sp. MZ8, Thermococcus sp. MZ9, Thermococcus sp. MZ10, Thermococcus sp. MZ11, Thermococcus sp. MZ12, Thermococcus sp. MZ13, Thermococcus sp. NS85- T, Thermococcus sp. P6, Thermococcus sp. Pd70, Thermococcus sp. Pd85, Thermococcus sp. PK, Thermococcus sp. PK(2011), Thermococcus sp. Rt3, Thermococcus sp. SB611, Thermococcus sp. SN531, Thermococcus sp. SRB55 1, Thermococcus sp. SRB70 1, Thermococcus sp. SRB70 10, Thermococcus sp. SY113, Thermococcus sp. Tc-1-70, Thermococcus sp. Tc-1-85, Thermococcus sp. Tc- 1-95, Thermococcus sp. Tc-2-85, Thermococcus sp. Tc-2-95, Thermococcus sp. Tc-365-70, Thermococcus sp. Tc-365-85, Thermococcus sp. Tc-365-95, Thermococcus sp. Tc-4-70, Thermococcus sp. Tc-4-85, Thermococcus sp. Tc-I-70, Thermococcus sp. Tc-I-85, Thermococcus sp. Tc-S-70, Thermococcus sp. Tc-S-85, Thermococcus sp. Tc55_l, Thermococcus sp. Tc55_12, Thermococcus sp. Tc70-4C-I, Thermococcus sp. Tc70-4C-S, Thermococcus sp. Tc70-7C-I, Thermococcus sp. Tc70-7C-S, Thermococcus sp. Tc70-CRC-I, Thermococcus sp. Tc70-CRC-S, Thermococcus sp. Tc70-MC-S, Thermococcus sp. Tc70-SC-I, Thermococcus sp. Tc70-SC-S, Thermococcus sp. Tc70-vw, Thermococcus sp. Tc70_l, Thermococcus sp. Tc70_10, Thermococcus sp. Tc70_l l, Thermococcus sp. Tc70_12, Thermococcus sp. Tc70_20, Thermococcus sp. Tc70_6, Thermococcus sp. Tc70_9,Thermococcus sp. Tc85-0 age SC, Thermococcus sp. Tc85-4C-I, Thermococcus sp. Tc85-4C-S, Thermococcus sp. Tc85-7C-S, Thermococcus sp. Tc85-CRC-I, Thermococcus sp. Tc85-CRC-S, Thermococcus sp. Tc85-MC-I, Thermococcus sp. Tc85-MC-S, Thermococcus sp. Tc85-SC-I, Thermococcus sp. Tc85-SC-ISCS, Thermococcus sp. Tc85-SC-S, Thermococcus sp. Tc85_l, Thermococcus sp. Tc85_10, Thermococcus sp. Tc85_l l, Thermococcus sp. Tc85_12, Thermococcus sp. Tc85_13, Thermococcus sp. Tc85_19, Thermococcus sp. Tc85_2, Thermococcus sp. Tc85_20, Thermococcus sp. Tc85_9, Thermococcus sp. Tc95-CRC-I, Thermococcus sp. Tc95-CRC-S, Thermococcus sp. Tc95-MC-I, Thermococcus sp. Tc95-MC-S, Thermococcus sp. Tc95-SC-S, Thermococcus sp. TK1, Thermococcus sp. TKM 55-W7-A, Thermococcus sp. TM1, Thermococcus sp. TP-33, Thermococcus sp. TP-37, Thermococcus sp. TS3, Thermococcus sp. TVG2, or Thermococcus sp. vpl97.

19. The recombinant primase of claim 15-17, wherein the Pyrococcus comprises Pyrococcus abyssi, Pyrococcus abyssi GE5, Pyrococcus furiosus, Pyrococcus furiosus COMI, Pyrococcus furiosus DSM 3638, Pyrococcus kukulkanii, Pyrococcus sp 12-1, Pyrococcus sp. NA2, Pyrococcus sp. ST04, Pyrococcus yayanosii, Pyrococcus yayanosii CHI, or Pyrococcus sp. ST04.

