Method for producing nucleoside triphosphate (NTP)

Abstract

The present invention relates to a novel enzymatic method for producing nucleoside triphosphates (NTP), 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

Technical Field

[0001] This invention relates to a novel enzymatic method for producing nucleoside triphosphates (NTPs), particularly modified NTPs. The NTPs generated by the method of this invention can be used in methods for producing therapeutic polynucleotides (including oligonucleotides). Background Technology

[0002] Nucleoside triphosphates (NTPs) are fundamental building blocks of 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 the primary 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 through cell fermentation and purification methods.

[0003] NTPs modified with sugar, base, and / or phosphate groups are valuable laboratory tools and also useful in therapeutics, such as in the preparation of nucleotide-based therapeutics (e.g., therapeutic oligonucleotides and mRNA), because they help enhance affinity for targets, improve nuclease resistance, alter cycling characteristics, and modulate immune properties (Kulkarnie et al, Nature Nanotechnology, 2021, 16, 630-643).

[0004] Some wild-type polymerases and engineered polymerases can utilize modified NTPs to produce modified polynucleotides complementary to the template sequence (Milisavljevic et al., Org. Biomol. Chem, 2018, 16, 5800-5807; and Sabat et al., Front Chem., 2023, 11, 1161462). Additionally, terminal deoxynucleotidyl transferases (TdTs) can utilize modified NTPs to produce modified oligonucleotides in a template-independent manner (WO2019 / 121500).

[0005] However, modified NTPs are difficult to generate and expensive to produce, especially for large-scale production. Synthetic reaction schemes for generating modified NTPs are listed in Marcel Hollenstein's 2012 review paper (Molecules, 2012, 17, 13569-13591), and include the Yoshikawa scheme, the Ludwig-Eckstein method, the Borch method, and other strategies (see also Burgess and Cook, Chem. Rev. 2000, 100, 2047-2059).

[0006] Recently, biocatalytic methods for the production of specific NTPs have been investigated. MK-1454, a cyclic dinucleotide stimulator (STING) agonist of the interferon gene, is being developed as an immuno-oncology therapeutic. MK-1454 has been diastereoselectively assembled from two non-natural nucleoside thiomonophosphates via a one-pot cascade reaction, in which two thiotriphosphate nucleotides are simultaneously generated biocatalystically, followed by coupling and cyclization catalyzed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the synthesis of thiotriphosphates, three kinases were engineered to develop a non-natural cofactor recycling system (McIntosh et al., Nature, 2022, 603, 439-444).

[0007] However, there is a need for simpler and more cost-effective methods to generate NTPs, especially modified NTPs, which can be used on a large scale, particularly in the preparation of polynucleotide therapeutics. Invention Overview

[0009] In a first aspect of the invention, the use of polyphosphate kinase (PPK) in the production of sugar-modified and / or α-phosphate-modified nucleoside triphosphates is provided, wherein the sugar-modified and / or α-phosphate-modified nucleoside triphosphates are produced from correspondingly modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

[0010] In a second aspect of the invention, a method is provided for screening a set of putative and known PPKs to identify enzymes capable of producing corresponding modified nucleoside triphosphates using sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates as substrates. The method comprises: a) identifying sequences encoding the set of putative and known PPKs; b) synthesizing the PPKs; c) purifying the PPKs; d) mixing each of the purified PPKs with the sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates to form a reaction mixture; and e) analyzing the presence of the corresponding modified nucleoside triphosphate in each reaction mixture to determine which enzyme(s) produce the corresponding modified nucleoside triphosphate.

[0011] In a third aspect of the invention, an enzymatic method for producing sugar-modified and / or α-phosphate-modified nucleoside triphosphates is provided, the method comprising incubating PPK with correspondingly modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates under suitable conditions.

[0012] In a fourth aspect of the invention, the use of modified nucleoside triphosphates obtained by the method of the invention in the preparation of polynucleotides is provided.

[0013] In a fifth aspect of the invention, the use of PPK to generate stereospecific α-phosphate-modified nucleoside triphosphates from α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates is provided.

[0014] In a sixth aspect of the invention, an engineered PPK having at least 90% sequence identity with SEQ ID NO: 22 is provided.

[0015] In a seventh aspect of the invention, a method for preparing polynucleotides is provided, the method comprising incubating a nucleoside triphosphate, a suitable enzyme, and optionally a template under suitable conditions, wherein the nucleoside triphosphate is prepared by incubating a corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating preferred catalytic reactions associated with ATP generation in the PPK2 subfamily.

[0017] Figure 2 The chromatogram from Example 5 depicts the 2'OMe-pGMP peak and the absence of the 2'OMe-pGDP and 2'OMe-pGTP peaks in the negative control sample [top trace]; and the reduction of the 2'OMe-pGMP peak in the PPK (SEQ ID NO: 31) sample, and two new peaks corresponding to the formation of 2'OMe-pGDP and 2'OMe-pGTP [bottom trace].

[0018] Figure 3 This is a chromatogram from Example 12, showing the stereoselectivity preferences of three different PPKs in the formation of 5'-(α-thio)triphosphoric acid. The PPK of SEQ ID NO: 8 [bottom trace] produces approximately 50:50 S... p :R p Diastereomers. PPK [middle and top traces] with SEQ ID NO: 34 and 47 have opposite stereoselective preferences, one of which favors S. p Diastereomers, and another favorable factor for R p Diastereomers. Invention Details

[0020] definition

[0021] As used herein, “Ambo” is a prefix to indicate that a molecule having two (or more) chiral elements exists as a mixture of two racemic diastereomers in an unspecified proportion (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.

[0022] As used in the context of enzymes in this article, “derived from” identifies the starting enzyme on which the engineering modification is based and / or the gene encoding such an enzyme.

[0023] As used herein, “enzymatic synthesis” of the production of polynucleotides (including oligonucleotides) means the preparation of said polynucleotides using enzymes (e.g., polymerases, ligases, transferases, phosphatases, kinases (including polyphosphate kinases), and nucleases (e.g., endonucleases). These enzymes may be wild-type enzymes, mutant enzymes, or engineered enzymes. Mutant or engineered enzymes capable of acting on modified nucleotide or oligonucleotide substrates are within the scope of this disclosure. “Enzymatic method” refers to a method relating to enzymatic synthesis.

[0024] As used herein, "engineered enzyme" refers to an enzyme in which the natural or native form of the enzyme, referred to as "wild-type," has been modified in a manner not found in nature. Modifications may include, for example, one or more deletions, insertions, and / or substitutions of amino acids in the wild-type sequence of the enzyme.

