Methods of producing nucleotide triphosphates (NTPS)

Abstract

The invention relates to a novel enzymatic process for producing nucleoside triphosphates (NTPs), in particular modified NTPs using polyphosphate kinase 2. The NTPs generated by the methods of the invention may be used in methods of producing polynucleotides, including oligonucleotides, for use in therapy.

Description

[0001] METHODS OF PRODUCING NUCLEOTIDE TRIPHOSPHATES (NTPs)

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a novel enzymatic process for producing nucleoside triphosphates (NTPs), in particular modified NTPs. The NTPs generated by the methods of the invention may be used in methods of producing polynucleotides, including oligonucleotides, for use in therapy.

[0004] BACKGROUND TO THE INVENTION

[0005] Nucleoside triphosphates (NTPs) are the fundamental building blocks for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Naturally occurring NTPs include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP) and uridine triphosphate (UTP). ATP is a major source of cellular energy and GTP is frequently used as a cofactor for enzymes and proteins. Commercial supplies of naturally occurring NTPs are typically obtained using cell fermentation and purification methods.

[0006] NTPs with modifications to the sugar, base and / or phosphate groups are invaluable laboratory tools and are also useful therapeutically, for example in the manufacture of nucleotide- based therapeutics such as therapeutic oligonucleotides and mRNA, since they contribute to enhanced affinity to targets, improved nuclease resistance, altered circulation characteristics and modulation of immunological properties (Kulkarni et al, Nature Nanotechnology, 2021, 16, 630-643).

[0007] Some wild-type polymerases, as well as engineered polymerases, are able to utilise modified NTPs to produce modified polynucleotides complementary to a template sequence (Milisavljevic et al., Org. Biomol. Chem, 2018, 16, 5800-5807; and Sabat et al., Front Chem., 2023, 11, 1161462). In addition, terminal deoxynucleotidyl transferase (TdT) enzymes can utilise modified NTPs and produce modified oligonucleotides in a template independent manner (W02019 / 121500).

[0008] Modified NTPs, however, are difficult and expensive to produce, particularly at large-scale. Synthetic reaction schemes for producing modified NTPs are set out in the 2012 review paper by Marcel Hohenstein (Molecules, 2012, 17, 13569-13591) and include the Yoshikawa Protocol, Ludwig- Eckstein method, the Borch approach, as well as other strategies (see also, Burgess and Cook, Chem. Rev. 2000, 100, 2047-2059).

[0009] More recently biocatalytic methods for specific NTP production have been investigated. MK- 1454 is a cyclic dinucleotide stimulator of interferon genes (STING) agonist that is being developed as an immune-oncology therapeutic. From two non-natural nucleotide monothiophosphates, MK- 1454 has been assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system (McIntosh et al., Nature, 2022, 603, 439-444).

[0010] There is, however, a need for simpler and more cost-effective means of generating NTPs, in particular modified NTPs, that can be used at large-scale, in particular in the manufacture of polynucleotide therapeutics.

[0011] SUMMARY OF THE INVENTION

[0012] In a first aspect of the invention, use of polyphosphate kinase (PPK) to produce sugar- modified and / or o-phosphate-modified nucleoside triphosphate from correspondingly modified nucleoside monophosphate or nucleoside diphosphate, and polyphosphate, is provided.

[0013] In a second aspect of the invention, a method of screening a panel of putative and known PPKs to identify an enzyme capable of using a sugar-modified and / or o-phosphate-modified nucleoside monophosphate or nucleoside diphosphate as a substrate, to produce a correspondingly modified nucleoside triphosphate, is provided. The method comprises: a) identifying sequences encoding said panel of putative and known PPKs; b) synthesizing said PPKs; c) purifying said PPKs; d) mixing each of said purified PPKs with said sugar-modified and / or o-phosphate-modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphate to form a reaction mixture; and e) analysing each of the reaction mixtures for the presence of correspondingly modified nucleoside triphosphate to determine which enzyme(s) produce correspondingly modified nucleoside triphosphates.

[0014] In a third aspect of the invention, an enzymatic method of producing sugar-modified and / or o-phosphate modified nucleoside triphosphates is provided, comprising incubating a PPK with correspondingly modified nucleoside monophosphates or nucleoside diphosphates and polyphosphate under suitable conditions.

[0015] In a fourth aspect of the invention, use of modified nucleoside triphosphates, obtained by the methods of the invention, in the manufacture of a polynucleotide is provided.

[0016] In a fifth aspect of the invention, use of PPK to produce stereospecific o-phosphate-modified nucleoside triphosphate from an o-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, and polyphosphate, is provided.

[0017] In a sixth aspect of the invention, an engineered PPK having at least 90% sequence identity to SEQ ID NO:22 is provided.

[0018] In a seventh aspect of the invention, a method of manufacturing a polynucleotide is provided comprising incubating nucleoside triphosphates, a suitable enzyme, and optionally a template, under suitable conditions, wherein the nucleoside triphosphates are made by incubating the corresponding nucleoside monophosphates and / or nucleoside diphosphates, PPK and polyphosphate under suitable conditions.

[0019] DESCRIPTION OF DRAWINGS / FIGURES

[0020] FIG. 1 is a schematic depicting the preferred catalysed reaction(s) related to ATP generation of PPK2 subfamilies.

[0021] FIG.2 is a chromatogram from Example 5 depicting the 2'OMe-pGMP peak and absence of 2'OMe-pGDP and 2'OMe-pGTP peaks for the negative control sample [top trace]; and a diminished 2'OMe-pGMP peak for the PPK (SEQ ID NO:31) sample, together with two new peaks corresponding to the formation of 2'OMe-pGDP and 2'OMe-pGTP [bottom trace].

[0022] FIG.3 is a chromatogram from Example 12, which illustrates the stereoselective preference of three different PPKs when forming 5'-(o-thio)triphosphate. The PPK of SEQ ID NO:8 [bottom trace] produced approximately 50:50 diastereoisomers. PPKs having SEQ ID NOs:34 and 47 [middle and top traces] had opposite stereoselective preferences with one favouring the SPdiastereoisomer and the other favouring the Rp diastereoisomer.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] DEFINITIONS

[0025] "Ambo" as used herein is a prefix to indicate that a molecule with two (or more) chiral elements is present as a mixture of the two racemic diastereoisomers in unspecified proportions (Pure and Appl. Chem., 68, 12, 2193-2222, 1996). See also 'Nomenclature and Symbolism for Amino Acids and Peptides', Pure Appl. Chem., 56, 595-624,1984; and 'Nomenclature of Tocopherols and Related Compounds', Pure Appl. Chem., 54, 1507-1510, 1982.

[0026] "Derived from" as used herein in the context of enzymes identifies the originating enzyme, and / or the gene encoding such enzyme, upon which engineering was based.

[0027] "Enzymatic synthesis" as used herein in relation to the production of polynucleotides, including oligonucleotides, means using enzymes, e.g. polymerases, ligases, transferases, phosphatases, kinases, including polyphosphate kinases, and nucleases e.g. endonucleases, to make said polynucleotides. These enzymes may be wild-type enzymes, mutant enzymes or engineered enzymes. Within the scope of the present disclosure are mutant enzymes or engineered enzymes capable of acting on modified nucleotide or oligonucleotide substrates. An "enzymatic method" is a method that involves enzymatic synthesis.

[0028] "Engineered enzyme" as used herein refers to an enzyme in which the natural or native form of the enzyme, known as the "wild-type", has been modified in a manner that would not otherwise exist in nature. The modification may comprise, for example, one or more deletions, insertions and / or substitutions of amino acid(s) in the wild-type sequence of the enzyme.

[0029] "Modified nucleotide", "modified nucleoside monophosphate, diphosphate or triphosphate" or "modified polynucleotide or oligonucleotide" as used herein means a nucleotide residue, nucleoside monophosphate / diphosphate / triphosphate, or polynucleotide or oligonucleotide, that contains at least one aspect of its chemistry that differs from a naturally occurring nucleotide residue, nucleoside monophosphate / diphosphate / triphosphate or polynucleotide or oligonucleotide. A modified polynucleotide or oligonucleotide contains at least one modified nucleotide. Modifications can occur in any part of the nucleotide residue, such as modification of the sugar moiety C'sugar- modified"), modification of the nucleobase ("nucleobase-modified"), and / or modification of the phosphate / backbone C'a-phosphate modified). One or more of the same or different modified nucleotide residues can form part of a modified polynucleotide or oligonucleotide.

[0030] The term "messenger RIMA" (mRNA) refers to one particular type of RNA molecule. In vivo, transcription of DNA usually results in the so-called premature RNA which has to be processed into so-called messenger RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g. in eukaryotic organisms, comprises a variety of different post-transcriptional modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of mRNA. The mature mRNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5' cap, a 5' untranslated region (5' UTR), an open reading frame, a 3' untranslated region (3' UTR) and a homopolymeric tail, e.g., a poly-A or a poly-C sequence. In the context of the present disclosure, the mRNA can be an artificial molecule, i.e., a molecule not occurring in nature. This means that the mRNA in the context of the present disclosure may comprise a combination of a 5' UTR, open reading frame, 3' UTR and poly-A sequence, which does not occur in this combination in nature. mRNA in the context of the present disclosure includes at least one modified nucleotide residue.

[0031] "Oligonucleotide", or "oligo" for short, as used herein means a polymer of nucleotide residues. The term "oligonucleotide" is usually used for shorter sequences than the term "polynucleotide", generally in the range of 3 to 30 nucleotides. These may incorporate deoxyribonucleotides (wherein the resulting oligonucleotide is DNA), ribonucleotides (wherein the resulting oligonucleotide is RNA), modified nucleotides, or a mixture thereof. Oligos can be singlestranded, such as antisense molecules, or double-stranded, such as siRNA molecules.

[0032] "Percent identity" or "% identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST / KERR) or software (e.g. DNASTAR Lasergene or GenePAST / KERR). Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence disclosed herein.

[0033] "Percent identity" or"% identity" between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST / KERR) or software (e.g. DNASTAR Lasergene or GenePAST / KERR). Importantly, a query amino acid sequence may be described by an amino acid sequence disclosed herein, in particular in one or more of the claims, for example SEQ ID NO:22.

[0034] The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. In the case of nucleic acid sequences, such alterations include at least one nucleotide residue deletion, substitution or insertion, wherein said alterations may occur at the 5'- or 3'-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the nucleotide residues in the query sequence or in one or more contiguous groups within the query sequence. In the case of amino acid sequences, such alterations include at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acid residues in the query sequence or in one or more contiguous groups within the query sequence.

[0035] "Polynucleotide" as used herein is a polymer of nucleotide residues. A single polynucleotide molecule can, for example, comprise 14 or more nucleotide residues in a chain structure. DNA and RNA are examples of polynucleotides. Polynucleotides include oligonucleotides. Polynucleotides are useful therapeutically, for example, in the production of therapeutic mRNA (which can be used as mRNA vaccines), antisense oligonucleotides, siRNAs, miRNAs, aptamers, CRISPR guide RNAs, and oligonucleotides that recruit and guide DNA and RNA editing enzymes, such as A to I RNA baseediting oligonucleotides (AIMers).

[0036] "Polyphosphate kinase", "PolyP kinase" or"PPK" as used herein is an enzyme capable of producing a nucleoside triphosphate from polyphosphate and a nucleoside monophosphate and / or nucleoside diphosphate. PPK may also be capable of producing a modified nucleoside triphosphate from polyphosphate and a correspondingly modified nucleoside monophosphate and / or diphosphate.

[0037] "Polymerase" as used herein means an enzyme that catalyses the joining, i.e., covalent joining, of a nucleotide to the 3'-OH of another nucleotide or oligonucleotide or polynucleotide e.g. by formation of a phosphodiester bond between the 3' end of one nucleotide or oligonucleotide or polynucleotide and the 5' end of another nucleotide. Accordingly, polymerase activity is 5' to 3'. The polymerase can include DNA and / or RNA polymerases. The polymerase may be a wild-type enzyme, mutant enzyme or engineered enzyme.

[0038] "Primer" as used herein means a polynucleotide or oligonucleotide sequence that is used as a starting point for synthesising a segment polynucleotide or oligonucleotide of the disclosure. A polymerase may require a primer, e.g., DNA polymerase may require a primer. A primer may comprise at least 3 nucleotides. The primer can be bound to the template before the polymerase catalyses the joining of nucleotides. The primer may not be removed. The primer can form part of the polynucleotide or oligonucleotide template. For example, the template may comprise a hairpin loop which comprises a primer. The primer can form part of the polynucleotide or oligonucleotide product.

[0039] "Product polynucleotide or oligonucleotide" as used herein means a desired polynucleotide or oligonucleotide having a specific sequence, also referred to herein as a "target polynucleotide", "target oligonucleotide" or "target oligo".

[0040] "Protecting group" as used herein is a group that is attached to an otherwise reactive 3' hydroxyl position of a nucleoside sugar to prevent unwanted side reactions in a given reaction scheme. A protecting group assists with selectivity of the desired reaction and is removed once the desired reaction has taken place.

[0041] "RNA" is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer consisting of nucleotide monomers. These nucleotides are usually adenosinemonophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers or analogues thereof, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e., ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e., the order of the bases linked to the sugar / phosphate-backbone, is called the RNA-sequence. The term "RIMA" generally refers to a molecule or to a molecular species selected from the group consisting of long-chain RNA, coding RNA, non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNAs, riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and a Piwi-interacting RNA (piRNA). Optionally in the context of the disclosure is any type of therapeutic RNA.

[0042] "Stopper" as used herein, also known as "block" or "3'-flanking oligonucleotide" means a polynucleotide or oligonucleotide sequence that stops or prevents polymerase from joining further nucleotides. A stopper may terminate polymerase extension. The stopper may comprise a 5' phosphate, 5' thiophosphate (which may result in a phosphorothioate bond), 5' amidophosphate (which may result in a phosphoramidate bond), 5' diamidophosphate (which may result in a phosphorodiamidate bond), 5' amidothiophosphate, 5' amidodithiophosphate, 5' diamidothiophosphate or 5' dithiophosphate (which may result in a phosphorod ith ioate bond).

[0043] "Template" as used herein means a polynucleotide or oligonucleotide that comprises a sequence complementary to a single-stranded polynucleotide or oligonucleotide product. The template can comprise a sequence which is 100% complementary to the sequence of the target (or product) polynucleotide or oligonucleotide. The template can consist of a sequence that is 100% complementary to the sequence of the target (or product) polynucleotide or oligonucleotide. The template can be a longer sequence compared to the product sequence. The template can comprise sequences which are not used to produce the polynucleotide or oligonucleotide product. When the template is longer in sequence compared to the product sequence a stopper can be used to control production and / or length of the polynucleotide or oligonucleotide product. When the template is longer in sequence compared to the product sequence a primer can be used to control production and / or length of the polynucleotide or oligonucleotide product. When the template is longer in sequence compared to the product sequence it may comprise a hairpin loop. The hairpin loop may act as a primer. The template may comprise a segment of the product in a hairpin loop. In such instances, the product may be released from the template via cleavage with a nuclease, a nickase, a DNAzyme, or chemical methods. Unless otherwise specified, as used herein, the term "complementary" means 100% complementary.

[0044] "Therapeutic oligonucleotide" or "therapeutic oligo" as used herein means an oligonucleotide that has a therapeutic application, e.g., in the prevention or treatment of a condition or disease in a human or animal. Such an oligo typically contains one or more modified nucleotide residues or linkages. Therapeutic oligos act via one of several different mechanisms, including, but not limited to, antisense, splice-switching or exon-skipping, immunostimulation and RIMA interference (RNAi), e.g., via microRNA (miRNA) or small interfering RNA (siRNA), and recruitment and guiding of DNA and RNA editing enzymes, for example an A to I RNA base-editing oligonucleotide (AIMer). A therapeutic oligo may be an aptamer. Therapeutic oligonucleotides will usually, but not always, have a defined sequence. A therapeutic oligonucleotide is an example of a therapeutic polynucleotide.