20. The recombinant primase of any one of claims 1-19, wherein the recombinant primase has nucleotidyl transferase activity for nucleotide donor dATP, dGTP, dCTP, dTTP, 2’-F-ATP, ddATP, ddGTP, ddCTP, ddTTP.

21. The recombinant primase of claim 20, wherein the acceptor comprises a modified oligonucleotide acceptor.

22. The recombinant primase of claim 5, comprising a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 316.

23. The recombinant primase of claim 22, comprising a primase domain comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 28, 32, 34, 40, 42, 46, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

24. The recombinant primase of claim 22 or 23, wherein the amino acid sequence of the primase domain comprises one or more amino acid differences relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 28, 32, 34, 40,42, 46, 54, 56, 58, 60, 62, or 316, or a reference sequence corresponding to SEQ ID NO: 28, 32, 34, 40, 42, 46, 48, 54, 56, 58, 60, 62, or 316.

25. The recombinant primase of claim 24, wherein the amino acid sequence of the primase domain comprises at least an amino acid difference at amino acid position 9, 18, 19, 34, 36, 40, 41, 42, 43, 44, 45, 46, 47, 52, 63, 64, 69, 71, 75, 80, 81, 82, 83, 84, 85, 89, 90, 92, 101, 105, 106, 109, 111, 113, 119, 124, 128, 136, 137, 142, 157, 159, 171, 184, 187, 191, 192, 195, 197, 200, 201, 203, 206, 208, 209, 210, 211, 224, 225, 247, 252, 273, 292, 297, 314, 327, 337, 340, 341, 344, 346, 348, 351, 355, 358, 370, 375, 377, 378, 383, 391, 399, 402, 411, 416, 419, 422, and 423, or combinations thereof, wherein the amino acid differences are relative to the reference sequence corresponding to the sequence from residue 12 to the carboxy terminus of SEQ ID NO: 60, or the reference sequence corresponding to SEQ ID NO: 60, or equivalent positions thereof.

26. The recombinant primase of claim 24, wherein the amino acid sequence of the primase domain comprises at least an amino acid difference or amino acid residue 9A, 18A, 19G / I / V. 34E, 36T, 40C / L, 41G / L, 42R, 43R / V. 44N, 45S, 46C / G / H / R, 47G, 52K / N, 63G / I / V, 64C / S, 69R / V. 71G / L / R, 75C / M / S, 80L / V, 81A / R, 82G, 83S, 84G / L, 85G / L / P, 89R, 90N / T / V, 92L / T, 101L, 105A / G / R, 106M / S, 109M / R, 1 HR, 113K, 119E, 124L, 128F, 136D / P, 137G / K, 142D / G / R, 157D, 159E, 171W, 184G / P, 187R, 191V, 192P, 195M, 197A, 200G / Q, 201T, 203L, 206S / T, 208F, 209G, 210G / P / Q, 21 IP, 224N, 225P, 247R, 252E, 273A, 292R, 297D, 314N, 327A, 337L, 340G, 341V, 344H, 346V, 348T, 35 IN, 355D, 358E, 370G, 375H, 377L, 378S, 383G, 391R, 399D, 402P, 41 IL, 416G, 419H, 422G, or 423V, or any combinations thereof, wherein the amino acid difference is relative to SEQ ID NO: 60.

27. The recombinant primase of any one of claims 22-26, having at least one improved property as compared to a reference recombinant primase having template independent nucleotidyl transferase activity.

28. The recombinant primase of claim 27, wherein the improved property comprises increased incorporation of ddATP or 2’-O-methyl-ATP to an oligonucleotide acceptor.

29. The recombinant primase of any one of claims 22-28, wherein the recombinant primase comprises a sequence corresponding to residues 12 to carboxy terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314, or the sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 64-314.

30. The recombinant primase of any one of claims 1-29, wherein the recombinant primase is a purified preparation.

31. A recombinant polynucleotide comprising a polynucleotide sequence encoding a recombinant primase of any one of claims 1-29.