[0025] As used herein, “modified nucleotide,” “modified nucleoside monophosphate, diphosphate, or triphosphate,” or “modified polynucleotide or oligonucleotide” means a nucleotide residue, nucleoside monophosphate / diphosphate / triphosphate, or polynucleotide or oligonucleotide that contains at least one aspect of its chemical properties that differs from those of naturally occurring nucleotide residues, nucleoside monophosphate / diphosphate / triphosphate, or polynucleotide or oligonucleotide. A modified polynucleotide or oligonucleotide contains at least one modified nucleotide. Modification can occur at any part of the nucleotide residue, such as modification of the sugar moiety (“sugar-modified”), modification of the nucleobase (“nucleobase-modified”), and / or modification of the phosphate / backbone (“α-phosphate-modified”). One or more identically or differently modified nucleotide residues can form part of a modified polynucleotide or oligonucleotide.

[0026] The term "messenger RNA" (mRNA) refers to a specific type of RNA molecule. In vivo, DNA transcription typically results in so-called mature RNA, which must be processed into so-called messenger RNA, often abbreviated as mRNA. In eukaryotes, for example, the processing of mature RNA involves a variety of different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called mRNA maturation. Mature mRNA typically provides a nucleotide sequence that can be translated into an amino acid sequence of a specific peptide or protein. Typically, mature mRNA contains a 5' cap, a 5' untranslated region (5'UTR), an open reading frame, a 3' untranslated region (3'UTR), and a homocoupling, such as a poly-A or poly-C sequence. In the context of this disclosure, mRNA can be an artificial molecule, i.e., a molecule that does not exist in nature. This means that mRNA in the context of this disclosure can contain a combination of a 5'UTR, an open reading frame, a 3'UTR, and a poly-A sequence that does not exist in nature. In the context of this disclosure, mRNA includes at least one modified nucleotide residue.

[0027] As used herein, “oligonucleotide” or simply “oligo” refers to a polymer of nucleotide residues. The term “oligonucleotide” is generally used for shorter sequences than the term “polynucleotide,” typically ranging from 3 to 30 nucleotides. These can include deoxyribonucleotides (where the resulting oligonucleotide is DNA), ribonucleotides (where the resulting oligonucleotide is RNA), modified nucleotides, or mixtures thereof. Oligonucleotides can be single-stranded, such as antisense molecules, or double-stranded, such as siRNA molecules.

[0028] The "percentage of identity" or "identity %" between the query nucleic acid sequence and the subject nucleic acid sequence is a percentage value of "identity" calculated over the entire length of the query sequence using appropriate algorithms (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GeneQuest, EMBOSS needle, or EMBOSS infoalign) after pairwise global sequence alignment using appropriate algorithms (e.g., Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR). Importantly, the query nucleic acid sequence can be described by the nucleic acid sequence description disclosed herein.

[0029] The "percentage of identity" or "% of identity" between the query amino acid sequence and the subject amino acid sequence is a percentage value of "identity" calculated over the entire length of the query sequence using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GeneQuest, EMBOSS needle, or EMBOSS infoalign) after pairwise global sequence alignment using a suitable algorithm (e.g., Needleman-Wunsch, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GeneQuest, EMBOSS needle, or EMBOSS infoalign). Importantly, the query amino acid sequence may be described by the amino acid sequence disclosed herein, particularly in one or more claims, such as SEQ ID NO: 22.

[0030] The query sequence may be 100% identical to the subject sequence, or compared to the subject sequence, the query sequence may include at most an integer number of amino acid or nucleotide changes such that the identity percentage 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 changes include at least one nucleotide residue deletion, substitution, or insertion, said change may occur at the 5' or 3' end of the query sequence or anywhere between those end positions, individually scattered among nucleotide residues in the query sequence or in one or more consecutive groups within the query sequence. In the case of amino acid sequences, such changes include at least one amino acid residue deletion, substitution (including conserved and non-conserved substitutions), or insertion, said change may occur at the N-terminal or C-terminal end of the query sequence or anywhere between those end positions, individually scattered among amino acid residues in the query sequence or in one or more consecutive groups within the query sequence.

[0031] As used herein, a “polynucleotide” is a polymer of nucleotide residues. A single polynucleotide molecule may contain, for example, 14 or more nucleotide residues in a chain structure. DNA and RNA are examples of polynucleotides. Polynucleotides include oligonucleotides. Polynucleotides are useful for therapeutic purposes, for example, in the production of therapeutic mRNAs (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 base-editing oligonucleotides (AIMer).

[0032] As used herein, “polyphosphate kinase,” “PolyP kinase,” or “PPK” is an enzyme capable of producing nucleoside triphosphates from polyphosphates and nucleoside monophosphates and / or nucleoside diphosphates. PPK can also produce modified nucleoside triphosphates from polyphosphates and correspondingly modified nucleoside monophosphates and / or diphosphates.

[0033] As used herein, "polymerase" refers to an enzyme that catalyzes the linking (i.e., covalent linking) of a nucleotide to the 3'-OH of another nucleotide, oligonucleotide, or polynucleotide, for example, by forming a phosphodiester bond between the 3' end of one nucleotide, oligonucleotide, or polynucleotide and the 5' end of another nucleotide. Therefore, polymerase activity ranges from 5' to 3'. Polymerases can include DNA and / or RNA polymerases. Polymerases can be wild-type enzymes, mutant enzymes, or engineered enzymes.

[0034] As used herein, "primer" means a polynucleotide or oligonucleotide sequence used as a starting point for synthesizing the fragment polynucleotide or oligonucleotide disclosed herein. Polymerases may require primers; for example, DNA polymerases may require primers. Primers may contain at least three nucleotides. Primers may bind to the template before the polymerase catalyzes the ligation of nucleotides. Primers may not be removed. Primers may form part of a polynucleotide or oligonucleotide template. For example, the template may contain a hairpin loop that contains the primer. Primers may form part of a polynucleotide or oligonucleotide product.

[0035] As used herein, “product polynucleotide or oligonucleotide” means a desired polynucleotide or oligonucleotide having a specific sequence, and is also referred to herein as “target polynucleotide,” “target oligonucleotide,” or “target oligo.”

[0036] As used herein, a "protecting group" is a group attached to another reactive 3' hydroxyl position of a nucleotide sugar to prevent undesirable side effects in a given reaction scheme. Protecting groups contribute to the selectivity of the desired reaction and are removed once the desired reaction has occurred.

[0037] "RNA" is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotide monomers. These nucleotides are typically adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers or their analogues, which are linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (i.e., ribose) of the first adjacent monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers, that is, the sequence of bases linked to the sugar / phosphate backbone, is called the RNA sequence. The term "RNA" generally refers to molecules or molecular classes selected from the group consisting of: long 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 RNA, riboswitch, immune-stimulating 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 Piwi-interacting RNA (piRNA). Optionally, in the context of this disclosure, it refers to any type of therapeutic RNA.