[0045] "Therapeutic polynucleotide" as used herein means a polynucleotide that has a therapeutic application, e.g., in the prevention or treatment of a condition or disease in a human or animal. Such a polynucleotide typically contains one or more modified nucleotide residues or linkages. Therapeutic polynucleotides act via one of several different mechanisms, including, but not limited to, antisense, splice-switching or exon-skipping, immunostimulation, RNA interference (RNAi), e.g., miRNA or siRNA, and recruitment and guiding of DNA and RNA editing enzymes, for example an A to I RNA base-editing oligonucleotide (AIMer). An aptamer is an example of a therapeutic polynucleotide. An mRNA vaccine is also an example of a therapeutic polynucleotide. Therapeutic polynucleotides will usually, but not always, have a defined sequence. Therapeutic polynucleotides include therapeutic oligonucleotides and therapeutic RNA.

[0046] "Therapeutic RNA" is RNA that has a therapeutic application, e.g., in the prevention or treatment of a condition or disease in a human or animal. Such RNA is suitable for use in the human or animal body for a medical purpose, e.g., it has a clinical grade, particularly when it comes to parameters such as purity, integrity, as well as concerning the underlying production methods that must comply with current good manufacturing practice (cGMP) conditions.

[0047] "Transferase" as used herein means an enzyme that catalyses template independent joining of one nucleotide to another nucleotide or oligonucleotide. A transferase as described herein includes a terminal nucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase. TdT is a specialised DNA polymerase that is expressed in immature, pre-B, pre-T-lymphoid cells where it enables V-D-J antibody gene junctional diversity. TdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. A transferase as described herein includes a non-naturally occurring or mutant TdT. Within the scope of the disclosure is a transferase capable of joining an unmodified nucleotide to an unmodified oligonucleotide, a transferase capable of joining an unmodified nucleotide to a modified oligonucleotide, a transferase capable of joining a modified nucleotide to an unmodified oligonucleotide, as well as a transferase capable of joining a modified nucleotide to a modified oligonucleotide. "Phosphoramidate (PN)" refers generally to a group having the structure -P(O)2NRaRb, wherein Ra and Rb are independent substituents, or together represent a double bond to an atom, e.g., carbon. For example PN may refer to PO2N(R3)2or PO2R4as defined herein.

[0048] "The term "pharmaceutically acceptable" refers to those compounds (including salts), materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0049] STATEMENT OF THE INVENTION

[0050] The inventors have surprisingly found that polyphosphate kinases (PPKs) are suitable and effective enzymes for producing modified nucleoside triphosphates (NTPs). NTPs produced by the methods of the disclosure provide a simpler and more cost-effective alternative to state-of-the-art methods. In particular, the inventors have used PPKs to produce sugar-modified and / or o- phosphate-modified nucleoside triphosphates from correspondingly modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphate.

[0051] Inorganic polyphosphate, otherwise known as polyphosphate or polyP, is a polymer, usually linear, of tens to hundreds of phosphate residues (Pi) linked by high-energy phosphoanhydride bonds, such as those in adenosine triphosphate (ATP). However, unlike ATP or other phosphate sources currently used in NTP production, polyP is a much more stable form of phosphate. It has been proposed that polyP served as an energy source in a pre-ATP world and was initially utilized by early single celled organisms. PolyP has been found in all living organisms from bacteria to higher eukaryotes and has numerous biological functions: as well as serving as a means of storing energy, it is also a reservoir for Pi, a chelator of metal ions, a channel for DNA entry, a regulator of stress and survival, and a supportive component in gene regulation and enzyme function (Motomura et al, Applied and Environmental Microbiology, 2014, 80(8), 2602-2608).

[0052] PPKs have traditionally been divided into two families, PPK1 and PPK2, due to differences in amino acid sequence and kinetic properties. PPK1 preferentially catalyses ATP-driven polyP formation, whereas PPK2 preferentially catalyses polyP-driven nucleotide phosphorylation, which is important for survival of microbial cells under conditions of stress or pathogenesis. The PPK2 family is further divided into the following subfamilies: class I - single-domain enzymes catalysing phosphorylation of nucleoside diphosphates; class II - mainly two-domain enzymes catalysing phosphorylation of nucleoside monophosphates; and class III - single-domain enzymes catalysing both nucleoside monophosphate and nucleoside diphosphate phosphorylation (see Figure 1).

[0053] Known PPKs can be found in INTERPRO (EMBL-EBI) entry IPR022488, and include those characterised by Nocek et al (ACS Catal. 2018, 8, 11, 10746-10760).

[0054] Putative PPKs may be identified by mining sequence databases. Sequence databases include Universal Protein Resource (UniProt), UniParc, European Nucleotide Archive, GenBank, Reference Sequence Database (RefSeq), Protein Information Resource (PIR), Protein Data Bank (PDB), Pfam, and InterPro. Suitable bioinformatic tools include Basic Local Alignment Search Tool (BLAST), Clustal Omega, ClustalW, HMMER, InterProScan, UniProt Tools, CD-HIT, MMseqs2, and CLANS.

[0055] Recently, PPKs have been utilized as ATP recycling systems in the industrial implementation of ATP-dependent enzymes, thus avoiding the addition of stoichiometric quantities of cofactor (Tavanti et al, Green Chemistry, 2021, 23, 828-837).

[0056] In the present disclosure, PPKs may be used to produce a solely sugar-modified nucleoside triphosphate with no modifications to the base or o-phosphate, i.e., the base is a naturally occurring base and the o-phosphate has no modifications. The solely sugar-modified nucleoside triphosphate may comprise a 2'-sugar modification, a 3'-sugar modification, or both a 2' and a 3' sugar modification. Alternatively, PPKs may be used to produce a solely o-phosphate-modified nucleoside triphosphate with no modifications to the base or sugar, i.e., the base is a naturally occurring base and the sugar is naturally occurring, e.g., ribose or deoxyribose. PPKs may also be used to produce a sugar-modified and / or o-phosphate-modified nucleoside triphosphate, which additionally has a modified base, i.e., the base is not naturally occurring.

[0057] In an aspect of the disclosure, there is provided a method of producing a compound of formula (II): or salt thereof, or pharmaceutically acceptable salt thereof; by reacting a compound of formula (I): or salt thereof, or pharmaceutically acceptable salt thereof; with PPK and polyphosphate, wherein:

[0058] Xi is selected from the group consisting of: OH, SH, CH2, NR32, CH3, BHs", Se and R4;

[0059] X2 is 0 or S;

[0060] R1is selected from the group consisting of: H, PO3, PO2S, PO2N(R3)2, PO2R4, POS2, PO2CH3, PO2BH3, PO2Se, N-methyl-anthraniloyl (MANT), acetyl (Ac), benzyl (Bn), benzoyl (Bz), allyl (All), 2- cyanoethyl (CE), azidomethyl (AzM), cyanovinyl (Cv), methoxymethyl (MOM), methoxyethoxymethyl (MEM), methoxymethyl phenyl methyl (MPM), diphenylmethyl (DPM), tetrahydropyranyl (THP), dimethoxytrityl (DMTr), methoxycarbonylmethyl (MOMcl), ethoxycarbonyl (EOC), benzyl carbonyl (Cbz), trifluoromethanesulfonate (triflate), methanesulfonate (mesylate), p-toluenesulfonate (tosylate), pivaloyl (Piv), ortho-nitrobenzyl (o-NB), bromo-methyl-coumarinylmethyl (Bhc), (7- (Diethylamino)coumarin-4-yl)methyl (DEACM), 6-Nitroveratryloxycarbonyl (NVOC), and 6-Bromo-7- hydroxycoumarin-4-ylmethyl (Bhc-ol);

[0061] R2is selected from the group consisting of: H, OH, O-CH3 (OMe), O-CH2-CH3 (Oet), O-CH2- CH2-CH3, O-CH2-CH2-O-CH3 (MOE), F, O-CH2-CO-NH-CH3, O-CH2-CH2-O-Nme2, O-methylene bridge with the 4' carbon, and O-constrained ethylene bridge with the 4' carbon;

[0062] R3is independently H or R4is selected from the group consisting of: and the Base is a naturally occurring base or a modified base; wherein Xi is not 0- if X2 is 0, Ri is H and R2 is H or OH.

[0063] As would be understood by the skilled person, in Formula (I) and (II), the OH groups may exist as O’, and the SH groups may exist as S’ depending on the conditions. Thus, in Formula (I) and (II), where relevant, OH and O’ are used interchangeably and SH and S’ are used interchangeably.

[0064] In an embodiment, the base is selected from the group consisting of: adenine (A), uracil (U), cytosine (C), guanine (G), thymine (T), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), 5-fluorocytosine (5fC), 5-Bromouracil (5BrU), 2-aminopurine (2AP), inosine (I), pseudouracil (I1), Nl-methylpseudouracil (mil1), 1-ethylpseudouracil, 2-thio-l-methyl-l-deaza-pseudouracil, 2-thio-l- methyl-pseudouracil, 2-thio-5-aza-uracil, 2-thio-dihydropseudouracil, 2-thio-dihydrouracil, 2-thio-pseudouriacil, 4-methoxy-2-thio-pseudouracil, 4-methoxy-pseudouracil, 4-thio-l-methyl- pseudouracil, 4-thio-pseudouracil, 5-aza-uracil, dihydropseudouracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-0-methyl uracil (2moll), 7-deazaguanine (7-deaza-G), 8-oxoguanine (8-oxo-G), 5- propynyluracil (pU), 5-propynylcytosine (pC), N6-methyladenine (m6A), N4-acetylcytosine (ac4C), 06-methylguanine (06meG), hypoxanthine (H), xanthine (X), isocytosine (iC), isoguanine (iG), queuine (Q), 2-thiouracil (s2U), 4-thiouracil (s4U), 5-iodouracil (5Iu), 2-thiocytosine (s2C), 5- chlorouracil (5CIU) and isouracil.

[0065] Xi is selected from the group consisting of: OH, SH, CH2, NR32, CH3, BHs’, Se and R4. As would be understood by the skilled person, if Xi is OH, it may be OH or O’ depending on the conditions, thus OH and O’ are used interchangeably herein. Xi may be SH or S’ depending on the conditions, thus SH and S’ are used interchangeably herein. If Xi is Se, it may be Se- depending on the conditions, thus Se is interchangeable with Se’ herein.

[0066] In an embodiment, Xi is selected from the group consisting of: OH, SH, and N; X2 is 0 or S; R1is selected from the group consisting of: H, PO3, PO2S, PO2NR32, PO2R4, POS2, MANT, Ac, Bz, Bn, DMTr and Cbz; R2is selected from the group consisting of: H, OH, OMe, MOE, OEt, and F; and the base is selected from the group consisting of: A, U, C, G, T, 5mC, I, 4J, and mil1. In an embodiment, Xi is selected from the group consisting of: OH, SH, NR32 or R4; X2 is 0 or S; R1is selected from the group consisting of: H, PO3, PO2S, and MANT; R2is selected from the group consisting of: H, OH, OMe, MOE, and F; and the base is selected from the group consisting of: A, U, C, G, T, 5mC, I, IP, and mBP.

[0067] In an embodiment, Xi is OH. In an embodiment, Xi is SH. In an embodiment, Xi is NR32. In an embodiment, Xi is R4.

[0068] In an embodiment, X2 is 0. In an embodiment, X2 is S.

[0069] In an embodiment, R1is H. In an embodiment, R1is PO3. In an embodiment, R1is MANT.

[0070] In an embodiment, R2is H. In an embodiment, R2is OH. In an embodiment, R2is OMe. In an embodiment, R2is MOE. In an embodiment, R2is F.

[0071] In an embodiment, the base is A. In an embodiment, the base is U. In an embodiment, the base is C. In an embodiment, the base is G. In an embodiment, the base is T. In an embodiment, the base is 5mC. In an embodiment, the base is I. In an embodiment, the base is 1J. In an embodiment, the base is mil1.

[0072] In an embodiment, Xi is OH, X2 is 0, R1is PO3 and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is OH, X2 is 0, R1is MANT and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is OH, X2 is 0, R1is H and R2is OMe, MOE, or F. In an embodiment, Xi is SH, X2 is 0, R1is PO3 and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is SH, X2 is 0, R1is MANT and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is SH, X2 is 0, R1is H and R2is H, OH, OMe, MOE, or F.

[0073] In an embodiment, Xi is OH, X2 is S, R1is PO3 and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is OH, X2 is S, R1is MANT and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is OH, X2 is S, R1is H and R2is OMe, MOE, or F. In an embodiment, Xi is SH, X2 is S, R1is PO3 and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is SH, X2 is S, R1is MANT and R2is H, OH, OMe, MOE, or F. In an embodiment, Xi is SH, X2 is S, R1is H and R2is H, OH, OMe, MOE, or F.

[0074] In an embodiment, Xi is OH, X2 is 0, R1is PO3, and R2is OMe. In an embodiment, Xi is OH, X2 is 0, R1is PO3, and R2is MOE. In an embodiment, Xi is OH, X2 is 0, R1is PO3, and R2is F. In an embodiment, Xi is OH, X2 is S, R1is PO3, and R2is OMe. In an embodiment, Xi is OH, X2 is S, R1is PO3, and R2is MOE. In an embodiment, Xi is OH, X2 is S, R1is PO3, and R2is F.

[0075] In an embodiment, Xi is OH, X2 is 0, R1is MANT, and R2is OMe. In an embodiment, Xi is OH, X2 is 0, R1is MANT, and R2is MOE. In an embodiment, Xi is OH, X2 is 0, R1is MANT, and R2is F. In an embodiment, Xi is OH, X2 is S, R1is MANT, and R2is OMe. In an embodiment, Xi is OH, X2 is S, R1is MANT, and R2is MOE. In an embodiment, Xi is OH, X2 is S, R1is MANT, and R2is F.

[0076] In an embodiment, Xi is OH, X2 is 0, R1is H, and R2is OMe. In an embodiment, Xi is OH, X2 is 0, R1is H, and R2is MOE. In an embodiment, Xi is OH, X2 is 0, R1is H, and R2is F. In an embodiment, Xi is OH, X2 is S, R1is H, and R2is OMe. In an embodiment, Xi is OH, X2 is S, R1is H, and R2is MOE. In an embodiment, Xi is OH, X2 is S, R1is H, and R2is F.

[0077] In an embodiment, Xi is SH, X2 is 0, R1is PO3, and R2is H. In an embodiment, Xi is SH, X2 is 0, R1is PO3, and R2is OH. In an embodiment, Xi is SH, X2 is 0, R1is PO3, and R2is OMe. In an embodiment, Xi is SH, X2 is 0, R1is PO3, and R2is MOE. In an embodiment, Xi is SH, X2 is 0, R1is PO3, and R2is F. In an embodiment, Xi is SH, X2 is S, R1is PO3, and R2is H. In an embodiment, Xi is SH, X2 is S, R1is PO3, and R2is OH. In an embodiment, Xi is SH, X2 is S, R1is PO3, and R2is OMe. In an embodiment, Xi is SH, X2 is S, R1is PO3, and R2is MOE. In an embodiment, Xi is SH, X2 is S, R1is PO3, and R2is F.

[0078] In an embodiment, Xi is SH, X2 is 0, R1is MANT, and R2is H. In an embodiment, Xi is SH, X2 is 0, R1is MANT, and R2is OH. In an embodiment, Xi is SH, X2 is 0, R1is MANT, and R2is OMe. In an embodiment, Xi is SH, X2 is 0, R1is MANT, and R2is MOE. In an embodiment, Xi is SH, X2 is 0, R1is MANT, and R2is F. In an embodiment, Xi is SH, X2 is S, R1is MANT, and R2is H. In an embodiment, Xi is SH, X2 is S, R1is MANT, and R2is OH. In an embodiment, Xi is SH, X2 is S, R1is MANT, and R2is OMe. In an embodiment, Xi is SH, X2 is S, R1is MANT, and R2is MOE. In an embodiment, Xi is SH, X2 is S, R1is MANT, and R2is F.