32. The recombinant polynucleotide of claim 31, wherein(a) the polynucleotide sequence has least 60%, 65%, 70%, 75%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to the sequence from residue 34 to the 3 ’-terminal nucleotide of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37 / 38, 39 / 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 315, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 25, 27, 29, 31, 33, 35, 37 / 38, 39 / 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 315, wherein the recombinant polynucleotide encodes a recombinant primase having template -independent terminal nucleotidyl transferase activity;(b) the polynucleotide sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to the sequence from residue 34 to 3 ’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, or to a reference polynucleotide sequence corresponding an odd numbered SEQ ID NO. of SEQ ID NOs: 63-313, wherein the recombinant polynucleotide encodes a recombinant primase having template-independent terminal nucleotidyl transferase activity.

33. The recombinant polynucleotide of claim 31 or 32, wherein the polynucleotide sequence is codon-optimized for expression of the encoded recombinant primase.

34. An expression vector comprising a recombinant polynucleotide of any one of claims 31- 33.

35. A host cell comprising an expression vector of claim 34.

36. The host cell of claim 35, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

37. A method of producing a recombinant primase in a host cell comprising culturing a host cell of claim 35 or 36, under suitable culture conditions such that the encoded recombinant primase is produced.

38. The method of claim 37, further comprising recovering the recombinant primase from the culture and / or host cells, and / or further comprising purifying the recombinant primase.

39. A composition comprising a recombinant primase of any one of claims 1-30.

40. The composition of claim 39, wherein the recombinant primase is immobilized on a support medium.

41. The composition of claim 39 or 40, further comprising one or more of a nucleotide acceptor and / or a nucleotide donor.

42. The composition of claim 53, wherein the nucleotide acceptor comprises a polynucleotide acceptor, oligonucleotide acceptor, or an initiating nucleotide acceptor.

43. The composition of claim 41 or 42, wherein the nucleotide donor comprises a 3’- blocking group or a chain terminator nucleotide donor.

44. The composition of claim 43, wherein the 3 ’-blocking group comprises a reversible 3’- blocking group or reversible chain terminating nucleotide donor comprising a 3 ’-blocking group.

45. The composition of any one of claims 39-44, further comprising a pyrophosphatase.

46. A method of oligonucleotide synthesis comprising reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a recombinant primase of any one of claims 1-30 under suitable reaction conditions for the extension of the nucleotide acceptor by attachment of the nucleotide donor to the nucleotide acceptor by the primase.

47. The method of claim 46, wherein the nucleotide acceptor comprises (a) a polynucleotide acceptor, an oligonucleotide acceptor, or an initiating nucleotide acceptor, or (b) an oligonucleotide acceptor of at least 2, 3, 4, 5, 6, 8, 9, 10 nucleotides in length.

48. The method of claim 46 or 47, wherein the polynucleotide or oligonucleotide acceptor is DNA, RNA, or a mixture of DNA and RNA.

49. The method of any one of claims 46-48, wherein the initiating nucleotide acceptor comprises NTP, NDP, NMP, or a nucleoside.

50. The method of any one of claims 46-49, wherein the nucleotide donor comprises a blocking group or the terminating nucleotide donor comprises a 3 ’-blocking group to form a 3 ’-blocked extended polynucleotide or extended oligonucleotide.

51. The method of claim 50, wherein the 3’-blocking group comprises a reversible 3’- blocking group.

52. The method of claim 51, further comprising inactivating the recombinant primase or separating the recombinant primase from the reaction solution containing the 3 ’-blocked extended polynucleotide or extended oligonucleotide.

53. The method of claim 52, further comprising removing or cleaving the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide.

54. The method of claim 53, further comprising inactivating or separating the deblocking agent from the unblocked extended polynucleotide or oligonucleotide.

55. The method of any one of claims 51- 54, further comprising one or more cycles of extension with a nucleotide donor; separation of 3 ’-blocked extended polynucleotide or oligonucleotide from the recombinant primase or inactivation of the recombinant primase; removing or cleaving the reversible 3 ’-blocking group with a deblocking agent; and separating the unblocked extended polynucleotide or oligonucleotide, wherein each cycle uses a new nucleotide donor.

56. The method of claim 55, wherein the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide,wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

57. The method of any one of claims 46-56, further comprising cleaving pyrophosphate with a pyrophosphatase in the reaction with the recombinant primase.