[0038] As used herein, a terminator (also known as a "blocker" or "3' flanking oligonucleotide") refers to a polynucleotide or oligonucleotide sequence that terminates or prevents polymerase from further linking nucleotides. A terminator can terminate polymerase elongation. Terminators may include 5' phosphate, 5' thiophosphate (which can form a thiophosphate ester bond), 5' aminophosphate (which can form a phosphoramide ester bond), 5' diaminophosphate (which can form a phosphoryl diamine ester bond), 5' aminothiophosphate, 5' aminodithiophosphate, 5' diaminothiophosphate, or 5' dithiophosphate (which can form a dithiophosphate ester bond).

[0039] As used herein, “template” means a polynucleotide or oligonucleotide containing a sequence complementary to a single-stranded polynucleotide or oligonucleotide product. A template may contain a sequence that is 100% complementary to the sequence of a target (or product) polynucleotide or oligonucleotide. A template may consist of sequences that are 100% complementary to the sequence of a target (or product) polynucleotide or oligonucleotide. The template may be a longer sequence than the product sequence. The template may contain sequences not used to produce the polynucleotide or oligonucleotide product. When the template sequence is longer than the product sequence, a terminator may be used to control the production and / or length of the polynucleotide or oligonucleotide product. When the template sequence is longer than the product sequence, a primer may be used to control the production and / or length of the polynucleotide or oligonucleotide product. When the template sequence is longer than the product sequence, it may contain a hairpin loop. A hairpin loop may be used as a primer. The template may contain a product fragment within a hairpin loop. In this case, the product may be released from the template by cleavage with a nuclease, nickase, DNase, or chemical method. Unless otherwise stated, as used herein, the term “complementary” means 100% complementary.

[0040] As used herein, "therapeutic oligonucleotide" or "therapeutic oligo" means, for example, an oligonucleotide that has a therapeutic application in the prevention or treatment of conditions or diseases in humans or animals. Such oligos typically contain one or more modified nucleotide residues or bonds. Therapeutic oligos act via one of several different mechanisms, including but not limited to antisense, splicing or exon skipping, immune stimulation, and RNA interference (RNAi), for example, via microRNA (miRNA) or small interfering RNA (siRNA), and recruitment and guidance of DNA and RNA editing enzymes, such as A-to-I RNA base-editing oligonucleotides (AIMer). Therapeutic oligos can be aptamers. Therapeutic oligonucleotides may typically, but not always, have a defined sequence. Therapeutic oligonucleotides are examples of therapeutic polynucleotides.

[0041] As used herein, "therapeutic polynucleotide" refers to a polynucleotide that has therapeutic applications, for example, in the prevention or treatment of conditions or diseases in humans or animals. Such polynucleotides typically contain one or more modified nucleotide residues or bonds. Therapeutic polynucleotides act via one of several different mechanisms, including but not limited to antisense, splicing or exon skipping, immune stimulation, RNA interference (RNAi), such as miRNA or siRNA, and recruitment and guidance of DNA and RNA editing enzymes, such as A-to-I RNA base-editing oligonucleotides (AIMer). Aptamers are examples of therapeutic polynucleotides. mRNA vaccines are also examples of therapeutic polynucleotides. Therapeutic polynucleotides may generally, but not always, have a defined sequence. Therapeutic polynucleotides include therapeutic oligonucleotides and therapeutic RNA.

[0042] "Therapeutic RNA" is RNA that has therapeutic applications, for example, in the prevention or treatment of conditions or diseases in humans or animals. Such RNA is suitable for use in humans or animals for medical purposes, for example, it has a clinical grade, especially when it involves, for example, purity, integrity, and basic manufacturing methods that must comply with current Good Manufacturing Practices (cGMP) conditions.

[0043] As used herein, "transferase" means an enzyme that catalyzes the template-independent ligation of one nucleotide to another nucleotide or oligonucleotide. Transferases described herein include terminal nucleotide transferases (TdT), also known as DNA extranucleotide transferases (DNTT) or terminal transferases. TdT is a specialized DNA polymerase expressed in immature pre-B and pre-T lymphocytes, where it enables VDJ antibody gene ligation diversity. TdT catalyzes the addition of nucleotides to the 3' end of a DNA molecule. Transferases described herein include non-naturally occurring or mutated TdT. Transferases capable of ligating unmodified nucleotides to unmodified oligonucleotides, transferases capable of ligating unmodified nucleotides to modified oligonucleotides, transferases capable of ligating modified nucleotides to unmodified oligonucleotides, and transferases capable of ligating modified nucleotides to modified oligonucleotides are all within the scope of this disclosure.

[0044] "Phosphoramide (PN)" generally refers to a group having the structure -P(O)₂NRaRb, where Ra and Rb are independent substituents or together represent a double bond of an atom (e.g., carbon). For example, PN can refer to PO₂N(R) as defined herein. 3 )2 or PO2R 4 .

[0045] The term “pharmaceutically acceptable” refers to compounds (including salts), materials, compositions, and dosage forms that are suitable for use in human and animal tissue contact without excessive toxicity, irritation, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0046] Invention Description

[0047] The inventors have surprisingly discovered that polyphosphate kinase (PPK) is a suitable and efficient enzyme for producing modified nucleoside triphosphates (NTPs). The NTPs produced by the methods disclosed herein offer a simpler and more cost-effective alternative to prior art methods. Specifically, the inventors have used PPK to produce corresponding sugar-modified and / or α-phosphate-modified nucleoside triphosphates from modified nucleoside monophosphates or diphosphates and polyphosphates.

[0048] Inorganic polyphosphates, also known as polyphosphates or polyPs, are polymers of dozens to hundreds of phosphate residues (Pi) linked by high-energy phosphate anhydride bonds (such as those in adenosine triphosphate), and are generally linear. However, unlike ATP or other phosphate sources currently used in NTP production, polyPs are a more stable form of phosphate. It has been proposed that polyPs served as an energy source in the pre-ATP world and were initially utilized by early single-celled organisms. PolyPs have been found in all living organisms, from bacteria to higher eukaryotes, and have many biological functions: as a means of storing energy, it is also a reservoir of Pis, a chelator of metal ions, a channel for DNA entry, a regulator of stress and survival, and a supporting component in gene regulation and enzyme function (Motomura et al, Applied and Environmental Microbiology, 2014, 80(8), 2602-2608).

[0049] Due to differences in amino acid sequence and kinetic properties, PPKs have traditionally been divided into two families, PPK1 and PPK2. PPK1 preferentially catalyzes ATP-driven polyP formation, while PPK2 preferentially catalyzes polyP-driven nucleotide phosphorylation, which is important for the survival of microbial cells under stress or pathological conditions. The PPK2 family is further divided into the following subfamilies: Class I – single-domain enzymes that catalyze the phosphorylation of nucleoside diphosphates; Class II – double-domain enzymes that primarily catalyze the phosphorylation of nucleoside monophosphates; and Class III – single-domain enzymes that catalyze the phosphorylation of both nucleoside monophosphates and nucleoside diphosphates (see [link to relevant documentation]). Figure 1 ).