[0079] In an embodiment, Xi is SH, X2 is 0, R1is H, and R2is H. In an embodiment, Xi is SH, X2 is 0, R1is H, and R2is OH. In an embodiment, Xi is SH, X2 is 0, R1is H, and R2is OMe. In an embodiment, Xi is SH, X2 is 0, R1is H, and R2is MOE. In an embodiment, Xi is SH, X2 is 0, R1is H, and R2is F. In an embodiment, Xi is SH, X2 is S, R1is H, and R2is H. In an embodiment, Xi is SH, X2 is S, R1is H, and R2is OH. In an embodiment, Xi is SH, X2 is S, R1is H, and R2is OMe. In an embodiment, Xi is SH, X2 is S, R1is H, and R2is MOE. In an embodiment, Xi is SH, X2 is S, R1is H, and R2is F.

[0080] In an embodiment, when R2is OH, it is ribose. In an embodiment, when R2is OH, it is 2'- arabinose (ANA).

[0081] In an embodiment, when R2is H, it is 2'-deoxyribose.

[0082] In an embodiment, when R2is F, it is 2'-deoxy-2'-fluororibose (FNA). In an embodiment, when R2is F, it is 2'-deoxy-2'-fluoro-beta-D-arabinose (FANA).

[0083] In an embodiment of the disclosure, a PPK may utilise one or more of the following substrates: locked nucleic acid (LNA), constrained ethyl (cEt) and 1,5 dianhydrohexitol (HNA) nucleosides.

[0084] In an embodiment, the nucleoside triphosphate produced by the PPK-catalysed reaction is any one of the following:

[0085]

[0086] wherein PN-1 = or salt thereof, or pharmaceutically acceptable salt thereof.

[0087] In an aspect of the disclosure, use of a PPK to produce a sugar-modified and / or o- phosphate-modified nucleoside triphosphate from a sugar-modified and / or an o-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, and polyphosphate is provided.

[0088] In an embodiment, the PPK is a PPK2. In an embodiment, the PPK2 is a PPK2-III.

[0089] In an embodiment, the PPK comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:22. In an embodiment, the PPK consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:22. In an embodiment, the PPK comprises SEQ ID NO:22. In an embodiment, the PPK is SEQ ID NO:22.

[0090] In an embodiment, the PPK is obtained by a screening method disclosed herein.

[0091] In an aspect of the disclosure, an enzymatic method of producing sugar-modified and / or o- phosphate modified nucleoside triphosphates, comprising incubating a PPK with correspondingly modified nucleoside monophosphates or nucleoside diphosphates and polyphosphate under suitable conditions, is provided.

[0092] In an embodiment, the incubating is with a divalent cation. In an embodiment, the divalent cation is Mg2+, Mn2+, Cd2+or Co2+. In an embodiment, the divalent cation is Mg2+. In an embodiment, the divalent cation is Mn2+. In an embodiment, the divalent cation is Cd2+. In an embodiment, the divalent cation is Co2+.

[0093] In an embodiment, the incubating is with magnesium chloride, manganese chloride, cadmium chloride, cobalt chloride, magnesium acetate, manganese acetate, cadmium acetate, or cobalt acetate. In an embodiment, the incubating is with magnesium chloride, manganese chloride, magnesium acetate, or manganese acetate. In an embodiment, the incubating is with magnesium chloride or magnesium acetate. In an embodiment, the incubating is with magnesium chloride. In an embodiment, the incubating is with manganese chloride or manganese acetate. In an embodiment, the incubating is with manganese chloride.

[0094] Sugar modifications include modification at the 2' and 3' positions. 3'-sugar modifications may act as protecting groups in certain reactions.

[0095] 2'-sugar modifications include phosphate (PO), O-methyl (OMe), O-methoxyethyl (MOE), fluoro (FNA), O-ethyl (OEt), O-propyl (OProp), 2'-arabino (ANA), 2'-deoxy-2'-fluoro-beta-D-arabino (FANA), O-[2-(methylamino)-2-oxoethyl] (NMA), and 2'-O-[2-{(N,N-dimethylamino)oxy}-ethyl] (DMAOE). In an embodiment, the 2'-sugar modification is selected from the group consisting of: OMe, MOE, FNA, OEt, and FANA. In an embodiment, the 2'-sugar modification is selected from the group consisting of: OMe, MOE, and FNA. In an embodiment, the 2'-sugar modification is OMe. In an embodiment, the 2'-sugar modification is MOE. In an embodiment, the 2'-sugar modification is FNA. In an embodiment, the 2'-sugar modification is FANA. In an embodiment, the 2'-sugar modification is OEt.

[0096] In an embodiment, the 2'-sugar modification is a bridge between the 2' oxygen and 4' carbon sugar (ribose) positions. In an embodiment, the bridge is a methylene bridge, i.e. a locked nucleic acid sugar. In an embodiment, the bridge is a constrained ethyl bridge.

[0097] 3'-sugar modifications include H, PO, phosphoroth ioate (PS), thiophosphate, phosphoramidate (PN), phosphorodithioate, methyl phosphonate, boranophosphate, phosphoroselenoate, MANT, Ac, Bn, Bz, All, CE, AzM, Cv, MOM, MEM, MPM, DPM, THP, DMTr, MOMcl, EOC, Cbz, triflate, mesylate, tosylate, Piv, o-NB, Bhc, DEACM, NVOC, and Bhc-ol. In an embodiment, the 3'-sugar modification is selected from the group consisting of: H, PO, PS, PN, phosphorodithioate, MANT, Ac, Bn, Bz, and Cbz. In an embodiment, the 3'-sugar modification is selected from the group consisting of: H, PO, PS, PN, phosphorodithioate, and MANT. In an embodiment, the 3'-sugar modification is selected from the group consisting of: H, PO, PS, and MANT. In an embodiment, the 3'-sugar modification is PO. In an embodiment, the 3'-sugar modification is MANT. In an embodiment, the 3'-sugar modification is H. When the 3'-sugar modification is PO and acting as a protecting group, a phosphatase may be used to remove the PO protecting group.

[0098] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-sugar modification. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-0Me, 2'-MOE or 2'-FNA. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) does not comprise a 2'-sugar modification, i.e., the 2' position is H or OH. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-H. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-OH.

[0099] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 3'-sugar modification. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 3'-sugar modification and a 2'-H. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 3'-sugar modification and a 2'-OH. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 3'-PO, 3'-PS, 3'-MANT or 3'-hydrogen. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 3'-PO, 3'-PS, 3'-MANT or 3'-hydrogen; and a 2'-H. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 3'-PO, 3'-PS, 3'-MANT or 3'- hydrogen; and a 2'-OH. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) does not comprise a 3'-sugar modification, i.e., the 3' position is OH.

[0100] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-sugar modification and a 3'- sugar modification. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-H, 2'- OH, 2'-0Me, 2'-MOE or 2'-FNA, and a 3'-PO, 3'-PS, 3'-MANT or 3'-hydrogen.

[0101] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-0Me and a 3'-PO. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-0Me and a 3'-PS. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-0Me and a 3'-MANT. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-0Me and a 3'-hydrogen.

[0102] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-MOE and a 3'-PO. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-MOE and a 3'-PS. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-MOE and a 3'-MANT. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-MOE and a 3'-hydrogen.

[0103] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-FNA and a 3'-PO. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-FNA and a 3'-PS. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-FNA and a 3'-MANT. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-FNA and a 3'-hydrogen.

[0104] In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an o-phosphate. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a naturally occurring base. In an embodiment, the sugar-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an o-phosphate and a naturally occurring base.

[0105] In an embodiment, any one or more of the aforementioned sugar modifications may be combined with an o-phosphate modification and / or a base modification. o-phosphate modifications include PS, PN, methyl phosphonate, boranophosphate, and phosphoroselenoate. In an embodiment, the o-phosphate modification is PS, for example a compound of formula (Ila) below: or salt thereof, or pharmaceutically acceptable salt thereof; wherein X2, R1, R2and Base are as described above.

[0106] In an embodiment, the o-phosphate modification is PN, for example a compound of formula (lib) or (lie) below: or salt thereof, or pharmaceutically acceptable salt thereof; wherein X2, R1, R2, R3, R4and Base are as described above.

[0107] It is to be understood that the references herein to a compound of Formula (I) or (II) or a salt thereof includes a compound of the Formula as a free acid, or as a salt thereof, for example as a pharmaceutically acceptable salt thereof. Because of its potential use in medicine, it will be appreciated that a salt of a compound of Formula (I) and (II) is preferably pharmaceutically acceptable. Pharmaceutically acceptable salts include, amongst others, those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those listed in P H Stahl and C G Wermuth, editors, Handbook of Pharmaceutical Salts; Properties, Selection and Use, Second Edition Stahl / Wermuth: Wiley- VCH / VHCA, 2011. Examples of pharmaceutically acceptable base addition salts of compounds of Formula (I) and (II) include sodium and lithium. Non-pharmaceutically acceptable salts are also within the scope of the present invention.

[0108] In an embodiment, the o-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-H. In an embodiment, the o-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a 2'-OH. In an embodiment, the o-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises ribose or deoxyribose.

[0109] In an embodiment, the o-phosphate-modified nucleoside triphosphate comprises a modified sugar. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) also comprises a sugar modification. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) also comprises a 2'- sugar modification. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) also comprises a 3'- sugar modification. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) also comprises a 2'- sugar modification and a 3'-sugar modification.

[0110] In an embodiment, the a-phosphate-modified nucleoside triphosphate comprises an a- thiophosphate and also comprises a sugar modification. In an embodiment, the a-phosphate- modified nucleoside triphosphate comprises an a-thiophosphate and also comprises a 2'-sugar modification. In an embodiment, the a-phosphate-modified nucleoside triphosphate comprises an a- thiophosphate and also comprises a 3'-sugar modification. In an embodiment, the a-phosphate- modified nucleoside triphosphate comprises an a-thiophosphate and also comprises a 2'-sugar modification and a 3'-sugar modification.

[0111] In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate and a 2'-0Me. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate and a 2'-MOE. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate and a 2'-FNA.

[0112] In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate and a 3'-PO. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate and a 3'-PS. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate and a 3'-MANT. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a- thiophosphate and a 3'-hydrogen.

[0113] In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'- OMe, and a 3'-PO. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an o- thiophosphate, a 2'-0Me, and a 3'-PS. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an o-thiophosphate, a 2'-0Me, and a 3'-MANT. In an embodiment, the a-phosphate- modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'-0Me, and a 3'-hydrogen.

[0114] In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'- MOE, and a 3'-PO. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a- thiophosphate, a 2'-MOE, and a 3'-PS. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'-MOE, and a 3'-MANT. In an embodiment, the a-phosphate- modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'-MOE, and a 3'-hydrogen.

[0115] In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'- FNA, and a 3'-PO. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a- thiophosphate, a 2'-FNA, and a 3'-PS. In an embodiment, the a-phosphate-modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'-FNA, and a 3'-MANT. In an embodiment, the a-phosphate- modified nucleoside triphosphate (and corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises an a-thiophosphate, a 2'-FNA, and a 3'-hydrogen.

[0116] NTPs comprising a-phosphate modifications have phosphorus chiral centres. For example, the formation of a phosphoanhydride bond to an a-thiophosphate creates a chiral centre at the a- thiophosphate phosphorus resulting in two diastereomers: and SPdiastereomer and an RPdiastereomer.

[0117] As will be apparent from the examples herein (see, for example, Fig. 3), different wild-type PPKs exert different control over stereochemistry under the same reaction conditions. The diastereomeric ratio generated by a PPK may be 1, indicating no stereopreference for phosphorylation, i.e. a 50:50 mixture of both diastereomers (S>. ^). A different PPK may have a preference for generating the SPdiastereomer, whereas another PPK may have a preference for generating the Rp diastereomer.

[0118] Stereocontrol may also be manipulable by changing the divalent cation.

[0119] In an aspect of the disclosure, use of PPK to produce stereospecific o-phosphate-modified nucleoside triphosphate from o-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, and polyphosphate, is provided.

[0120] In an embodiment, the PPK produces about a 50:50 ratio of the Sp diastereomer to the RPdiastereomer. In an embodiment, the PPK produces about a 55:45 ratio of the RPdiastereomer to the 5^ diastereomer. In an embodiment, the PPK produces about a 55:45 ratio of the 5^ diastereomer to the RPdiastereomer.

[0121] In an embodiment, the PPK produces about a 55:45, 60:40, 65:35, 70:30, 75:25 or 80:20 ratio of the Sp diastereomer to the RPdiastereomer.

[0122] In an embodiment, the PPK produces about a 55:45, 60:40, 65:35, 70:30, 75:25 or 80:20 ratio of the RPdiastereomer to the Sp diastereomer.

[0123] In an embodiment, the PPK produces substantially all 5^ diastereomer. In an embodiment, S> / ^ is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2 or 99:1. In an embodiment, S>:RPis greater than 95:5. In an embodiment, S>:Rp is greater than 99:1.

[0124] In an embodiment, the PPK produced substantially all Rp diastereomer. In an embodiment, ^ / S> is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2 or 99:1. In an embodiment, p.’Sp is greater than 95:5. In an embodiment, Rp:S> is greater than 99:1.

[0125] In an embodiment, the stereospecific o-phosphate-modified nucleoside triphosphate is a stereospecific PS-modified nucleoside triphosphate.

[0126] In an embodiment, the stereospecific PS-modified nucleoside triphosphate is a nucleoside 5'- ( 3>)-[o-thio]triphosphate. In an embodiment, the stereospecific PS-modified nucleoside triphosphate is a nucleoside 5'-(S>)-[o-thio]triphosphate.

[0127] In an embodiment, sugar-modified and / or o-phosphate-modified triphosphate comprises a naturally occurring base. In an embodiment, sugar-modified and / or o-phosphate-modified nucleoside triphosphate also comprises a base modification.

[0128] In an embodiment, the base is selected from the group consisting of: A, U, C, G, T, m5C, hm5C, 5fC, 5BrU, 2AP, I, ip, mil1, 1-ethylpseudouracil, 2-thio-l-methyl-l-deaza-pseudouracil, 2- thio-l-methyl-pseudouracil, 2-thio-5-aza-uracil, 2-thio-dihydropseudouracil, 2-thio-dihydrouracil, 2-thio-pseudouriacil, 4-methoxy-2-thio-pseudouracil, 4-methoxy-pseudouracil, 4-thio-l-methyl- pseudouracil, 4-thio-pseudouracil, 5-aza-uracil, dihydropseudouracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-0-methyl uracil, 7-deaza-G, 8-oxo-G, pU, pC, m6A, ac4C, 06meG, H, X, iC, iG, Q, s2U, s4U, 5Iu, s2C, 5CIU and isouracil.

[0129] In an embodiment, the base is selected from the group consisting of: A, U, C, G, T, m5C, hm5C, 5fC, 5BrU, 2AP, I, ip, ml , 7-deaza-G, 8-oxo-G, pU, pC, m6A, ac4C, 06meG, H, X, iC, iG, Q, s2U, 5Iu, s2C and 5CIU.

[0130] In an embodiment, the base is selected from the group consisting of: A, U, C, G, T, m5C and I. In an embodiment, the base is selected from the group consisting of: A, U, C, G, and T. In an embodiment, the base is selected from the group consisting of: A, U, C, G, and m5C. In an embodiment, the base is selected from the group consisting of: A, U, C, and G.

[0131] In an embodiment, the base is A. In an embodiment, the base is U. In an embodiment, the base is C. In an embodiment, the base is G. In an embodiment, the base is T. In an embodiment, the base is 5mC. In an embodiment, the base is I. In an embodiment, the base is 1J. In an embodiment, the base is mil1.

[0132] In an embodiment, the sugar-modified nucleoside triphosphate comprises a 2'-sugar modification and a base modification. In an embodiment, the sugar-modified nucleoside triphosphate comprises a 3'-sugar modification and a base modification. In an embodiment, the sugar-modified nucleoside triphosphate comprises a 2'-sugar modification, a 3'-sugar modification, and a base modification. In an embodiment, the sugar-modified nucleoside triphosphate comprises a 2'-sugar modification, a 3'-sugar modification, a base modification, and an o-phosphate modification.

[0133] In an embodiment, the o-phosphate-modified nucleoside triphosphate also comprises a base modification. In an embodiment, the o-phosphate-modified nucleoside triphosphate also comprises a base modification and a 2'-sugar modification. In an embodiment, the o-phosphate-modified nucleoside triphosphate also comprises a base modification and a 3'-sugar modification. In an embodiment, the o-phosphate-modified nucleoside triphosphate also comprises a base modification, a 2'-sugar modification and a 3'-sugar modification.