58. The method of any one of claims 46-49, wherein the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor is attached to a support medium and the recombinant primase and nucleotide donor are reacted with the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor attached to a support medium.

59. The method of claim 58, wherein the nucleotide donor comprises a 3’-blocking group or the terminating nucleotide donor comprises a 3 ’-blocking group to form a 3 ’-blocked extended polynucleotide or extended oligonucleotide attached to the support medium.

60. The method of claim 59, wherein the 3 ’-blocking group comprises a reversible 3’- blocking group.

61. The method of claim 60, further comprising separating the recombinant primase and nucleotide donor from the 3 ’-blocked extended polynucleotide or extended oligonucleotide attached to the support medium.

62. The method of claim 61, further comprising removing or cleaving the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide attached to the support medium.

63. The method of claim 62, further comprising inactivating or separating the deblocking agent from the unblocked extended polynucleotide or oligonucleotide attached to the support medium.

64. The method of any one of claims 58-63, further comprising one or more cycles of: extension with a nucleotide donor; separation of the recombinant primase from the 3 ’-blocked extended polynucleotide or oligonucleotide attached to the support medium; removing or cleaving the reversible 3 ’-blocking group with a deblocking agent to form the unblocked extended polynucleotide or extend oligonucleotide attached to the support medium; and separating the unblocked extended polynucleotide or oligonucleotide attached to the support medium from the deblocking agent, wherein each cycle uses a new nucleotide donor.

65. The method of claim 64, wherein the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

66. The method of any one of claims 58-65, further comprising a pyrophosphatase in the reaction with the recombinant primase.

67. The method of any one of claims 46-49, wherein the recombinant primase is immobilized on a support medium, and the polynucleotide acceptor, oligonucleotide acceptor, or initiating nucleotide acceptor reacted with the nucleotide donor is in solution in presence of the immobilized recombinant primase.

68. The method of claim 67, wherein the immobilized recombinant primase is retained in a first chamber.

69. The method of claim 67 or 68, wherein the nucleotide donor comprises a 3 ’-blocking group or the terminating nucleotide donor comprises a 3 ’-blocking group to form a 3 ’-blocked extended polynucleotide or extended oligonucleotide in solution.

70. The method of claim 69, wherein the 3 ’-blocking group comprises a reversible 3’- blocking group.

71. The method of claim 69 or 70, further comprising separating the 3 ’-blocked extended polynucleotide or extended oligonucleotide in solution from the recombinant primase immobilized on the support medium.

72. The method of claim 71, wherein the separating is by removing the 3’-blocked extended polynucleotide or extended oligonucleotide in solution from the first chamber containing the recombinant primase immobilized on the support medium.

73. The method of claim 67 or 72, further comprising removing or cleaving the 3 ’-blocking group with a deblocking agent to form an unblocked extended polynucleotide or oligonucleotide in solution.

74. The method of claim 73, wherein the deblocking with the deblocking agent is carried out in a second chamber.

75. The method of claim 74, further comprising inactivating or separating the deblocking agent from the unblocked extended polynucleotide or oligonucleotide in solution.

76. The method of any one of claims 67- 75, further comprising one or more cycles of: extension with a nucleotide donor; separation of the recombinant primase immobilized on a support medium from the 3 ’-blocked extended polynucleotide or oligonucleotide; removing or cleaving the reversible 3 ’-blocking group with a deblocking agent to form the unblocked extended polynucleotide or extend oligonucleotide; and separating the unblocked extended polynucleotide or oligonucleotide from the deblocking agent, wherein each cycle uses a new nucleotide donor.

77. The method of claim 76, wherein the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor to form an extended polynucleotide or oligonucleotide, wherein at least the extended portion of the polynucleotide or oligonucleotide has a defined nucleotide sequence.

78. The method of any one of claims 67-77, further comprising a pyrophosphatase in the reaction with the recombinant primase.

79. The method of claim 78, wherein the pyrophosphatase is immobilized on a support medium, or wherein the recombinant primase and pyrophosphatase are co-immobilized on a support medium.

80. The method of any one of claims 46-79, wherein the suitable reaction conditions for the reaction with recombinant primase comprises a temperature ranging from about 50 °C to about 95 °C, and / or a pH of about 7-9.

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