[0050] Known PPKs can be found in INTERPRO (EMBL-EBI) entry IPR022488, including those characterized by Noceket al (ACS Catal. 2018, 8, 11, 10746-10760).

[0051] The inferred PPK can 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 Database (PDB), Pfam, and InterPro. Suitable bioinformatics tools include Basic Local Alignment Search (BLAST), Clustal Omega, ClustalW, HMMER, InterProScan, UniProt tools, CD-HIT, MMseqs2, and CLANS.

[0052] Recently, PPK has been used as an ATP recycling system in the industrial implementation of ATP-dependent enzymes, thereby avoiding the addition of stoichiometric amounts of cofactors (Tavanti et al, Green Chemistry, 2021, 23, 828-837).

[0053] In this disclosure, PPK can be used to produce nucleoside triphosphates with only a single sugar modification and no modification to the base or α-phosphate, i.e., the base is a naturally occurring base and the α-phosphate is unmodified. The single sugar modification of the nucleoside triphosphate can include 2'-sugar modification, 3'-sugar modification, or both 2'-sugar modification and 3'-sugar modification. Alternatively, PPK can be used to produce nucleoside triphosphates with only a single α-phosphate modification and no modification to the base or sugar, i.e., the base is a naturally occurring base and the sugar is naturally occurring, such as ribose or deoxyribose. PPK can also be used to produce nucleoside triphosphates with sugar modification and / or α-phosphate modification, which additionally have modified bases, i.e., the bases are not naturally occurring.

[0054] In this disclosure, compounds of formula (II) are provided:

[0055]

[0056] Or its salt, or a pharmaceutically acceptable salt thereof;

[0057] By making compound of formula (I):

[0058]

[0059] Or its salts, or pharmaceutically acceptable salts thereof, react with PPK and polyphosphates.

[0060] in:

[0061] X1 is selected from the following groups: OH, SH, CH2, NR 3 2. CH3, BH3 - ,Se and R 4 ;

[0062] X2 is O or S;

[0063] R 1 Choose from the following groups: H, PO3, PO2S, PO2N (R) 3 2. PO2R 4 POS2, PO2CH3, PO2BH3, PO2Se, N-methyl-anthraniloyl MANT), Acetyl (Ac), Benzyl (Bn), Benzoyl (Bz), Allyl (All), 2-Cyanoethyl (CE), Azoxymethyl (AzM), Cyanovinyl (Cv), Methoxymethyl (MOM), Methoxyethoxymethyl (MEM), Methoxymethylphenylmethyl (MPM), Diphenylmethyl (DPM), Tetrahydropyranyl (THP), Dimethoxytriphenylmethyl (DMTr), Methoxycarbonylmethyl (MOMcl), Ethoxycarbonyl (EOC), Benzylcarbonyl (Cbz), Triflate, Mesylate, Tosylate, Trimethylacetyl (Piv), o-Nitrobenzyl (o-NB), Bromo-methyl-coumarin methyl (Bhc), (7-(diethylamino)coumarin-4-yl)methyl (DEACM), 6-Nitroveratroloxycarbonyl (NVOC), and 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc-ol);

[0064] R 2 Choose from the following groups: 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-restricted ethylene bridge with the 4' carbon;

[0065] R 3 Independently is H or

[0066] ;

[0067] R4 Choose from the following groups:

[0068] , , , , and ;

[0069] Furthermore, the bases are either naturally occurring bases or modified bases;

[0070] If X2 is O, R1 is H and R2 is H or OH, then X1 is not O-.

[0071] As will be understood by those skilled in the art, in formulas (I) and (II), the OH group can serve as O - It exists, and the SH group can act as S. - Existence depends on the conditions. Therefore, in equations (I) and (II), under the relevant conditions, OH and O - They are interchangeable, and SH and S - They can be used interchangeably.

[0072] In the implementation scheme, the bases are 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 (Ψ), N1-methylpseudouracil Pyrimidine (m1Ψ), 1-ethylpseudorazine, 2-thio-1-methyl-1-deazo-pseudorazine, 2-thio-1-methyl-pseudorazine, 2-thio-5-aza-uracil, 2-thio-dihydropseudorazine, 2-thio-dihydrouracil, 2-thio-pseudorazine, 4-methoxy-2-thio-pseudorazine, 4-methoxy-pseudorazine, 4-thio-1-methyl-pseudorazine 4-Thio-pseudorazine, 5-aza-uracil, dihydropseudorazine, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyluracil (2moU), 7-denitroguanine (7-denitro-G), 8-oxoguanine (8-oxo-G), 5-propynyluracil (pU), 5-propynylcytosine (pC), N6-methyladenine (m6A), N 4-Acetylcytosine (ac4C), O6-methylguanine (O6meG), hypoxanthine (H), xanthine (X), isocytosine (iC), isoguanine (iG), queuine (Q), 2-thiouracil (s2U), 4-thiouracil (s4U), 5-iodouracil (5Iu), 2-thiocytosine (s2C), 5-chlorouracil (5ClU), and isouriacil.

[0073] X1 is selected from the following groups: OH, SH, CH2, NR 3 2. CH3, BH3 - ,Se and R 4 As those skilled in the art will understand, if X1 is OH, then it can be OH or O. - This depends on the conditions, therefore OH and O - These terms can be used interchangeably in this document. X1 can be SH or S. - This depends on the conditions, therefore SH and S - These terms are used interchangeably in this article. If X1 is Se, then it can be Se. - This depends on the conditions, therefore Se in this article can be related to Se. - exchange.

[0074] In the implementation scheme, X1 is selected from the group consisting of: OH, SH, and N; X2 is O or S; R 1 Choose from the following groups: H, PO3, PO2S, PO2NR 3 2. PO2R 4, POS2, MANT, Ac, Bz, Bn, DMTr and Cbz; R 2 Choose from the following groups: H, OH, OMe, MOE, OEt, and F; and choose from the following groups of bases: A, U, C, G, T, 5mC, I, Ψ, and m1Ψ.

[0075] In the implementation plan, X1 is selected from the group consisting of: OH, SH, NR. 3 2 or R 4 X2 is O or S; R 1 Choose from the following groups: H, PO3, PO2S, and MANT; R 2 Choose from the following groups: H, OH, OMe, MOE, and F; and choose from the following groups of bases: A, U, C, G, T, 5mC, I, Ψ, and m1Ψ.