[0134] Nucleoside triphosphates that may be produced by the methods of the disclosure include: modified deoxyadenosine triphosphate, modified deoxycytidine triphosphate, modified deoxyguanosine triphosphate, modified deoxythymidine triphosphate, modified deoxyuridine triphosphate, modified adenosine triphosphate, modified cytidine triphosphate, modified guanosine triphosphate, modified thymidine triphosphate, modified uridine triphosphate, 2'-deoxyadenosine-5'- (o-thio)-triphosphate, 2'-deoxycytidine-5'-(o-thio)-triphosphate, 2'-deoxyguanosine-5'-(o-thio)- triphosphate, 2'-deoxythymidine-(o-thio)-triphosphate, 2'-deoxyuridine-(o-thio)-triphosphate, 2'- adenosine-5'-(o-thio)-triphosphate, 2'-cytidine-5'-(o-thio)-triphosphate, 2'-guanosine-5'-(o-thio)- triphosphate, 2'-thymidine-(o-thio)-triphosphate and 2'-uridine-(o-thio)-triphosphate. In an embodiment, the nucleoside triphosphate produced by the methods of the disclosure is selected from the group consisting of: 2'-OMe-,3'-PO3-CTP; 2'-OMe-,3'-PO3-CTP-o-PN-l (S>); 2'- OMe-,3'-PO3-CTP-a-S (RP) 2'-{R )-F-,3'-PO3-ATP-a-S ( / 3>); 2'-{R )-F-,3'-PO3-GTP-a-S ( / 3>); 2'-MOE-, UTP-a-PN-1 (S>); 2'-OMe-,3'-PC>3-CTP-a-S (ambo); 2'-OMe-,3'-PO3-GTP; 2'-OMe-,3'-PO3-UTP; 2'- OMe-,3'-PO3-ATP; 2'-(A )-F-,3'-PO3-GTP; 2'-(A )-F-,3'-PO3-CTP; 2'-OMe-,3'-PO3-UTP-a-S (ambo); 2'- (A)-F-,3'-PO3-CTP-a-S (ambo); 2'-OMe-,3'-PO3-ATP-o-S (ambo); 2'-(A )-F-,3'-PO3-UTP-o-S (ambo); 2'-OMe-,3'-PO3-CTP; 2'-{R )-F-,3'-PO3-UTP; 2'-( A )-F-,3'-PO3-ATP; 2'-OMe-,3'-PO3-GTP-o-S (ambo); 2'-MOE-,3'-PO3-GTP-O-S (ambo); 2'-MOE-,3'-PO3-MeCTP-a-S (ambo); 2'-MOE-,3'-PO3-ATP-o-S (ambo); 2'-deoxy-,3'-PO3-GTP-o-S (ambo); 2'-deoxy-,3'-PO3-TTP-o-S (ambo); 2'-deoxy-,3'-PC>3-ATP- o-S (ambo); 2'-deoxy-,3'-PO3-MeCTP-o-S (ambo); and 2'-MOE-,3'-PO3-MeUTP-o-S (ambo).

[0135] In an aspect of the disclosure, a method of screening a panel of putative and known PPKs to identify an enzyme capable of using a sugar-modified and / or o-phosphate-modified nucleoside monophosphate or nucleoside diphosphate as a substrate, to produce a correspondingly modified nucleoside triphosphate, is provided. The screening method comprises: a) identifying sequences encoding said panel of putative and known PPKs; b) synthesizing said PPKs; c) purifying said PPKs; d) mixing each of said purified PPKs with said sugar-modified and / or o-phosphate-modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphate to form a reaction mixture; and e) analysing each of the reaction mixtures for the presence of correspondingly modified nucleoside triphosphate to determine which enzyme(s) produce correspondingly modified nucleoside triphosphates.

[0136] In an embodiment, the sequences in step a) are identified using one or more sequence databases and bioinformatic tools. In an embodiment, the sequence database is selected from the group consisting of: UniProt, UniParc, European Nucleotide Archive, GenBank, RefSeq, PIR, PDB, Pfam, and InterPro. In an embodiment, the sequence database is UniProt. In an embodiment, the sequence database is InterPro. In an embodiment, the bioinformatic tool are selected from the group consisting of: BLAST, Clustal Omega, ClustalW, HMMER, InterProScan, UniProt Tools, CD-HIT, MMseqs2, and CLANS. In an embodiment, the bioinformatic tool is CD-HIT. In an embodiment, the sequence database is UniProt and the bioinformatic tool is CD-HIT.

[0137] In an embodiment, the sequences in step a) encode proteins that are between 150 and 800 amino acid residues in length. In an embodiment, the sequences in step a) encode proteins that are between 150 and 600 amino acid residues in length. In an embodiment, the sequences in step a) encode proteins that are between 600 and 700 amino acid residues in length.

[0138] In an embodiment, PPKs are synthesized in step b) with an N-terminal hexa-histidine tag and are purified in step c) using nickel nitriloacetate.

[0139] In an embodiment, step d) comprises incubating said sugar-modified and / or o-phosphate- modified nucleoside monophosphates or nucleoside diphosphates, polyphosphate and PPK at 25 °C. In an embodiment, the incubating at 25 °C is for 24 hours. In an embodiment, the reaction is quenched after incubating at 25 °C for 24 hours. In an embodiment, the reaction is quenched with EDTA at 80 °C for 15 minutes. In an embodiment, the polyphosphate is sodium hexametaphosphate.

[0140] In an embodiment, the reaction mixture is centrifuged, and the supernatant is analysed for the presence of the correspondingly modified nucleoside triphosphate. In an embodiment, the reaction mixture is centrifuged for 15 minutes at 4000 g. In an embodiment, the supernatant is analysed using HPLC or UPLC.

[0141] In an embodiment, the enzyme capable of producing the correspondingly modified nucleoside triphosphate is further engineered to increase the % conversion to the correspondingly modified nucleoside triphosphate. Methods of engineering enzymes are known to persons skilled in the art and include mutagenesis and / or DNA shuffling, as described in Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and U.S. Patent 6,537,746. Other procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (Zhao, et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell, et al., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black, et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesis and directed evolution techniques useful for the purposes herein are also described in the following references: Ling, et al., 1997, "Approaches to DNA mutagenesis: an overview," Anal. Biochem. 254(2): 157-78; Vidal, et al., 2023, "A primer to directed evolution: current methodologies and future directions", RSC Chem Biol, 4:271-291; Currin, et al., 2021, "The evolving art of creating genetic diversity: From directed evolution to synthetic biology", Biotechnology Advances, 50:107762; Currin et al., 2015, "Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently", Chem Soc rev, 44:1172-1239; Dale, et al., 1996, "Oligonucleotide- directed random mutagenesis using the phosphoroth ioate method," Methods Mol. Biol. 57:369-74; Smith, 1985, "In vitro mutagenesis," Ann. Rev. Genet. 19:423-462; Botstein, et al., 1985, "Strategies and applications of in vitro mutagenesis," Science 229:1193-1201; Carter, 1986, "Site- directed mutagenesis," Biochem. J. 237:1-7; Kramer, et al., 1984, "Point Mismatch Repair," Cell 38:879-887; Wells, et al., 1985, "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites," Gene 34:315-323; Minshull, et al., 1999, "Protein evolution by molecular breeding," Curr Opin Chem Biol 3:284-290; Christians, et al., 1999, "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling," Nature Biotech 17:259-264; Crameri, et al., 1998, "DNA shuffling of a family of genes from diverse species accelerates directed evolution," Nature 391:288-291; Crameri, et al., 1997, "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotech 15:436-438; Zhang, et al., 1997, "Directed evolution of an effective fructosidase from a galactosidase by DNA shuffling and screening," Proc Natl Acad Sci USA 94:45-4-4509; Crameri, et al., 1996, "Improved green fluorescent protein by molecular evolution using DNA shuffling,' Nature Biotech 14:315-319; and Stemmer, 1994, "Rapid evolution of a protein in vitro by DNA shuffling," Nature 370:389-391.

[0142] In an aspect of the disclosure, an engineered PPK having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% sequence identity to SEQ ID NO:22 is provided. A PPK having 100% sequence identity to SEQ ID NO:22 is not an engineered PPK.

[0143] NTPs produced by the methods of the disclosure may be used in the production of polynucleotides, including oligonucleotides.

[0144] A polynucleotide may be entirely composed of nucleotide residues as found in nature {i.e. all "natural nucleotides" or "naturally occurring nucleotides") or may contain at least one modified nucleotide residue, or at least one linkage between nucleotide residues that has been modified.

[0145] Examples of naturally occurring nucleotides include deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, deoxyuridine monophosphate, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, thymidine monophosphate and uridine monophosphate.

[0146] A modified nucleotide is not a naturally occurring nucleotide {i.e. it is a non-natural nucleotide). A modified nucleotide may be a naturally occurring nucleotide that has been modified, for example chemically modified. A modified nucleotide may comprise a modified phosphate group, a modified sugar, and / or a modified nucleobase. It is acknowledged that certain modifications occur sporadically in nature, i.e. in naturally occurring nucleotides, such as 2'-OMe or C5 pyrimidine modifications, however, in the present disclosure these are considered modified nucleotides. Modifications in the sugar moiety can include a modification at the 2'-position of the sugar moiety, a modification at the 3'-position of the sugar moiety, a bicyclic sugar, or a 4'-CH(CH3)-O-2' group, and combinations thereof. For example, the modification at the 2' position of the sugar moiety may comprise 2'-F, 2'-0Me, or 2'-MOE.

[0147] Examples of modified nucleobases include, a modified cytosine, such as a 5-methyl cytosine, a 5-methyl pyrimidine, a 7-deazaguanosine and an abasic nucleotide. Further examples of modified nucleobases include, m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-0-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2'-0-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6- threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6. -hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (l,2'-0-dimethylinosine); m3C (3-methylcytidine); Cm (2T-0-methylcytidine); s2C (2- thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-0-dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mIG (1-methylguanosine); m2G (N2- methylguanosine); m7G (7-methylguanosine); Gm (2'-0-methylguanosine); m22G (N2,N2- dimethylguanosine); m2Gm (N2,2'-0-dimethylguanosine); m22Gm (N2,N2,2'-0-trimethylguanosine); Gr(p) (2'-0-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxy wybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7- deazaguanosine); G (archaeosine); D (dihydrouridine); m5Um (5,2'-0-dimethyluridine); s4U (4- thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-0-methyluridine); acp3U (3-(3-amino- 3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5- oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5- carbamoylmethyl-2'-0-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L- Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2- thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); rn62Am (N6,N6,0-2- trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); £5Cm (5-formyl-2'-0- methylcytidine); mIGm (l,2'-0-dimethylguanosine); m'Am (1,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl -2 -thiouridine)); imG- 14 (4-demethyl guanosine); imG2 (isoguanosine); and ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7- substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(CI-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5- (hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(CI-C6 )- alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8- azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8- substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6- chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deaza purine, 7- deaza-7-substituted purine and 7-deaza -8-substituted purine. For instance, the polynucleotide may include one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5- methylcytosine residues.

[0148] Backbone modifications include phosphoroth ioate, phosphora midate, phosphorodiamidate, phosphorodithioate and / or methylphosphonate. Zero, one, several or every internucleoside linkage may be a modified internucleoside linkage. The polynucleotides of the disclosure may include only phosphodiester linkages between nucleosides, but in other examples may contain phosphoroth ioate, phosphoramidate, phosphorodiamidate, phosphorodithioate and / or methylphosphonate linkages.

[0149] Polynucleotides, including oligonucleotides, may be single-stranded or double-stranded. A polynucleotide or an oligonucleotide of the disclosure may be conjugated to another molecule, e.g. N-Acetylgalactosamine (GalNAc) or multiples thereof (GalNAc clusters).

[0150] Modified nucleoside triphosphates known in the art include 2-amino-6-chloropurineriboside- 5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'- amino-2'-deoxycytidine-triphosphate, 2-thiocytid ine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-Fluorothymidine-5'-triphosphate, 2'-0-Methyl-inosine-5'-triphosphate 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'- triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo- 2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-lodo-2'-deoxycytidine-5'- triphosphate, 5-iodouridine-5'-triphosphate, 5-lodo-2'-deoxyuridine-5'-triphosphate, 5- methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'- triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6- azauridine-5'-triphosphate, 6-chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'- triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine- 5'-triphosphate, benzimidazole-riboside-5'-triphosphate, Nl-methyladenosine-5'-triphosphate, N1 - methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'- triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, and xanthosine-5'- triphosphate.

[0151] Base-modified nucleotides known in the art include 5-methylcytidine-5'-triphosphate, 7- deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'- triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5- taurinomethyluridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 - taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl-pseudouridine, 4-thio-l -methyl- pseudouridine, 2-thio-l -methyl-pseudouridine, 1 -methyl-1 -deaza-pseudouridine, 2-thio-l -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1 -methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-th io-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l -methyl-pseudoisocytidine, 4-thio-l -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl-1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l -methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8- aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 - methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8- aza-guanosine, 6-th io-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 - methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8- oxo-guanosine, 1 -methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6- thio-guanosine, 5'-0-(l -thiophosphate)-adenosine, 5'-0-(l -thiophosphate)-cytidine, 5'-0-(l - thiophosphate)-guanosine, 5'-0-(l -thiophosphate)-uridine, 5'-0-(l -thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo- uridine, N1 -methyl-pseudouridine, 5,6-dihydrouridine, alpha -thio-uridine, 4-th io-urid ine, 6-aza- uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha -thioguanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1 - methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6- chloro-purine, N6-methyl-adenosine, alpha -thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiou ridine , 4'- thiouridine, 5- methyluridine, 2-thio-l -methyl-1 -deaza-pseudouridine, 2-thio-l -methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2- thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2'-0-methyl uridine, pseudouridine (y), N1 - methylpseudouridine, 5-methylcytosine, and 5-methoxyuridine.

[0152] In an embodiment, the polynucleotide is a therapeutic polynucleotide. In an embodiment, the therapeutic polynucleotide is a therapeutic RNA. In an embodiment, the therapeutic polynucleotide is a therapeutic mRNA.

[0153] In an embodiment, the polynucleotide is an oligonucleotide. In an embodiment, the oligonucleotide is selected from the group consisting of: an antisense oligonucleotide, siRNA, miRNA, aptamer, CRISPR guide RNA, and an oligonucleotide used to recruit and guide DNA and RNA editing enzymes. In an embodiment, the oligonucleotide used to recruit and guide DNA and RNA editing enzymes is an A to I RNA base-editing oligonucleotide (AIMer). In an embodiment, the oligonucleotide is a therapeutic oligonucleotide.

[0154] In an embodiment, the polynucleotide is manufactured via an enzymatic method, i.e. via enzymatic synthesis, also referred to herein as enzymatically. In an embodiment, the enzyme is a polymerase. In an embodiment, the polymerase is a template-dependent polymerase. In an embodiment, the polymerase is a template-independent polymerase. In an embodiment, the enzyme is a transferase. In an embodiment, the enzyme is a terminal deoxynucleotidyl transferase (TdT). In an aspect of the disclosure, a method of manufacturing a polynucleotide is provided, comprising incubating nucleotide triphosphates, a suitable enzyme, and optionally a template, under suitable conditions, wherein the nucleotide triphosphates are made by incubating the corresponding nucleoside monophosphates and / or nucleoside diphosphates, PPK and polyphosphate under suitable conditions.

[0155] In an aspect of the disclosure, a method of manufacturing a polynucleotide comprising the following steps is provided: a) providing nucleoside triphosphates that have been made using corresponding nucleoside monophosphates and / or nucleoside diphosphates, PPK, and polyphosphate under suitable conditions; and b) using said nucleoside triphosphates and a suitable enzyme under suitable conditions to produce a polynucleotide.

[0156] In an embodiment, one or more or all of the NTPs are naturally occurring NTPs.

[0157] In an embodiment, one or more or all of the NTPs comprise a sugar, base and / or o- phosphate modification. In an embodiment, one or more or all of the NTPs comprise base modification. In an embodiment, one or more or all of the NTPs comprise a sugar modification. In an embodiment, one or more or all of the NTPs comprise a 2' sugar modification. In an embodiment, one or more or all of the NTPs comprise a 3' sugar modification. In an embodiment, one or more or all of the NTPs comprise an o-phosphate modification.

[0158] In an embodiment, the polynucleotide is mRNA. In an embodiment, one or more or all of the NTPs comprise a base modification. In an embodiment, the NTPs provided comprise or consist of naturally occurring NTPs. In an embodiment, the NTPs are naturally occurring NTPs, except that uridine triphosphate is replaced with pseudouridine triphosphate. In an embodiment, the NTPs provided are naturally occurring NTPs, except that uridine triphosphate is replaced with Nl-methyl pseudouridine triphosphate.

[0159] In an embodiment, the enzyme used in step b) is a polymerase. In an embodiment, the polymerase is a template-dependent polymerase and a suitable template is included in step a). In an embodiment, the polymerase is a template-independent polymerase. In an embodiment, the enzyme used in step b) is a transferase. In an embodiment, the enzyme used in step b) is a terminal deoxynucleotidyl transferase (TdT).

[0160] Enzymes disclosed herein, in particular PPKs, may be prepared by any of a number of conventional techniques. For example, PPKs may be purified from cells that naturally express them or produced in recombinant expression systems.

[0161] A number of different expression systems and purification regimes can be used to generate the PPKs of the disclosure. Generally, host cells are transformed with a recombinant expression vector encoding the desired PPK. The expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system. A wide range of host cells can be employed, including Prokaryotes (including Gram negative or Gram positive bacteria, for example Escherichia coii, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergiius sp.), or higher Eukaryotes, including insect cells and cell lines of mammalian origin (for example, CHO, NSO, PER.C6, HEK293, and HeLa). In an embodiment, the vector is a pET28a vector.

[0162] The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism e.g, plant or animal). The host cell may be a non-human host cell. In an embodiment, the host cell is a bacterial cell. In an embodiment, the host cell is a gram-positive bacterial cell. In an embodiment, the host cell is E. coii. In an embodiment, the host cell is E. co / / BL21(DE3).

[0163] Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.

[0164] The cells can be cultured under conditions that promote expression of the PPK using a variety of equipment such as shake flasks, spinner flasks, and bioreactors. The polypeptide is recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations comprised of various filtration and chromatographic processes developed to selectively concentrate and isolate the PPK. In a particular embodiment, hexa-histidine is fused to the N-terminal of the PPK and the PPK is purified using immobilised metal affinity chromatography. In a particular embodiment, hexa-histidine is fused to the N-terminal of the PPK and the PPK is purified using nickel nitiloacetate (Ni-NTA).

[0165] EXAMPLES

[0166] Example 1: Selection of Polyphosphate Kinases (PPK)

[0167] INTERPRO (EMBL-EBI) provides functional analysis of proteins by classifying them into familiesand predicting domains and important sites. (Paysan-Lafosse et al, Nucleic Acids Research, 2023, 51, D418-D427). INTERPRO entry IPR022488 corresponds to polyphosphate kinase-2-related proteins, i.e., putative PPK2s as opposed to characterized PPK2s, and was used as a starting point (28,000 proteins).

[0168] Sequences of 150 amino acids in length to 600 amino acids in length were filtered using a standard python script and sequences having at least 90% sequence identity to each other were clustered using CD-HIT V4.6.8 (11,779 clusters). Characterized PPK2-III sequences (Nocek et al, ACS Catal. 2018, 8, 11, 10746-10760) and in-house PPK2 sequences were inserted into the data set without additional clustering in order to preserve sequence diversity.

[0169] Multiple sequence alignments were performed with MAFFT (a multiple sequence alignment program) version 7.520 (2023 / March) and a phylogenetic tree file was generated and visualized using Christian Zmasek's Archaeopteryx software (https: / / sites.google.com / site / cmzmasek / christian- zmasek / software) VO.9928. Clustering of PPK2s belonging to different classes was checked manually by searching for relevant sequences in the generated tree (i.e., using the characterized PPK2-III and known PPK2 I and II sequences). 4185 PPK2-III family members were identified for further study.

[0170] The PPK2-III family member sequences were retrieved using UNIPROT. Fragments and poorly annotated sequences were removed by column sorting and filtering leaving 4002 sequences of potential interest. UNIPROT was then used to separate the sequences into three groups: those originating from archaea (34 sequences), metagenome (38 sequences) and bacteria (3930 sequences).

[0171] Additional clustering of bacterial sequences was carried out using CD-HIT (3 steps: 80%, 70% and 60% sequence identity across sequences) resulting in 686 clusters. Representative sequences were aligned with Clustal Omega, a phylogenetic tree file was generated and visualized using the Archaeopteryx software. Phylogenetic analysis separated the sequences into four main clades and a further phylogenetic tree was created for each of these clades. The least populated clades from each of the four trees were removed and, where available, sequences within clades from known extremophilic organisms were selected as the representative example for the clade. A final 95 genes were selected after trimming sequences for maximum sequence diversity. Gene synthesis failures and sequences that were not produced in a sufficiently soluble form reduced the PPK sequences to 66.

[0172] Example 2: 5' phosphorylation of 2'-hydroxyl adenosine 5'-monophosphates (AMP) by PPK to yield 2'-hydroxyl adenosine 5'-tri phosphates (ATP) (natural PPK reaction)

[0173] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to a hexa-histidine tag (6xHis). Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. co / / BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using nickel nitriloacetate (Ni-NTA) and used directly.

[0174] Reactions were set up as per table 1, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of ethylenediaminetetraacetic acid (EDTA) (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new polymerase chain reaction (PCR) plate. Reactions were then analysed by high performance liquid chromatography (HPLC) for the presence of adenosine 5'-triphosphate (ATP). Product peaks were assigned using liquid chromatography-mass spectrometry (LCMS).

[0175] Table 1: Reaction set-up

[0176] Table 2: Results from reactions

[0177]

[0178]

[0179] Conversion (%) to product - % area of 5'-tri- & 5'-tetraphosphate peaks vs. 5'-mono-, 5'-di-, 5'-tri- and 5'-tetraphosphate peaks at wavelength 256 nm.

[0180] Conclusion

[0181] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs tested (SEQ ID NOs.1-5, 7-9, 11-31, 33-41, 43-45, 47-57, and 59-66) will synthesize adenosine 5'-triphosphates from adenosine 5'-monophosphates by phosphorylation at the 5'-phosphate, with thirteen wild-type PPKs (SEQ ID NOs:ll, 12, 15, 21, 23, 26, 38, 41, 49-51, 54, and 65) converting about three-quarters or more of substrate to product under the specified reaction conditions.

[0182] Example 3: 5' phosphorylation of 2'- methoxy modified, 3'- phosphate protected adenosine 5'-monophosphates by PPK to yield 2'-methoxy-adenosine 3'-phosphate-5'- triphosphates.

[0183] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0184] Reactions were set up as per table 3, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'- methoxy-adenosine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0185] Table 3: Reaction set-up

[0186]

[0187] Table 4: Results from reactions

[0188]

[0189]

[0190] Conversion (%) to product - % area of 5'-tri- & 5'-tetraphosphate peaks vs. 5'-mono-, 5'-di-, 5'-tri- and 5'-tetraphosphate peaks at wavelength 256 nm.

[0191] Conclusion

[0192] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs.ll, 14, 15, 19-23, 25-27, 29-31, 36-38, 40, 49-52, 65, and 66) will synthesize 2'-methoxy modified, 3'-phosphate protected adenosine 5'-triphosphates from 2'- methoxy modified, 3'-phosphate protected adenosine 5'-monophosphates by phosphorylation at the 5' phosphate, with two wild-type PPKs working particularly well (SEQ ID NO:22, 94.3% conversion; and SEQ ID NO: 52, 86.5% conversion).

[0193] Example 4: 5' phosphorylation of 2 '-fluoro, 3'-phosphate protected adenosine 5'- monophosphates by wild-type PPK to yield 2'-fluoro, 3'-phosphate protected adenosine 5'-tri phosphates

[0194] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0195] Reactions were set up as per table 5, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-fluoro- adenosine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0196] Table 5: Reaction set-up

[0197] Table 6: Results from reactions

[0198]

[0199] 5' -tetraphosphate peaks at wavelength 256 nm.

[0200] Conclusion From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs.ll, 15, 19, 21, 22, 25, 27, 29, 31, 36, 38, 49, 52, 59, and 66) will synthesize 2'-fluoro, 3'-phosphate protected adenosine nucleotide 5'-triophosphates from 2'- methoxy modified, 2'-fluoro, 3'-phosphate protected adenosine nucleotide 5'-monophosphates by phosphorylation at the 5' phosphate. One wild-type PPK, SEQ ID NO:22, worked exceptionally well and converted 100% of substrate to product under the specified reaction conditions.

[0201] Example 5: 5' phosphorylation of 2'-methoxy modified, 3'- phosphate protected guanosine 5'-monophosphates by PPK to yield 2'-methoxy guanosine 3'-phosphate-5' triphosphates

[0202] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0203] Reactions were set up as per table 7, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'- methoxy-guanosine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0204] Table 7: Reaction set-up

[0205] Results and conclusion

[0206] From the described experiment and resulting conversion data it has been shown that several wild-type PPKs (SEQ ID NOs.19, 22, 31, and 52) will synthesize 2'-methoxy modified, 3'-phosphate protected guanosine 5'-triphosphates from 2'-methoxy modified, 3'-phosphate protected guanosine 5'-monophosphates by phosphorylation at the 5' phosphate (substrate to product % conversion of 5.8%, 99%, 28.6% and 4.0% respectively). A representative chromatogram is shown in Fig. 2. One wild-type PPK, SEQ ID NO:22, worked exceptionally well and converted 99% of substrate to product under the specified reaction conditions.

[0207] Example 6: 5' phosphorylation of 2'-methoxy modified, 3'- phosphate protected uridine 5'- monophosphates by PPK to yield 2'-methoxy uridine 3'-phosphate-5' triphosphates

[0208] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0209] Reactions were set up as per table 8, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'- methoxy-uridine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0210] Table 8: Reaction set-up

[0211] Results and conclusion

[0212] From the described experiment and resulting conversion data it has been shown that a single wild-type PPK (SEQ ID NO:22) will synthesize 2'-methoxy modified, 3'-phosphate protected uridine 5'-triphosphates from 2'-methoxy modified, 3'-phosphate protected uridine 5'- monophosphates by phosphorylation at the 5' phosphate (39.6% conversion of substrate to 5'- triphosphate product and 43.1% to 5'-tri- and 5'-tetra phosphate product). Example 7: 5' phosphorylation of 2 '-fluoro modified, 3'- phosphate protected cytidine 5'- monophosphates by PPK to yield 2'-fluoro cytidine 3'- phosphate- 5' triphosphates

[0213] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0214] Reactions were set up as per table 9, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-fluoro- cytidine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0215] Table 9: Reaction set-up

[0216] Results and conclusion

[0217] From the described experiment and resulting conversion data it has been shown that a single wild-type PPK (SEQ ID NO:22) will synthesize 2'-fluoro modified, 3'-phosphate protected cytidine 5'-triphosphates from 2'-fluoro modified, 3'-phosphate protected cytidine 5'- monophosphates by phosphorylation at the 5' phosphate (22.6% conversion of substrate to product).

[0218] Example 8: 5' phosphorylation of 2' methoxy-ethyl modified methyl cytidine 5'- monophosphates by PPK to yield methyl cytidine 2'- methoxy-ethyl methyl cytidine 3'- phosphate-5' triphosphates Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0219] Reactions were set up as per table 10, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-methoxy-ethyl-methylcytidine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0220] Table 10: Reaction set-up

[0221] Results and conclusion

[0222] From the described experiment and resulting conversion data it has been shown that several wild-type PPKs (SEQ ID NOs.22, 31, 49) will synthesize, albeit at a low substrate to product % conversion (5.4%, 2.4% and 0.9%, respectively), 2'-methoxy-ethyl-methyl cytidine, 3'-phosphate protected, 5'-triphosphates from 2'-methoxy-ethyl methyl cytidine, 3'-phosphate protected, 5'- monophosphates by phosphorylation at the 5' phosphate.

[0223] Example 9: 5' phosphorylation of 2'-methoxy modified, 3'- phosphate protected cytidine 5'-monophosphates by PPK to yield 2'-methoxy modified, 3'-phosphate protected cytidine 5 '-tri phosphates

[0224] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0225] Reactions were set up as per table 11, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-methoxy-cytidine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0226] Table 11: Reaction set-up

[0227] Results and conclusion

[0228] From the described experiment and resulting conversion data it has been shown that several wild-type PPKs (SEQ ID NOs: 22, 29, 31) will synthesize 2'-methoxy modified, 3'-phosphate protected cytidine 5'-tri phosphates from 2'-methoxy modified, 3'-phosphate protected cytidine 5'- monophosphates by phosphorylation at the 5' phosphate (substrate to product % conversion of 10.4%, 0.3% and 38.1% respectively).

[0229] Example 10: 5' phosphorylation of 2'-methoxy modified, 3'-phosphate protected adenosine 5'-monothiophosphates by PPK to yield 2'-methoxy adenosine 3'-phosphate- 5' a-thio-triphosphates.

[0230] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0231] Reactions were set up as per table 12, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-methoxy-adenosine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0232] Table 12: Reaction set-up

[0233] Table 13: Results from reactions

[0234]

[0235]

[0236] *Conversion (%) to product - % area of 5'-a-thio-tri- & 5'-a-thio -tetraphosphate peaks vs. 5'-mono-, 5'- di-, 5'-tri- and 5'-tetra-5'-a-thio phosphate peaks at wavelength 256 nm.

[0237] Conclusion

[0238] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 1-4, 10-15, 18-23, 25-27, 29-31, 36-42, 47, 49-54, 56, 57, 59, 60, 65, and 66) will synthesize 2'-methoxy modified, 3'-phosphate protected adenosine 5'-o-thio- triphosphates from 2'-methoxy modified, 3'-phosphate protected adenosine 5'-th iophosphates by phosphorylation at the 5' thiophosphate.

[0239] Example 11: 5' phosphorylation of 2'-methoxy modified, 3'-phosphate protected cytidine 5'-monothiophosphates by PPK to yield 2'-methoxy cytidine 3'-phosphate-5' a- thio-triphosphates.

[0240] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0241] Reactions were set up as per table 14, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-methoxy-cytidine 3'-phosphate-5'-triphosphate. Product peaks were assigned using LCMS.

[0242] Table 14: Reaction set-up

[0243] Results and conclusion

[0244] From the described experiment and resulting conversion data it has been shown that several wild-type PPKs (SEQ ID NOs. 22, 31, 37, 59) will synthesize 2'-methoxy modified, 3'-phosphate protected cytidine 5'-o-thio-triphosphates from 2'-methoxy modified, 3'-phosphate protected cytidine 5'-th iophosphates by phosphorylation at the 5' thiophosphate (substrate to product % conversion of 3.3%, 6.2%, 11.9% and 17.0% respectively).

[0245] Example 12: 5' phosphorylation of adenosine 5'-monothiophosphates by PPK to yield adenosine 5'-(a-thio)triphosphate.

[0246] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0247] Reactions were set up as per table 15, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of adenosine 5'-(o-thio)triphosphate. Product peaks were assigned using LCMS.

[0248] Table 15: Reaction set-up

[0249]

[0250] The formation of a phosphoanhydride bond to an o-thiophosphate creates a chiral centre at the o-thiophosphate phosphorus resulting in 2 diastereomers. The diastereomers of adenosine 5'-(o- thio)diphosphate and adenosine 5'-(o-thio)triphosphate were resolvable on ultra-performance liquid chromatography (UPLC). Data for the diastereomeric ratio (first eluting peak / second eluting peak) and % composition relative to the mono-, di- and triphosphate is shown in table 16 below.

[0251] Table 16: Diastereomeric results from reactions

[0252] Conclusion

[0253] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 1-9, 11, 12, 15-23, 25-41, 44-54, 56, 57, 59 and 61-66) will synthesize adenosine 5'-(o-thio)-triphosphate from adenosine 5'-monothiophosphate by phosphorylation at the terminal 5' thiophosphate.