[0076] In the implementation scheme, X1 is OH. In the implementation scheme, X1 is SH. In the implementation scheme, X1 is NR. 3 2. In the implementation plan, X1 is R 4 .

[0077] In the implementation scheme, X2 is O. In the implementation scheme, X2 is S.

[0078] In the implementation plan, R 1 It is H. In the implementation plan, R 1 It is PO3. In the implementation plan, R 1 It's MANT.

[0079] In the implementation plan, R 2 It is H. In the implementation plan, R 2 It is OH. In the implementation scheme, R 2 It is OMe. In the implementation plan, R 2 It is MOE. In the implementation plan, R 2 It is F.

[0080] In the embodiment, the base is A. In the embodiment, the base is U. In the embodiment, the base is C. In the embodiment, the base is G. In the embodiment, the base is T. In the embodiment, the base is 5mC. In the embodiment, the base is I. In the embodiment, the base is Ψ. In the embodiment, the base is m1Ψ.

[0081] In the implementation scheme, X1 is OH, X2 is O, and R is R. 1 It is PO3 and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is OH, X2 is O, and R is... 1 It is MANT and R2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is OH, X2 is O, R 1 It is H and R 2 It is OMe, MOE, or F. In the implementation scheme, X1 is SH, X2 is O, R 1 It is PO3 and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is SH, X2 is O, R 1 It is MANT and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is SH, X2 is O, R 1 It is H and R 2 It is H, OH, OMe, MOE, or F.

[0082] In the implementation scheme, X1 is OH, X2 is S, and R. 1 It is PO3 and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is OH, X2 is S, R 1 It is MANT and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is OH, X2 is S, R 1 It is H and R 2 It is OMe, MOE, or F. In the implementation scheme, X1 is SH, X2 is S, R 1 It is PO3 and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is SH, X2 is S, R 1 It is MANT and R 2 It is H, OH, OMe, MOE, or F. In the implementation scheme, X1 is SH, X2 is S, R 1 It is H and R 2 It is H, OH, OMe, MOE, or F.

[0083] In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is PO3, and R 2 It is OMe. In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is PO3, and R 2 It is MOE. In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is PO3, and R 2 It is F. In the implementation scheme, X1 is OH, X2 is S, and R is R. 1 It is PO3, and R 2It is OMe. In the implementation scheme, X1 is OH, X2 is S, and R... 1 It is PO3, and R 2 It is MOE. In the implementation scheme, X1 is OH, X2 is S, R 1 It is PO3, and R 2 It is F.

[0084] In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is MANT, and R 2 It is OMe. In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is MANT, and R 2 It is MOE. In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is MANT, and R 2 It is F. In the implementation scheme, X1 is OH, X2 is S, and R is R. 1 It is MANT, and R 2 It is OMe. In the implementation scheme, X1 is OH, X2 is S, and R... 1 It is MANT, and R 2 It is MOE. In the implementation scheme, X1 is OH, X2 is S, R 1 It is MANT, and R 2 It is F.

[0085] In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is H, and R 2 It is OMe. In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is H, and R 2 It is MOE. In the implementation scheme, X1 is OH, X2 is O, and R... 1 It is H, and R 2 It is F. In the implementation scheme, X1 is OH, X2 is S, and R is R. 1 It is H, and R 2 It is OMe. In the implementation scheme, X1 is OH, X2 is S, and R... 1 It is H, and R 2 It is MOE. In the implementation scheme, X1 is OH, X2 is S, R 1 It is H, and R 2 It is F.

[0086] In the implementation plan, X1 is SH, X2 is O, and R is R. 1 It is PO3, and R 2 It is H. In the implementation scheme, X1 is SH, X2 is O, and R is R. 1 It is PO3, and R 2It is OH. In the implementation scheme, X1 is SH, X2 is O, and R is OH. 1 It is PO3, and R 2 It is OMe. In the implementation plan, X1 is SH, X2 is O, and R is R. 1 It is PO3, and R 2 It is MOE. In the implementation scheme, X1 is SH, X2 is O, and R is R. 1 It is PO3, and R 2 It is F. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is PO3, and R 2 It is H. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is PO3, and R 2 It is OH. In the implementation scheme, X1 is SH, X2 is S, R 1 It is PO3, and R 2 It is OMe. In the implementation plan, X1 is SH, X2 is S, R 1 It is PO3, and R 2 It is MOE. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is PO3, and R 2 It is F.

[0087] In the implementation plan, X1 is SH, X2 is O, and R is R. 1 It is MANT, and R 2 It is H. In the implementation scheme, X1 is SH, X2 is O, and R is R. 1 It is MANT, and R 2 It is OH. In the implementation scheme, X1 is SH, X2 is O, and R is OH. 1 It is MANT, and R 2 It is OMe. In the implementation plan, X1 is SH, X2 is O, and R is R. 1 It is MANT, and R 2 It is MOE. In the implementation scheme, X1 is SH, X2 is O, and R is R. 1 It is MANT, and R 2 It is F. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is MANT, and R 2 It is H. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is MANT, and R 2 It is OH. In the implementation scheme, X1 is SH, X2 is S, R 1 It is MANT, and R 2 It is OMe. In the implementation plan, X1 is SH, X2 is S, R 1 It is MANT, and R2 It is MOE. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is MANT, and R 2 It is F.

[0088] In the implementation plan, X1 is SH, X2 is O, and R is R. 1 It is H, and R 2 It is H. In the implementation scheme, X1 is SH, X2 is O, and R is R. 1 It is H, and R 2 It is OH. In the implementation scheme, X1 is SH, X2 is O, and R is OH. 1 It is H, and R 2 It is OMe. In the implementation plan, X1 is SH, X2 is O, and R is R. 1 It is H, and R 2 It is MOE. In the implementation scheme, X1 is SH, X2 is O, and R is R. 1 It is H, and R 2 It is F. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is H, and R 2 It is H. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is H, and R 2 It is OH. In the implementation scheme, X1 is SH, X2 is S, R 1 It is H, and R 2 It is OMe. In the implementation plan, X1 is SH, X2 is S, R 1 It is H, and R 2 It is MOE. In the implementation scheme, X1 is SH, X2 is S, and R is R. 1 It is H, and R 2 It is F.

[0089] In the implementation plan, when R 2 When it is OH, it is ribose. In the implementation scheme, R 2 It is OH, and it is 2'-arabinose (ANA).

[0090] In the implementation plan, when R 2 When it is H, it is 2'-deoxyribose.

[0091] In the implementation plan, when R 2 When it is F, it is 2'-deoxy-2'-fluororibose (FNA). In the implementation, when R 2 When it is F, it is 2'-deoxy-2'-fluoro-β-D-arabinose (FANA).