[0254] The diastereomeric ratio of the 5'-(o-thio)-triphosphate and 5'-(o-thio)-diphosphate generated may be 1 (indicating no stereopreference for phosphorylation) >1 (indicating a preference for generating the early eluting diastereomer) or <1 (indicating a preference for generating the later eluting diastereomer) depending on the PPK used (see Fig. 3).

[0255] Example 13: 5' phosphorylation of 2 '-deoxyadenosine 5'-monothiophosphates by PPK to yield 2'-deoxyadenosine 5'-(a-thio)triphosphates.

[0256] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0257] Reactions were set up as per table 17, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-deoxyadenosine 5'-(o-thio)-triphosphate. Product peaks were assigned using LCMS.

[0258] Table 17: Reaction set-up

[0259] Table 18: Results from reactions

[0260]

[0261]

[0262] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm.

[0263] Conclusion

[0264] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 2-4, 7-9, 11,12, 14-23, 25-31, 33-40, 45, 47-54, 56, 57 ,59, 61, 62, 64-66) will synthesize 2'-deoxyadenosine 5'-(a-thio)-triphosphate from 2'deoxyadenosine 5'- monothiophosphate by phosphorylation at the terminal 5' thiophosphate.

[0265] Example 14: 5' phosphorylation of 2'-deoxygua nosine 5'-monothiophosphates by PPKto yield 2'-deoxyguanosine 5'-(a-thio)triphosphates.

[0266] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0267] Reactions were set up as per table 19, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-deoxyguanosine 5'-(o-thio)-triphosphate. Product peaks were assigned using LCMS.

[0268] Table 19: Reaction set-up

[0269] Table 20: Results from reactions

[0270]

[0271] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm.

[0272] Conclusion

[0273] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 8, 9, 11, 15, 19-23, 25, 27, 29, 31, 32, 35, 37, 39, 45, 47, 49, 50, 54, 58, 61, and 64-66) will synthesize 2'-deoxyguanosine 5'-(a-thio)-tri phosphate from 2'deoxyguanosine 5'-monothiophosphate by phosphorylation at the terminal 5' thiophosphate.

[0274] Example 15: 5' phosphorylation of 2 '-deoxy cytidine 5'-monothiophosphates by polyphosphate kinase to yield 2'-deoxycytidine 5'-(a-thio)triphosphates.

[0275] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0276] Reactions were set up as per table 21, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-deoxycytidine 5'-(o-thio)-triphosphate. Product peaks were assigned using LCMS.

[0277] Table 21: Reaction set-up

[0278] Table 22: Results from reactions

[0279]

[0280]

[0281] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm.

[0282] Conclusion

[0283] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 9, 14-16, 19, 20, 22, 23, 25, 27, 29, 31, 37, 39, 44, 47, 59, and 65) will synthesize 2'-deoxycytidine 5'-(a-thio)-triphosphate from 2'deoxycytidine 5'- monothiophosphate by phosphorylation at the terminal 5' thiophosphate.

[0284] Example 16: 5' phosphorylation of 2'-fluoro-cytidine-5'-monophosphate by PPK to yield 2'-fluoro-cytidine-5'-triphosphate.

[0285] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0286] Reactions were set up as per table 23, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-fluoro-cytidine-5'-triphosphate. Product peaks were assigned using LCMS.

[0287] Table 23: Reaction set-up

[0288] Table 24: Results from reactions

[0289]

[0290]

[0291] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm. Conclusion

[0292] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 10, 11, 15, 19, 21-23, 25-27, 29, 31, 32, 37, 39, 40, 49, 54, 59, and 66) will synthesize 2'-fluoro-cytidine-5'-triphosphate from 2'-fluoro-cytidine-5'- monophosphate by phosphorylation at the terminal 5' thiophosphate.

[0293] Example 17: 5' phosphorylation of 2'-methoxy modified adenosine 5'-monophosphate by PPK to yield 2'-methoxy modified adenosine 5'-tri phosphate.

[0294] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0295] Reactions were set up as per table 25, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 2'-methoxy-adenosine 5'-triphosphate. Product peaks were assigned using LCMS.

[0296] Table 25: Reaction set-up

[0297] Table 26: Results from reactions

[0298]

[0299]

[0300]

[0301] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm.

[0302] Conclusion

[0303] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 1, 2, 4, 5, 7, 9, 11, 12, 14-23, 25-40, 43-45, 47-54, 56, 57, 59-62, and 64-66) will synthesize 2'-methoxy-adenosine 5'-tri phosphate from 2'-methoxy-adenosine 5'-monophosphate by phosphorylation at the terminal 5' thiophosphate.

[0304] Example 18: 5' phosphorylation of 3'-(N'-methyl-anthraniloyl) 2'-deoxyadenosine-5'- monophosphate by PPK to yield 3'-(N'-methyl-anthraniloyl) 2'-deoxyadenosine-5'- tri phosphate. Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0305] Reactions were set up as per table 27 , with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 3'-(N'-methyl-anthraniloyl) 2'-deoxyadenosine-5'-triphosphate. Product peaks were assigned using LCMS.

[0306] Table 27: Reaction set-up

[0307] Table 28: Results from reactions

[0308]

[0309]

[0310] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm.

[0311] Conclusion

[0312] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs. 2, 4, 6-9, 11, 12, 14-17, 19-23, 25-40, 42, 44, 45, 47-54, 56-62, and 64-66) will synthesize 3'-(N'-methyl-anthraniloyl) 2'-deoxyadenosine-5'-triphosphate from 3'-(N'-methyl-anthraniloyl) 2'-deoxyadenosine-5'-monophosphate by phosphorylation at the terminal 5' phosphate.

[0313] Example 19: 5' phosphorylation of 3'-(N'-methyl-anthraniloyl) 2'-deoxyguanosine-5'- monophosphate by PPK to yield 3'-(N'-methyl-anthraniloyl) 2'-deoxyguanosine-5'- tri phosphate.

[0314] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0315] Reactions were set up as per table 29, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (5 pL, 20 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 12 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 3'-(N'-methyl-anthraniloyl) 2'-deoxyguanosine-5'-triphosphate. Product peaks were assigned using LCMS.

[0316] Table 29: Reaction set-up

[0317] Table 30: Results from reactions

[0318]

[0319]

[0320]

[0321] Conversion (%) to product - % area of 5'-monophosphate & 5'-diphosphate peaks vs. Product 5'- triphosphate peak at wavelength 256 nm.

[0322] Conclusion

[0323] From the described experiment and resulting conversion data it has been shown that a number of wild-type PPKs (SEQ ID NOs. 2, 4-9, 11, 12, 14-40, 42, 44-47, 49-54, 56, 58, 59, 61, and 63-66) will synthesize 3'-(N'-methyl-anthraniloyl) 2'-deoxyguanosine-5'-triphosphate from 3'-(N'- methyl-anthraniloyl) 2'-deoxyguanosine-5'-monophosphate by phosphorylation at the terminal 5' phosphate.

[0324] Example 21: 5' phosphorylation of 3'-pivaloyl 2'-methoxyguanosine -5'-monophosphate by PPKto yield 3'-pivaloyl 2'-methoxyguanosine -5'-triphosphate.

[0325] Wild-type PPKs, as set out in SEQ ID NOs: 1-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0326] Reactions were set up as per table 31, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (2 pL, 100 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 20 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 3'-pivaloyl 2'-methoxyguanosine -5'-triphosphate.

[0327] Table 31: Reaction set-up

[0328] Table 32: Results from reactions

[0329]

[0330]

[0331]

[0332] Conversion (%) to product - % area of 5'-tri- vs. 5'-mono-, 5'-di- and 5'-tri phosphate peaks at wavelength 256 nm.

[0333] Conclusion

[0334] From the described experiment and resulting conversion data it has been shown that numerous wild-type PPKs (SEQ ID NOs.19, 20, 24, 32 and 36) will synthesize 3'-pivaloyl 2'- methoxyguanosine -5'-triphosphate from 3'-pivaloyl 2'-methoxyguanosine -5'-monophosphate by phosphorylation at the 5' phosphate, with one wild-type PPK working particularly well (SEQ ID NO:22, 72.5% conversion).

[0335] Example 22: 5' phosphorylation of 3'-pivaloyl 2'-methoxyuridine -5'-monophosphate by PPK to yield 3'-pivaloyl 2'-methoxyuridine -5'-triphosphate.

[0336] Wild-type PPKs, as set out in SEQ ID NOs:l-66, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. coH BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni- NTA and used directly.

[0337] Reactions were set up as per table 33, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (2 pL, 100 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 20 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of 3'-pivaloyl 2'-methoxyuridine -5'-triphosphate.

[0338] Table 33: Reaction set-up

[0339] Table 34: Results from reactions

[0340]

[0341]

[0342] Conversion (%) to product - % area of 5'-tri- vs. 5'-mono-, 5'-di- and 5'-tri phosphate peaks at wavelength 256 nm.

[0343] Conclusion From the described experiment and resulting conversion data it has been shown that only one wild-type PPK (SEQ ID NOs.24) will synthesize 3'-pivaloyl 2'-methoxyuridine -5'-triphosphate from 3'-pivaloyl 2'-methoxyuridine -5'-monophosphate by phosphorylation at the 5' phosphate (SEQ ID NO:22, 28.5% conversion).

[0344] Example 23: 5' phosphorylation of deoxyadenosine 5'-monothiophosphates by PPK to yield deoxyadenosine 5'-(a-thio)triphosphate.

[0345] Wild-type PPKs, as set out in SEQ ID NOs:22,31 and 34, were each fused at the N-terminus to 6xHis. Genes were synthesised, cloned into pET28a and protein encoding the gene was produced in E. co / / BL21(DE3) using standard cloning, expression, and extraction methods. PPKs were purified using Ni-NTA and used directly.

[0346] Reactions were set up as per table 35, with the addition of PPK last. Reactions were incubated at 25 °C for 24 hours. Reactions were then quenched by addition of EDTA (2 pL, 100 mM), heating to 80 °C for 15 minutes, and centrifuging at 4000 x g for 15 minutes. 20 pL of supernatant was then transferred to a new PCR plate. Reactions were then analysed by HPLC for the presence of deoxyadenosine 5'-(o-thio)triphosphate.

[0347] Table 35: Reaction set-up

[0348] The formation of a phosphoanhydride bond to an o-thiophosphate creates a chiral centre at the o-thiophosphate phosphorus resulting in 2 diastereomers. The diastereomers of deoxyadenosine 5'-(o-thio)diphosphate and deoxyadenosine 5'-(o-thio)tri phosphate were resolvable on ultraperformance liquid chromatography (UPLC). Data for the diastereomeric ratio (first eluting peak / second eluting peak) and % composition relative to the mono-, di- and triphosphate is shown in table 36 below.

[0349] Table 36: Diastereomeric results from reactions

[0350] Conclusion

[0351] From the described experiment and resulting conversion data it has been shown that wildtype PPKs (SEQ ID NOs. 22, 31 and 34) will synthesize deoxyadenosine 5'-(o-thio)-triphosphate from deoxyadenosine 5'-monothiophosphate by phosphorylation at the terminal 5' thiophosphate.

[0352] The diastereomeric ratio of the 5'-(o-thio)-triphosphate and 5'-(o-thio)-diphosphate generated may be 1 (indicating no stereopreference for phosphorylation) >1 (indicating a preference for generating the early eluting diastereomer) or <1 (indicating a preference for generating the later eluting diastereomer) depending on the PPK.

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[0422] SEO ID NO: 29

[0423] MDIDNYRVKPGKRVKLSDWATNDDAGLSKEEGQAQTAKLAGELAEWQERLYAEGKQSLLLILQARDAAGKDGA

[0424] VKKVIGAFNPAGVQITSFKQPSAEELSHDFLWRIHQKAPAKGYVGVFNRSQYEDVLVTRVYDMIDDKTAKRRLE HIRHFEELLTDNATRIVKVYLHISPEEQKERLQARLDNPGKHWKFNPGDLKDRSNWDKFNDVYEDALTTSTDDA PWYWPADRKWYRDLVLSHILLGALKDMNPQFPAIDYDPSKWIH

[0425] SEO ID NQ:30

[0426] MLRPDTTNQIRAGTDGGFSLSDHQPSWEGSGRVDGLTGAGLKKRAKKYVKAQRRLLAKAQEKLYADDRFSVLIL

[0427] LQAMDAAGKDSTIKQVMSGVNPQGCQVFSFKQPSAEELDHNFLWRYWRDVPERGRIGIFNRSYYEEVLWRVH PELVQRQRLPAAPNGSDFWAARFEDINAFERHMTRNGTVILKFFLNVSKEEQRQRFVRRLTKPKKLWKFSANDL YERAHWDDYQHAYEELITATSTEWAPWYVIPADNKWLMRALVSHVIVEAVEGLGVTYPKVPPGREAEFRKALN ALRAEGAASGKKGGIG

[0428] SEO ID NO:31

[0429] MINIYKIDKLNNFNLNNHKTDDYSLCKDKDTALELTQKNIQKIYDYQQKLYAEKKEGLIIAFQAMDAAGKDGTIR

[0430] EVLKALAPQGVHEKPFKSPSSTELAHDYLWRVHNAVPEKGEITIFNRSHYEDVLIGKVKELYKFQNKADRIDENT WDNRYEDIRNFEKYLYNNSVRIIKIFLNVSKKEQAERFLSRIEEPEKNWKFSDSDFEERVYWDKYQQAFEDAIN ATSTKDCPWYWPADRKWYMRYWSEIWKTLEEMNPKYPTVTKETLERFEGYRTKLLEEYNYDLDTIRPIEK

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[0432] MRLPDAVLQQCHYQHASALNLDPNRDFGLPMRKADARLEDLRKWLDRQQQLLWANNGPAILVWLQGPDCSG KDGAIRNSLRGLNPQGIRVSDFQKPTEQQRSEHFLKRYREQLPTAGMITVFNRTPYEGLVSDLADGDIDSNQAA ERLQQLLAFEEQLAEQNIHLLKIYLHISKSEQKKRLQQRFINPEKRWKISAADLIGHQNFEQIQANWNQAFAQSS SLSHPWHILPSHNKALRNLLICSLIAQKLEALDLQWPQPALPFAIEDLDLACSISG SEO ID NO:33

[0433] MAKTSFLQRFKVLPGKKIHLAEIDTDATPGYKNKQEAKADLADCLQRLRDLQYLLYAENKRSLLIVLQGLDASGK DGTIRH VLGYM NPQGCRVTSFKVPTEEEAAH DFLWRVH KAAPGRRQVAIFN RSHYEDVLWRVH KLVPKSVWE KRYDQINEFERLLADNGTHILKFFLHISPEEQLRRFRKRLENPKKRWKISEADYSERKYWHDYQQAYEDALSRCS RDHAPWFVIPADKKWFRNLAISQIWESLESLKMQWPNPQVDLDLIRLKYH EAVENS

[0434] SEO ID NO: 34

[0435] MTTIPEEMEDRIKAVMDIMRVPPDEKIDLEKDYDPGFTGKWMKKKEAKETLVRGIRLLAEMQNKLYAQDQYALL IVLQALDAAGKDSTIKHVMSGINPQGVDVHSFKTPSGEELDHDYLWRNFKALPARGRIGIFNRSYYEEVLWRVH PEFLASQKLPPILKDKHIWKRRFEEINNFEKYLVDNGIIVLKFFLYVSKEKQRERFLERALLPEKNWKFSTADMKER ARWDEYITAYEDM FN HTSTEWAPWYWPADH KWFTRLAVAAVIYNTM DKLNLAYPWSEQQKQALLVAEKELE NENGGPDDKAVRKVKAKAVASRKNKKKDKKKK

[0436] SEO ID NO:35

[0437] MIKKRYLKKIMVSPGSRVSLERYDTGWAQDDELKKVGKDAIKERAAEILEANRAALAESQELLWASNLYSFLIVL QGMDTSGKDGAIKHVMAGVNPQGCRVTSFKVPTSEETDHNYLWRYTRALPARGEIGIFNRSYYEDVLWKVHP EILDHQQMPPGKRGDKFWNARYDDINAFERHLVQNGTFILKFFLHISREEQKKRLMERLEDKEKYWKFSLSDLA ERRFWDDYQSAYEMMLSKTSTRIAPWYIIPADYKWIARSLIADIISTKIRALDLKYPEVSAETLKQLEGAREQLAR E

[0438] SEO ID NO:36

[0439] MKTNLIKDLWRPNMKVKLNEWDPDYDGGIFKKEVESTLNKDLLDRMSDLQYKLFADKNQSLLIILQGIDTSGKD STIRHVMGAFNPQSCKVISFKAPNDEELSHDYLWRIHKVTPAKGEIVIFNRSHYEDVIEARVQRLTPEDMWFERY RQINEFERYLYENNIKTVKLLLHISKDEQKKRLEDRIKDPSKHWKFSEADIIKRKYWDQYITAYEEAISRCSTPWA PWYIIPANTKWFRNFAIANIWKSLEDMKLRFPAPKIDLSKWIDD

[0440] SEO ID NO:37

[0441] MNIKTSEFKVPEGIAVDLSKLPTQVEPVYRSKKEYKELLAEQVAALSAQQQLQYADNSHSLLLIFQAMDAAGKDG VIKHVMSGVNPQGCQVFSFKHPSAQELDHDFLWRTTQCLPERGRIGIFNRSYYEEVLIVRVHPEILKNQKLPDSL LNDDAIWPNRYKSIVDFEQHMHRNGTKVIKFFLHLSKDEQRKRFIDRIDQPEKNWKFTVSDINERKHWTQYMK AYESCLTATSTKEAPWYWPADDKKNARLIVSQIILDAYKDMDMKYPETSEERRQELLSIREQLIND

[0442] SEO ID NO:38

[0443] MKEPILAVLPQGPVDLTAIPTDDTGDFDGNKADGEKALADLGPELSELQERLFAERTAGSTRRVLWLQGMDTS GKGGVLRHTVGLVDPQGVRITSFKAPTDEERAHDFLWRIEKALPQPGTIGVFDRSHYEDVLIARVRKFAGDEEIE RRYGAINDFEQRLVDDGVTIVKCMLHISAEEQKARLLARLDDPSKFYKFNPGDLDERALWPAYREAYEIALERTN TDAAPWHVIPSDKKWFRNLCVGQLLLEALRGLELQWPAADFDVEAQKARLRDEVSPE SEO ID NO: 39

[0444] MGKSKSSENDKPKKIKSPEKKLKNFQKLSPKERIEHAKKFSEQFCVGDGNSFQLKDYKSDIDFDLGKGTKSLVAD TLELGVDALATMQEILYAQDKWSLLLVFQAMDAAGKDGAIKHVLSGVNPQGCQVSSFKAPSSEELDHDFLWRC QKHLPERGRIGIFNRSYYEEVLWRVHEHILKAQKLAEELITENIWEERFEDIKNFEKYLRRNGTVWKFFLNVSKD EQKKRFIERIDDADKNWKFSTADAKERQHWDEYMHAYEDLIKNTSTKDSPWYVIPADNKAYARIAIASAVIHTL DEMGLEYPKVSDEKVAELNEIKKALLSEE

[0445] SEO ID N0:4Q

[0446] MKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLWLQAMDAGGKDGTI RWFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLWRVKNLVPQQVWQKRYR HIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEY APWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE

[0447] SEO ID NO:41

[0448] MPHRHRLDHAPFTVQPGQRVRLKDYDSRETAGFKNKSEAKKALLEDVSLLAEMQQLLWAAKEYSLLIVFQALDA AGKDGAIRHVMSGVNPQGVDVYSFKAPTEEERLHHFLWRPAKVMPARGRIAIFNRSYYEEVLWRVHPEWLERQ WVPPRYRGGHYEELWPERFEEINAFERLMSENGVAILKFFLNVSKAEQRERFLERVTDPEKHWKYSAADLRERG YWDMYQAAYEDMLSHTSTKHAPWHVIPADRKWFTRACVADIIAAKIESLDLSYPTVSEEEHSAIEAARRQLEAE SD

[0449] SEO ID NO:42

[0450] M KTTTPKPTEGQRLQSTAPSFDGCPDWQALADAARVAPGTH VH LM RDH N PGRHTPGLARDGGEYALAEAKTLL LELQDRFFASSGHALVWLQAIDAAGKDGTIKHVMSGLNPVGVDVYSFKEPTPIEAAHDYLWRYHRVLPERGRIA VFNRSHYEAVLTTRVHPEFLVPPVADADLHHLWHRRFQEIKEWERYLHDNGTVIVKLFLNLSKGEQKRRFLQRLE NPQKNWKFSVNDLAERTHWDDYQFAFQEMLSHTSTTWAPWYWPADHKWFSHLTTSAVLVQALRGMDPQYP ALTDSARERLTAAKLRLDSEPC

[0451] SEO ID NO:43

[0452] MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAM DAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVTRVHPEYLLS EQTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKER AHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSL

[0453] SEO ID NO:44

[0454] MASRIEDMETRIEKLLDIMRVPPGKKIDLQKDYDPGFTGKWVTKEEAEETLAKGVQLLAEMQDKLWAQNQYALL MILQALDAAGKDGAIKHVMSGVNPQGVDVHSFKGPSSEELDHDYLWRNFKTLPSRGRIGIFNRSYYEEVLWRV HPEFLASQQLPPALKDKRIWRRRFEEINNFEKYLVDNGIIWKIFLYVSKEVQKERFLERAMMPEKNWKFSVADM KERAHWDDYIAAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYNTMKNLNLAYPTVSEQQKQALLAAK

[0455] DELEREDGGLKDKAWKAETKAATDPQAPDASSAVTKGKAKKKGKKKNK

[0456] SEO ID NO:45

[0457] MANRIEELLDVLRVPSGKKINLKKDYDPDFTGKWVKKEEAKEALTEGIQILAEMQDKLWAQDQYALLIILQALDA

[0458] AGKDSTIKHVMSGVNPQGVDVHSFKVPSGEELDHDYLWRNFKALPARGRIGIFNRSYYEEVLAVRVHPEFLAGQ

[0459] KLPPSLKDKNIWNRRFEEINNFEKYLVDNGIIWKLFLYVSKETQKERFLERTLEPEKNWKFSAADMKERAFWDD

[0460] YMAAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYNTMKSLNLAYPTVSEEQKQALLVAKEELEHEDGG LKDKAVTRAEAKTAADPKTLAARSKVTKEEVKKNSTKGKKNK

[0461] SEO ID NO:46

[0462] MDVSGPRVRKYLRQFVADPTKKIRLKKYDTARVPRKELSAESDDEAKALLKDLLDKNRQELARAQDLLAASAQYS

[0463] ILIVLQGMDTSGKDGTIRHVMSGINPQGCAVHSFKVPGAEEHAHDFLWRYEKVLPERGMIGIFNRSYYEDVLW

[0464] HVHPDRMESLPAAIGPENAGFWEGRYKDINAFEKHLVRNGTWLKFFLHISRDEQKRRLLDRLSHQEKFWKFSF TDIAERQYWDQYQVAYEEMLSATSTEYAPWFIVPADHKWVARTWAEVIASAIEGLHLCYPVFPDEKIRQLEKAK AALERE

[0465] SEO ID NO:47

[0466] MDTLKDLRSLIVEPGRKFDLADRATALDLPGDPAKEEIEAELATLREQIGDIQDRMFTRHEQALLWLQGMDSAG

[0467] KDSTTKAVFTGVNPLGIRAASFSVPTPLERRHDYLWRIHAQVPQLGCIGVFNRSHYEDPVIRRVHGEIKNGAMK

[0468] QYLQQIAEFERLLTETGTTIVKCFLHIGFDEQRERLQERIDNPAKNWKFDPSDLQERRYWKAYQSAYSEALTSTS TEQAPWYWPADRKPARNLLVARLVLQALKGLSAPEPAPKPELKGLKVR

[0469] SEO ID NO:48

[0470] MARSTAYLKTYRVGRKLRLKDIDPGARPAASSSREADDARLAELAIEIDRLQDLLYANGSAGRPPKLLLVLQGMD

[0471] TSGKDGTARSVFRQCSPLGVRVAAFKAPTEVERAHDFLWRVHAVAPRAGEWVFNRSHYEDVLVPFVEGWIDA

[0472] AERQRRLAHINAFERLLHDSGTTIVKCFLHISKDEQRERLQARLDDPAKRWKFQVGDLETREKWKAYLAAYETA LAATSTACAPWH WPADSKSN RN LMIATLVAQALAGM KLKPPKPDFDPAAVRW

[0473] SEO ID NO:49

[0474] MLFKPERYLWRRQSAPDLRQCATRLPYEDKDSRDQLKREAKDLLEESQKELIESQHRLNVIDTHAVLLILQGMDA

[0475] AGKDGIIKHVLSGLNPLYCQITGFRAPSSIELEYPFLSRYVAHLPQRGHIGLFNRSWYEETVrVKIFPEFLHKQRIA

[0476] LEAINDRFWQHRFEDINHFEQHLTRNGIAIIKCFLHVSREEQRQRLLDRWKPEKFWKFDPFDLETRKRWDEFM

[0477] QAWNETLTATDSEHAPWHVIPADDKPMARALVARLLASTIDALPLKEKSFSETQLERIKEARMILENEKDDSAEH D SEO ID NO: 50

[0478] MNLKDYCFFGNKPFVIEQFDPAQTGKFQLKNEVEELHHTNLLKMQELQDRLYAENKEALLIIFQGMDASGKDSA

[0479] VKHVMGGVNPQGINVHNFKKPSSEELDHDYMWRSMRVIPERGKIGVFNRSYYEDVLVGKVHKLYQESYLPDRC KTDKIFEQRYQQIKNYERYLYENGIRVIKFFLHISKEEQKKRFLERIEDDQKNWKFSDSDMIERDYWEEYQRAYH DAINATATRIAPWYWPSDKKWFGHFIISETIIDTLEKINPQYPAVTRERKERLLEFRSKLLREEEQSSF

[0480] SEO ID NO:51

[0481] MQLDRYRVPPGQRVRLSNWPTDDDGGLSKAEGEALLPDLQQRLANLQERLYAESQQALLIVLQARDAGGKDGT

[0482] VKHVIGAFNPSGVQVSNFKVPTEEERAHDFLWRIHRQTPRLGMIGVFNRSQYEDVLVTRVHHLIDDQTAQRRLK HICAFESLLTDSGTRIVKFYLHISPEEQKKRLEARLADPSKHWKFNPGDLQERAHWDAYTAVYEDVLTTSTPAAP WYWPADRKWFRNLLVSQILVQTLEEMNPQFPAPAFNAADLRIV

[0483] SEO ID NO: 52

[0484] MDTSQFQVSNPDSYVLADAPTVAPSDFDREESKRRVKQLTKQIQALQEKMWAGRKHRLLWLQAIDAGGKDGT

[0485] VRSVFGRTNPQGVKVHSFKRPTEQELAHDYLWRVHPHVPGNGEITVFNRSHYEDVLWKVHGYAPLDWEKRY QHIRNFEQMLVDEGTTXIKIFLHISKDEQKKRFEARLNRPEKNWKFKKGDIKERAFWEQYHEVFEQMLKKTTTD DSPWYWPADNKKYRNEIISSLILETLESFKMQWPQPEEDLSDIVIE

[0486] SEO ID NO: 53

[0487] MIFNGENSVLAEELIAKFRIDEGQKVKLRDYDTGWKVSDEIAYFQKEEVKNKAKDILEKNREDLARGQSLLWANN

[0488] TYSLLVIIQGMDTAGKDSLIKHVMSGVNPQGCRVSSFDVPSKEELDHDYFWRCNLKLPKRGEIGIFNRSYYEEVLI TKVHPEILEGRHLPQKEFHDEFWKMRYEDINSYERHLSNSGTWLKFFLHISKEKQKERLLARFENESKYWKISPS DIAERKFWDNYIRAYEDMLNNTSTKSAPWYIIPADYKWVARTIVSDIIASKLISLHDSYPAVSEETLEKIKKAADM LKNE

[0489] SEO ID NO: 54

[0490] MKNEFKGLIVQGDKKFSLKNHKTDFTGGYNKEKAKDALVNSKIELSHLQEKLYASGKHSVLIIFQAMDAAGKDSA

[0491] IEHVMSGLNPQGCQVYSFKVPTSEEYQHDFLWRHYKALPERGRIGIHNRSHYENVLVCKVHPEYILSEDIPGITD VKKISKDFWKQRYQSIRNFEQHLTANGTVILKFFLNVSKDEQKQRFLDRIEDPSKNWKFSSGDIKERALWDKYM EAYQDAINETSTAESPWHIIPADKKWFARLAISEIIEDRLKNLELKFPVLGAEETLKLSETKNALLAE

[0492] SEO ID NO:55

[0493] MIPQDFLDEINPDRYIVPAGGNFHWKDYDPGDTAGLKSKVEAQELLAAGIKKLAAYQDVLYAQNIYGLLIIFQAM

[0494] DAAGKDSTIKHVMSGLNPQACRVYSFKAPSAEELDHDFLWRANRALPERGCIGIFNRSYYEEVLWRVHPDLLNR QQLPPETKTKHIWKERFEDINHYERYLTRNGILILKFFLHISKAEQKKRFLERISRPEKNWKFSIEDVRDRAHWDD YQQAYADVFRHTSTKWAPWHIIPANHKWFARLMVAHFIYQKLASLNLHYPMLSEAHREQLLEAKALLENEPDED SEO ID NO: 56

[0495] MVGKYRVDGKKSIKLKDFPTADKGIFKNKEEGLLKLGENIEIISELQNKLYAEDTYSLLIIFQAMDAAGKDGTIKHV

[0496] FSGINPQGFQIFNFKQPSREELDHTYMWRTSKSMPERGRIGVFNRSYYEDVLWRVHNLLSTQHLPKEKQYKDI WKRRFHNIREQERYLFENGVIPIKFFLNVSKEVQKERFLERIEDPSKNWKFSSADIEERKYWDDYQDAYEDAINH TSTSYAPWYVIPADKKWFAKFAVSEIIKETLESLNLKYPELNKTQKSELKNYKEILLKEK

[0497] SEO ID NO: 57

[0498] MGFCSIEFLMGAQMKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLW

[0499] LQAMDAGGKDGTIRWFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLWRVK NLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQ EAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE

[0500] SEO ID NO: 58

[0501] MKINSEDFIVSTAGRKKVNLKHRPTIVKPLYGSKKQYKEILNEHIEELSSLQELLFASNRYSLLVIFEGMDAAGKDS

[0502] AIKHVMSGVNPQGCQVFSFKRPTPIELQHDFLWRYWCLPERGRIGIFNRSYYEGVLAARVHPEVDLRSEGFLDQ DDGPAADEEKKKEEVWEQRYESIVNLEKHLYHNGTRIIKIFLHLSEEEQRKRFLERIDDPKRNWKFSLADVEERK FWHDYMKAYEDCLSATSTTTYAPWYVVPADNKENARLIISEIILETLNGLEMSYPKLDPKRLDELQSTRKKLLLNK

[0503] SEO ID NO: 59

[0504] MDTSEFLISNAEDFALSNWQVDPPAEFDEKQSKRTLKQNTKVLQSLQKLMWAERKQRVLWLQAIDAGGKDGT

[0505] IRSVFGGLNPQGVKVQSFKRPTKQELAHDYLWRVHKHVPGNGEIMVFNRSHYEDVLWKVHEYAPLDLVEKRYE HIRNFEQLLADEGTTIIKIFLHISKDEQKRRFEDRLNEPDKNWKFKKEDLKERAFWDQYQDAFETMIEQTSTKDA PWYWPSNNKKYRNEIISNLLIETLSGLNMEFPPAADGLDNIVID

[0506] SEO ID NO: 60

[0507] MSAIRLTDIPTRAPGNMDKKKTRKATHKLKKAIADLQNILYAEAKHSLLVILQGMDASGKDGAIRHVFNRINPQG

[0508] IQVTSFKVPTPEEAAHDFLWRIHAHAPARGMMHIFNRSHYEDVLVTRVHGWIDDDTAHRRMQHINHFEQLLLD HQTTILKFYLHISREEQAKRLQERLKNPKKRWKYNPSDLEEAQKWHDYLHYYEEVFATCSPEIPWIIVPADQNW YKEFVIAEAVHQALKNMQLRYPRRASLASAGHAHNRKK