[0092] In embodiments of this disclosure, PPK may utilize one or more of the following substrates: locked nucleic acid (LNA), restricted ethyl (cEt), and 1,5-disodehydrated hexadiol (HNA) nucleoside.

[0093] In the implementation scheme, the nucleoside triphosphate produced by the PPK-catalyzed reaction is any one of the following:

[0094]

[0095]

[0096]

[0097]

[0098] Where PN-1=

[0099] ;

[0100] Or its salt, or its pharmaceutically acceptable salt.

[0101] In this disclosure, the use of PPK to produce sugar-modified and / or α-phosphate-modified nucleoside triphosphates from sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates is provided.

[0102] In the implementation scheme, PPK is PPK2. In the implementation scheme, PPK is PPK2-III.

[0103] In an embodiment, PPK comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 22. In an embodiment, PPK consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 22. In an embodiment, PPK comprises SEQ ID NO: 22. In an embodiment, PPK is SEQ ID NO: 22.

[0104] In the implementation scheme, PPK is obtained through the screening method disclosed herein.

[0105] In this disclosure, an enzymatic method for producing sugar-modified and / or α-phosphate-modified nucleoside triphosphates is provided, the method comprising incubating PPK with the corresponding modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate under suitable conditions.

[0106] In this embodiment, incubation is carried out together with a divalent cation. In this embodiment, the divalent cation is Mg. 2+ Mn 2+ Cd 2+ or Co 2+ In the implementation scheme, the divalent cation is Mg. 2+ In the implementation scheme, the divalent cation is Mn. 2+ In the implementation scheme, the divalent cation is Cd. 2+ In the implementation scheme, the divalent cation is Co. 2+ .

[0107] In the implementation scheme, incubation is carried out together with magnesium chloride, manganese chloride, cadmium chloride, cobalt chloride, magnesium acetate, manganese acetate, cadmium acetate, or cobalt acetate. In the implementation scheme, incubation is carried out together with magnesium chloride, manganese chloride, magnesium acetate, or manganese acetate. In the implementation scheme, incubation is carried out together with magnesium chloride or magnesium acetate. In the implementation scheme, incubation is carried out together with magnesium chloride. In the implementation scheme, incubation is carried out together with manganese chloride or manganese acetate. In the implementation scheme, incubation is carried out together with manganese chloride.

[0108] Sugar modifications include modifications at the 2' and 3' positions. 3'-Sugar modifications can act as protecting groups in certain reactions.

[0109] 2'-Sugar modifications include phosphoric acid (PO), O-methyl (OMe), O-methoxyethyl (MOE), fluoro (FNA), O-ethyl (OEt), O-propyl (OProp), 2'-arabinose (ANA), 2'-deoxy-2'-fluoro-β-D-arabinose (FANA), O-[2-(methylamino)-2-oxoethyl] (NMA), and 2'-O-[2-{(N,N-dimethylamino)oxy}-ethyl] (DMAOE). In embodiments, the 2'-sugar modification is selected from the group consisting of: OMe, MOE, FNA, OEt, and FANA. In embodiments, the 2'-sugar modification is selected from the group consisting of: OMe, MOE, and FNA. In embodiments, the 2'-sugar modification is OMe. In embodiments, the 2'-sugar modification is MOE. In embodiments, the 2'-sugar modification is FNA. In embodiments, the 2'-sugar modification is FANA. In embodiments, the 2'-sugar modification is OEt.

[0110] In one embodiment, the 2'-sugar modification is a bridge between the 2' oxygen and the 4' carbon sugar (ribose) position. In another embodiment, the bridge is a methylene bridge, i.e., a locked nucleose. In yet another embodiment, the bridge is a restricted ethyl bridge.

[0111] 3'-Sugar modifications include H, PO, thiophosphate (PS), thiophosphate, phosphoramide (PN), dithiophosphate, methylphosphonate, borophosphate, selenophosphate, MANT, Ac, Bn, Bz, All, CE, AzM, Cv, MOM, MEM, MPM, DPM, THP, DMTr, MOMcl, EOC, Cbz, trifluoromethanesulfonate, methanesulfonate, p-toluenesulfonate, Piv, o-NB, Bhc, DEACM, NVOC, and Bhc-ol. In embodiments, the 3'-sugar modification is selected from the group consisting of: H, PO, PS, PN, dithiophosphate, MANT, Ac, Bn, Bz, and Cbz. In embodiments, the 3'-sugar modification is selected from the group consisting of: H, PO, PS, PN, dithiophosphate, and MANT. In embodiments, the 3'-sugar modification is selected from the group consisting of: H, PO, PS, and MANT. In embodiments, the 3'-sugar modification is PO. In one embodiment, the 3'-sugar modification is MANT. In another embodiment, the 3'-sugar modification is H. When the 3'-sugar modification is PO and acts as a protecting group, a phosphatase can be used to remove the PO protecting group.

[0112] In some embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) contain a 2'-sugar modification. In some embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) contain 2'-OMe, 2'-MOE, or 2'-FNA. In some embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) do not contain a 2'-sugar modification, i.e., the 2' position is H or OH. In some embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) contain a 2'-H. In some embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) contain a 2'-OH.

[0113] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 3'-sugar modification. In another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 3'-sugar modification and 2'-H. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 3'-sugar modification and 2'-OH. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 3'-PO, 3'-PS, 3'-MANT, or 3'-H. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 3'-PO, 3'-PS, 3'-MANT, or 3'-H; and 2'-H. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 3'-PO, 3'-PS, 3'-MANT, or 3'-hydrogen; and 2'-OH. In another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) does not contain 3'-sugar modification, i.e., the 3' position is OH.

[0114] In the embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise 2'-sugar modification and 3'-sugar modification. In the embodiments, the sugar-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise 2'-H, 2'-OH, 2'-OMe, 2'-MOE, or 2'-FNA, and 3'-PO, 3'-PS, 3'-MANT, or 3'-hydrogen.

[0115] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-PO. In another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-PS. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-MANT. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-hydrogen.

[0116] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-PO. In another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-PS. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-MANT. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-H.

[0117] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-PO. In another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-PS. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-MANT. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-H.

[0118] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-phosphate. In another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises a naturally occurring base. In yet another embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-phosphate and a naturally occurring base.

[0119] In the implementation scheme, any one or more of the above sugar modifications may be combined with α-phosphate modification and / or base modification.

[0120] α-phosphoric acid modification includes PS, PN, methylphosphonates, borophosphates, and selenophosphates. In the embodiments, the α-phosphoric acid modification is PS, such as compounds of formula (IIa):

[0121] ,

[0122] Or its salt, or its pharmaceutically acceptable salt

[0123] Among them, X2, R 1 R 2 And the bases as described above.