[0509] SEO ID NO:61

[0510] MSNGTRQPIVYPPHTRVDLRKFDPAATNGMSREEAEAELVGLRLRLNELQNMLYADARYALLWLQGIDAAGKD

[0511] GTINSVFEQVGPIGCSWSFKAPTPEELAHDYLWRYHRVMPERGHITIFNRSYYEAVLVERVKGIVPKATWEQRY EDINRLEEYLLRNGTWMKFFLAISKEEQRERLQERIDNPKKQWKFRLGDLEERKLWDEYLEAFEDMLERCNTKH APWHWPADRKWYRDWVARTLVEKLASLGLRYPPADPAVLGLKVD SEO ID NO: 62

[0512] MANRTEDMETRIEDVEARIEALMEFMRVPPGRKINLRKDYDPGFTGRWLKKAEAKETLARGIQMLAEMQDKLW

[0513] AQNQYALLMVLQALDAAGKDGTIKHVMSGVNPQGVDVYSFKAPSGEERDHDYLWRNFRALPARGDIGIFNRSY

[0514] YEEVLWRVHPEILAGQQLPPALKDEGIWKRRFEEINNFEKYLVDNGIIWKFFLYVSKEAQKKRFLKRTMLPEKN WKFSAADIKERAH WDDYLDAYEDM FN HTSTEWAPWYIVPADH KWFTRLAVAAVLYSTM KKLNLAYPTVSEQK KQALLVAKEELENEDGGKKDKAWKAKAKAAANKKVPASGREGTRNESKKKGKKNR

[0515] SEO ID NO: 63

[0516] MATPLEKLADIFRVPPGRNIDLRKEYDPGFTSDSVSKENAQKYLETGIQKLAEQQDKLYAQNTLALLINFQAMDA

[0517] AGKDSTIKQVMSGVNPQGVQVKSFKVPSAEELDHDYLWRTFLELPNRGHIGIFNRSYYEEVLWRVHPEFLERQ

[0518] QLPPKQKGEDIWKRRYGEINNFEKYLTDNGIWLKFFLNVSKEEQKKRFLERIEEPEKNWKFSANDVLERQYWD RYMDAYEEMFNNTSTPWAPWYIIPADHKWFTRFAWSVINYTIDGMNLAYPSVSAEQKEALQKAKEAMEQEET HPEKTTGTKKKRKR

[0519] SEO ID NO: 64

[0520] MKIRSKDFRVPPGEKLKLDEWPTLIKPAYKSKKAYAELLQEHVAELSTLQQLHYASGRYALLLIFQAMDAAGKDG

[0521] AIKHVMSGVNPQGCQVFSFKHPSATELEHDFLWRTNQCLPERGRIGIFNRSYYEEVLIVRVHPEILRSQGLPDGA

[0522] VDEKTVWKERYRSIVDLENHLYRNGTRIIKFFLHLSAEEQRKRFLERIDEPEKNWKFSLADIQERKFWPQYMQAY EACLGATSTKIAPWHWPADDKENARLIVSQIILDAFKALKMRYPETDAKRRQELLSIRHGLSEEGRRNFSPQ

[0523] SEO ID NO:65

[0524] MIKLIKDDRITITSGKNFKLKNFDTDFSLDFVSKESAGVIMQEHIQRIAALQERLYASDNHAILMIFQARDAAGKD

[0525] STIKHVMSGINPSGCQWSFKVPTSTEIEHDFLWRTAVQLPERGRIGIFNRSYYEEVLVTKVHPEIILNQRLPSVLT

[0526] EKDLDKKFWEHRYESIRDHEKHLSRNGFVILKFFLNVSKKEQKKRFLERIDQPDKNWKFSYGDIKERQYWDQYE DAFEQMIEETAEEHAPWFVIPSDKKWFAQLAVARILLETLESLDLKFPELGDEEKAVLEKAKMELESEKD

[0527] SEO ID NO: 66

[0528] MKAINTEDFKVTGKFDVKGVPTFIDVETSDKKIANSLEEVSIKLGKLQDKMYANNRYSVLICLQGMDTAGKDSLI

[0529] REVFKNFNARGVNVYSFKKPSSSELEHDYLWRHYIALPEKGKFAVFNRTHYENVLVTRVHPEFLMYENLPGIETV

[0530] EEIPADLWEQRYEQINNFEKHISANGTIIFKFFLHLGKDEQKKRLLRRLEKQSHNWKFSPGDLTERDLWDKYQQ YYEEAINKTSKEHAPWYVIPADKKETARYLVAKTILDTLKKYDDIKEPEVSEDVLANISLYRDQLMNEE

Claims

CLAIMS1. Use of a polyphosphate kinase (PPK) to produce a sugar-modified and / or o-phosphate- modified nucleoside triphosphate from a correspondingly modified nucleoside monophosphate or nucleoside diphosphate, and polyphosphate.

2. The use of claim 1, wherein the PPK is a PPK2.

3. The use of claim 1 or claim 2, wherein the PPK is a PPK2-III.

4. The use of any one of claims 1 to 3, wherein the PPK has a sequence that is at least 90% identical to SEQ ID NO:22.

5. The use of any one of claims 1 to 4, wherein the PPK has the sequence of SEQ ID NO:22.

6. A method of screening a panel of putative and known PPKs to identify an enzyme capable of using a sugar-modified and / or o-phosphate-modified nucleoside monophosphate or nucleoside diphosphate as a substrate, to produce a correspondingly modified nucleoside triphosphate, comprising: a) identifying sequences encoding said panel of putative and known PPKs; b) synthesizing said PPKs; c) purifying said PPKs; d) mixing each of said purified PPKs with said sugar-modified and / or o-phosphate- modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphate to form a reaction mixture; and e) analysing each of the reaction mixtures for the presence of correspondingly modified nucleoside triphosphate to determine which enzyme(s) produce correspondingly modified nucleoside triphosphates.

7. The method of claim 6, wherein the sequences in step a) are identified using sequence databases and bioinformatic tools.

8. The method of claim 6 or claim 7, wherein the sequences in step a) encode proteins that are between 150 and 600 amino acid residues in length.

9. The method of any one of claims 6 to 8, wherein the PPKs are synthesized in step b) with an N-terminal hexa-histidine tag and are purified in step c) using nickel nitriloacetate.

10. The method of any one of claims 6 to 9, wherein step d) comprises incubating said sugar-modified and / or o-phosphate-modified nucleoside monophosphates or nucleoside diphosphates, polyphosphate and PPK at 25 °C for 24 hours and quenching the reaction.

11. The method of claim 10, wherein the reaction is quenched with EDTA at 80 °C for 15 minutes.

12. The method of any one of claims 6 to 11, wherein the reaction mixture is centrifuged, and the supernatant is analysed for the presence of the correspondingly modified nucleoside triphosphate.

13. The method of claim 12, wherein the reaction mixture is centrifuged for 15 minutes at 4000 g.

14. The method of claim 12 or claim 13, wherein the supernatant is analysed using HPLC or UPLC.

15. The method of any one of claims 6 to 14, wherein the enzyme capable of producing the correspondingly modified nucleoside triphosphate is further engineered to increase the % conversion to the correspondingly modified nucleoside triphosphate.

16. An enzymatic method of producing sugar-modified and / or o-phosphate modified nucleoside triphosphates, comprising: incubating a PPK with correspondingly modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphate under suitable conditions.

17. The method of claim 16, further comprising incubating with a divalent cation.

18. The method of claim 17, wherein the divalent cation is Mg2+, Mn2+or Co2+.

19. The method of claim 16, further comprising incubating with magnesium chloride.

20. The method of any one of claims 16 to 19, wherein the PPK is selected using the method of any one of claims 6 to 15.

21. Use of modified nucleoside triphosphates obtained by the method of any one of claims 16 to 20 in the manufacture of a polynucleotide.

22. The use of claim 21, wherein the polynucleotide is a therapeutic polynucleotide.

23. The use of claim 22, wherein the polynucleotide is a therapeutic mRNA.

24. The use of claim 21 or claim 22, where in the polynucleotide is an oligonucleotide.

25. The use of claim 24, wherein the oligonucleotide is selected from the group consisting of: an antisense oligonucleotide, siRNA, miRNA, aptamer, CRISPR guide RNA, and an oligonucleotide used to recruit and guide DNA and RNA editing enzymes.

26. The use of claim 25, wherein the oligonucleotide used to recruit and guide DNA and RNA editing enzymes is an A to I RNA base-editing oligonucleotide (AIMer).

27. The use of any one of claims 21 to 26, wherein the manufacture of a polynucleotide is via an enzymatic method.

28. The use of claim 27 , wherein the enzyme is a polymerase.

29. The use of claim 28, wherein the polymerase is a template-dependent polymerase.

30. The use of claim 28, wherein the polymerase is a template-independent polymerase.

31. The use of claim 27 , wherein the enzyme is a terminal deoxynucleotidyl transferase (TdT).

32. The use or method of any one of the preceding claims, wherein the sugar modification is a 2'-sugar modification.

33. The use or method of claim 32, wherein the 2'-sugar modification is selected from the group consisting of: H, OH, O-CH3 (OMe), O-CH2-CH3 (OEt), O-CH2-CH2-CH3, O-CH2- CH2-O-CH3 (MOE), F, O-CH2-CO-NH-CH3, O-CH2-CH2-O-Nme2, O-methylene bridge with the 4' carbon, and O-constrained ethylene bridge with the 4' carbon;34. The use or method of claim 32, wherein the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate comprises a locked nucleic acid (LNA).

35. The use or method of claim 32, wherein the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate comprises a constrained ethyl (cEt).

36. The use or method of claim 33, wherein the 2'-sugar modification is selected from the group consisting of: 2'-OMe, 2'-MOE and 2'-FNA.

37. The use or method of claim 36, wherein the 2'-sugar modification is 2'-OMe.

38. The use or method of claim 36, wherein the 2'-sugar modification is 2'-MOE.

39. The use or method of claim 36, wherein the 2'-sugar modification is 2'-FNA.

40. The use or method of any one of the preceding claims, wherein the sugar modification is a 3'-sugar modification.

41. The use or method of claim 40, wherein the 3'-sugar modification is selected from the group consisting of: phosphate (PO), phosphoroth ioate (PS), phosphoramidate (PN), phosphorod ith ioate, methyl phosphonate, boranophosphate, phosphoroselenoate, N- methyl-anthraniloyl (MANT), acetyl (Ac), benzyl (Bn), benzoyl (Bz), allyl (All), 2- cyanoethyl (CE), azidomethyl (AzM), cyanovinyl (Cv), methoxymethyl (MOM), methoxyethoxymethyl (MEM), methoxymethyl phenyl methyl (MPM), di phenyl methyl (DPM), tetrahydropyranyl (THP), dimethoxytrityl (DMTr), methoxycarbonylmethyl (MOMcl), ethoxycarbonyl (EOC), benzyl carbonyl (Cbz), trifluoromethanesulfonate (triflate), methanesulfonate (mesylate), p-Toluenesulfonate (tosylate), pivaloyl (Piv), ortho-Nitrobenzyl (o-NB), bromo-methyl-coumarinylmethyl (Bhc), (7- (Diethylamino)coumarin-4-yl)methyl (DEACM), 6-Nitroveratryloxycarbonyl (NVOC), and 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc-ol).

42. The use or method of claim 41, wherein the 3'-sugar modification is PO or MANT.

43. The use or method of any one of the preceding claims, wherein the o-phosphate- modification is selected from the group consisting of: PS, PN, methyl phosphonate, boranophosphate, and phosphoroselenoate.

44. The use or method of any one of the preceding claims, wherein the o-phosphate- modification is stereospecific.

45. The use or method of claim 43 or claim 44, wherein the o-phosphate-modification is PS.

46. The use or method of claim 43 or claim 44, wherein the o-phosphate-modification is PN.

47. The use or method of any one of the preceding claims, wherein the base in the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate is selected from the group consisting of: adenine (A), uracil (U), cytosine (C), guanine (G), thymine (T), 5-methylcytosine (m5C), 5-hydroxy methylcytosine (hm5C), 5-fluorocytosine (5fC), 5-Bromouracil (5BrU), 2-aminopurine (2AP), inosine (I), pseudouracil Cl1), Nl- methylpseudouracil (ml ), 1-ethylpseudouracil, 2-thio-l-methyl-l-deaza-pseudouracil, 2-thio-l-methyl-pseudouracil, 2-thio-5-aza-uracil, 2-thio-dihydropseudouracil, 2-thio- dihydrouracil, 2-thio-pseudouriacil, 4-methoxy-2-thio-pseudouracil, 4-methoxy- pseudouracil, 4-thio-l-methyl-pseudouracil, 4-thio-pseudouracil, 5-aza-uracil, dihydropseudouracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyl uracil (2moll), 7-deazaguanine (7-deaza-G), 8-oxoguanine (8-oxo-G), 5-propynyluracil (pU), 5- propynylcytosine (pC), N6-methyladenine (m6A), N4-acetylcytosine (ac4C), 06- methylguanine (06meG), hypoxanthine (H), xanthine (X), isocytosine (iC), isoguanine (iG), queuine (Q), 2-thiouracil (s2U), 4-thiouracil (s4U), 5-iodouracil (5Iu), 2-thiocytosine (s2C), 5-chlorouracil (5CIU) and isouracil.

48. The use or method of claim 47, wherein the base in the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate is selected from the group consisting of: A, U, C, G, T, m5C, I, I1and mil1.

49. The use or method of claim 47 or claim 48, wherein the base in the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate is selected from the group consisting of: A, U, C, G, and T.

50. The use or method of claim 47 or claim 48, wherein the base in the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate is m5C.

51. The use or method of claim 47 or claim 48, wherein the base in the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate is ml .

52. Use of a PPK to produce a stereospecific o-phosphate-modified nucleoside triphosphate from an o-phosphate-modified modified nucleoside monophosphate or nucleoside diphosphate, and polyphosphate.

53. The use of claim 52, wherein the stereospecific o-phosphate-modified nucleoside triphosphate is a stereospecific PS-modified nucleoside triphosphate.

54. The use of claim 53, wherein there is about a 50:50 ratio of the 5^ diastereomer to the RPdiastereomer, about a 55:45 ratio of the SPdiastereomer to the RPdiastereomer, or about a 55:45 ratio of the RPdiastereomer to the SPdiastereomer.

55. The use of claim 53, wherein the 5P.7?Pratio is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2 or 99:1.

56. The use of claim 53, wherein the ^:S> ratio is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2 or 99:1.

57. An engineered PPK having at least 90% sequence identity to SEQ ID NO:22.

58. A method of manufacturing a polynucleotide comprising incubating nucleoside triphosphates, a suitable enzyme, and optionally a template, under suitable conditions, wherein the nucleoside triphosphates are made by incubating the corresponding nucleoside monophosphates and / or nucleoside diphosphates, PPK and polyphosphate under suitable conditions.

59. The method of claim 58, wherein the nucleoside triphosphates comprise or consist of naturally occurring nucleoside triphosphates.

60. The method of claim 58, wherein the nucleoside triphosphates comprise or consist of modified nucleoside triphosphates.

61. The method of claim 59, wherein the nucleoside triphosphates consist of naturally occurring nucleoside triphosphates, except that uridine triphosphate is replaced with Nl- methyl pseudouridine triphosphate.

62. The method of claim 60, wherein one or more or all of the nucleoside triphosphates are modified nucleoside triphosphates comprising a sugar, o-phosphate and / or base modification.

63. The method of claim 62 wherein the modified nucleoside triphosphate is a base-modified nucleoside triphosphate.

64. The method of any one of claims 58 to 63, wherein the enzyme is a polymerase.

65. The method of claim 64, wherein the polymerase is a template-dependent polymerase.

66. The method of claim 64, wherein the polymerase is a template-independent polymerase.

67. The method of any one of claims 58 to 63, wherein the enzyme is a terminal deoxynucleotidyl transferase (TdT).

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