[0124] In the embodiments, the α-phosphoric acid modification is PN, such as compounds of formula (IIb) or (IIc):

[0125] ,

[0126] Or its salt, or its pharmaceutically acceptable salt;

[0127] Among them, X2, R 1 R 2 R 3 R 4 And the bases as described above.

[0128] It should be understood that compounds of formula (I) or (II) or salts thereof mentioned herein include compounds of that formula as free acids, or as salts thereof, for example, as pharmaceutically acceptable salts thereof. Due to their potential use in pharmaceuticals, it should be understood that salts of compounds of formula (I) and (II) are preferably pharmaceutically acceptable. Pharmaceutically acceptable salts, among others, include those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those listed in PH Stahl and C GWermuth, 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 this invention.

[0129] In the embodiments, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-H. In the embodiments, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OH. In the embodiments, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises ribose or deoxyribose.

[0130] In the implementation scheme, the α-phosphate-modified nucleoside triphosphate contains a modified sugar.

[0131] In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) further comprise sugar modifications. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) further comprise 2'-sugar modifications. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) further comprise 3'-sugar modifications. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) further comprise both 2'-sugar and 3'-sugar modifications.

[0132] In one embodiment, the α-phosphate-modified nucleoside triphosphate comprises α-thiophosphate and further comprises sugar modification. In another embodiment, the α-phosphate-modified nucleoside triphosphate comprises α-thiophosphate and further comprises 2'-sugar modification. In yet another embodiment, the α-phosphate-modified nucleoside triphosphate comprises α-thiophosphate and further comprises 3'-sugar modification. In yet another embodiment, the α-phosphate-modified nucleoside triphosphate comprises α-thiophosphate and further comprises both 2'-sugar and 3'-sugar modification.

[0133] In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate and 2'-OMe. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate and 2'-MOE. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate and 2'-FNA.

[0134] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 3'-PO. In another embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 3'-PS. In yet another embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 3'-MANT. In yet another embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 3'-hydrogen.

[0135] In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-OMe, and 3'-PO. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-OMe, and 3'-PS. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-OMe, and 3'-MANT. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-OMe, and 3'-hydrogen.

[0136] In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-MOE, and 3'-PO. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-MOE, and 3'-PS. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-MOE, and 3'-MANT. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-MOE, and 3'-H.

[0137] In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-FNA, and 3'-PO. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-FNA, and 3'-PS. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-FNA, and 3'-MANT. In the embodiments, the α-phosphate-modified nucleoside triphosphates (and correspondingly modified nucleoside monophosphates or nucleoside diphosphates) comprise α-thiophosphate, 2'-FNA, and 3'-hydrogen.

[0138] NTPs containing α-phosphate modification possess a chiral phosphorus center. For example, the phosphoric anhydride bond formed with α-thiophosphate generates a chiral center on the phosphorus of the α-thiophosphate, producing two diastereomers: and S. p diastereomers and R p Diastereomers.

[0139] As can be seen from the embodiments described herein (see, for example) Figure 3 It is evident that different wild-type PPKs exhibit different stereochemical controls under the same reaction conditions. The ratio of diastereomers generated from PPK can be 1, indicating no stereopreferential treatment for phosphorylation, i.e., the two diastereomers (S... P :R p A 50:50 mixture of PPK. Different PPKs can produce S. p A preference for diastereomers, while another PPK may have the ability to generate R. p A preference for diastereomers.

[0140] 3D control can also be achieved by changing divalent cations.

[0141] In this disclosure, the use of PPK to generate stereospecific α-phosphate-modified nucleoside triphosphates from α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates is provided.

[0142] In the implementation plan, PPK produces S in an approximately 50:50 ratio. p diastereomers and R p Diastereomers. In the implementation scheme, PPK produces R in an approximately 55:45 ratio. p diastereomers and S p Diastereomers. In the implementation scheme, PPK produces S in an approximately 55:45 ratio. p diastereomers and R p Diastereomers.

[0143] In the implementation plan, PPK produces S in ratios of approximately 55:45, 60:40, 65:35, 70:30, 75:25, or 80:20. p diastereomers and R p Diastereomers.

[0144] In the implementation plan, PPK produces R in ratios of approximately 55:45, 60:40, 65:35, 70:30, 75:25, or 80:20. p diastereomers and S p Diastereomers.

[0145] In the implementation plan, PPK generates essentially all of the S p Diastereomers. In the implementation scheme, S P :R p Greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1. In the implementation plan, S P :R p Greater than 95:5. In the implementation plan, S P :R p Greater than 99:1.

[0146] In the implementation plan, PPK generates essentially all of the R p Diastereomers. In the implementation scheme, R p :S P Greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1. In the implementation plan, R p :S P Greater than 95:5. In the implementation plan, R p :S P Greater than 99:1.

[0147] In the implementation scheme, the stereospecific α-phosphate modified nucleoside triphosphate is a stereospecific PS modified nucleoside triphosphate.

[0148] In the implementation scheme, the stereospecific PS-modified nucleoside triphosphate is nucleoside 5'-(R P 5'-[α-thio]triphosphate. In the embodiments, the stereospecific PS-modified nucleoside triphosphate is nucleoside 5'-(S P )-[α-thio]triphosphate.

[0149] In one embodiment, the sugar-modified and / or α-phosphate-modified triphosphates contain naturally occurring bases. In another embodiment, the sugar-modified and / or α-phosphate-modified nucleoside triphosphates also contain base modifications.

[0150] In the implementation scheme, the bases are selected from the group consisting of: A, U, C, G, T, m5C, hm5C, 5fC, 5BrU, 2AP, I, Ψ, m1Ψ, 1-ethylpseudorazine, 2-thio-1-methyl-1-deazo-pseudorazine, 2-thio-1-methyl-pseudorazine, 2-thio-5-aza-uracil, 2-thio-dihydropseudorazine, 2-thio-dihydrouracil, 2-thio-pseudorazine, 4-methoxy-2-thio-pseudorazine Uracil, 4-methoxy-pseuuracil, 4-thio-1-methyl-pseuuracil, 4-thio-pseuuracil, 5-aza-uracil, dihydropseuuracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyluracil, 7-aza-G, 8-oxo-G, pU, pC, m6A, ac4C, O6meG, H, X, iC, iG, Q, s2U, s4U, 5Iu, s2C, 5ClU and isoururacil.

[0151] In the implementation scheme, the bases are selected from the group consisting of: A, U, C, G, T, m5C, hm5C, 5fC, 5BrU, 2AP, I, Ψ, m1Ψ, 7-denitro-G, 8-oxo-G, pU, pC, m6A, ac4C, O6meG, H, X, iC, iG, Q, s2U, 5Iu, s2C, and 5ClU.

[0152] In the implementation scheme, the bases are selected from the group consisting of: A, U, C, G, T, m5C, and I. In the implementation scheme, the bases are selected from the group consisting of: A, U, C, G, and T. In the implementation scheme, the bases are selected from the group consisting of: A, U, C, G, and m5C. In the implementation scheme, the bases are selected from the group consisting of: A, U, C, and G.

[0153] In the embodiment, the base is A. In the embodiment, the base is U. In the embodiment, the base is C. In the embodiment, the base is G. In the embodiment, the base is T. In the embodiment, the base is 5mC. In the embodiment, the base is I. In the embodiment, the base is Ψ. In the embodiment, the base is m1Ψ.

[0154] In one embodiment, the sugar-modified nucleoside triphosphate includes 2'-sugar modification and base modification. In another embodiment, the sugar-modified nucleoside triphosphate includes 3'-sugar modification and base modification. In yet another embodiment, the sugar-modified nucleoside triphosphate includes 2'-sugar modification, 3'-sugar modification, and base modification. In yet another embodiment, the sugar-modified nucleoside triphosphate includes 2'-sugar modification, 3'-sugar modification, base modification, and α-phosphate modification.

[0155] In some embodiments, the α-phosphate-modified nucleoside triphosphate further includes base modification. In some embodiments, the α-phosphate-modified nucleoside triphosphate further includes base modification and 2'-sugar modification. In some embodiments, the α-phosphate-modified nucleoside triphosphate further includes base modification and 3'-sugar modification. In some embodiments, the α-phosphate-modified nucleoside triphosphate further includes base modification, 2'-sugar modification, and 3'-sugar modification.

[0156] Nucleoside triphosphates that can be produced by the methods disclosed herein 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'-(α-thio)-triphosphate, and 2'-deoxycytidine-5'-(α-thio)-triphosphate. - Triphosphate, 2'-deoxyguanosine-5'-(α-thio)-triphosphate, 2'-deoxythymidine-(α-thio)-triphosphate, 2'-deoxyuridine-(α-thio)-triphosphate, 2'-adenosine-5'-(α-thio)-triphosphate, 2'-cytidine-5'-(α-thio)-triphosphate, 2'-guanosine-5'-(α-thio)-triphosphate, 2'-thymidine-(α-thio)-triphosphate and 2'-uridine-(α-thio)-triphosphate.

[0157] In the embodiments, the nucleoside triphosphates produced by the method of this disclosure are selected from the group consisting of: 2'-OMe-,3'-PO3-CTP; 2'-OMe-,3'-PO3-CTP-α-PN-1 (S P ); 2'-OMe-,3'-PO3-CTP-α-S (R P ); 2'-(R )-F-,3'-PO3-ATP-α-S (R P);2’-(R)-F-,3’-PO3-GTP-α-S (R P );2’-MOE-, 3’-PO3-5mCTP-α-S (R P );2’-MOE-,3’-PO3-ATP;2’-(R)-F-,3’-PO3-GTP;2’-(R)-F-,3’-PO3-CTP-α-S (R P );2’-OMe-,3’-PO3-UTP-α-S (R P );2’-OMe-,3’-PO3-UTP;2’-OMe-,3’-PO3-CTP-α-PN-1 (R P );2’-(R)-F-,3’-PO3-ATP;2’-(R)-F-,3’-PO3-GTP-α-S (R P );2’-(R)-F-,3’-PO3-UTP-α-PN-1 (S P );2’-MOE-,3’-PO3-5mCTP;2’-(R)-F-,3’-PO3-CTP-α-S (R P );2’-OMe-,3’-PO3-CTP-α-S (R P );2’-OMe-,3’-PO3-UTP;2’-OMe-,3’-PO3-UTP-α-PN-1 (R P );2’-MOE-,3’-PO3-5mCTP;2’-(R)-F-,3’-PO3-CTP;2’-deoxy-,3’-PO3-5mCTP-α-S (R P );2’-deoxy-,3’-PO3-(N3)-UTP-α-S (R P );2’-deoxy-,3’-PO3-ITP-α-S (R P );2’-OMe-,3’-PO3-UTP-α-PN-1 (R P );2’-(R)-F-,3’-PO3-CTP;2’-OMe-,3’-PO3-GTP-α-S (R P );2’-OMe-,3’-PO3-ATP-α-S (R P );2’-OMe-,3’-PO3-UTP-α-PN-1 (S P); 2'-OMe-,3'-PO3-CTP-α-S (ambo); 2'-OMe-,3'-PO3-GTP; 2'-OMe-,3'-PO3-UTP; 2'-OMe-,3'-PO3-ATP; 2'-(R )-F-,3'-PO3-GTP;2'-(R)-F-,3'-PO3-CTP;2'-OMe-,3'-PO3-UTP-α-S (ambo);2'-(R )-F-,3'-PO3-CTP-α-S(ambo);2'-OMe-,3'-PO3-ATP-α-S (ambo); )-F-,3'-PO3-UTP-α-S (ambo); 2'-OMe-,3'-PO3-CTP; 2'-(R 2'-(R)-F-,3'-PO3-UTP; 2'-(R)-F-,3'-PO3-ATP; 2'-OMe-,3'-PO3-GTP-α-S (ambo); 2'-MOE-,3'-PO3-GTP-α-S (ambo); 2'-MOE-,3'-PO3-MeCTP-α-S (ambo); 2'-MOE-,3'-PO3-ATP-α-S (ambo); 2'-deoxy-,3'-PO3-GTP-α-S (ambo); 2'-deoxy-,3'-PO3-TTP-α-S (ambo); 2'-deoxy-,3'-PO3-ATP-α-S (ambo); 2'-deoxy-,3'-PO3-MeCTP-α-S (ambo); and 2'-MOE-,3'-PO3-MeUTP-α-S (ambo).

[0158] In an aspect of the invention, a method is provided for screening a set of putative and known PPKs to identify enzymes capable of producing corresponding modified nucleoside triphosphates using sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates as substrates. The screening method comprises: a) identifying sequences encoding the set of putative and known PPKs; b) synthesizing the PPKs; c) purifying the PPKs; d) mixing each of the purified PPKs with the sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates to form a reaction mixture; and e) analyzing the presence of the corresponding modified nucleoside triphosphate in each reaction mixture to determine which enzyme(s) produce the corresponding modified nucleoside triphosphate.

[0159] In this implementation, the sequence in step a) is identified using one or more sequence databases and bioinformatics tools. In this implementation, the sequence database is selected from the group consisting of: UniProt, UniParc, European Nucleotide Archive, GenBank, RefSeq, PIR, PDB, Pfam, and InterPro. In this implementation, the sequence database is UniProt. In this implementation, the sequence database is InterPro. In this implementation, the bioinformatics tool is selected from the group consisting of: BLAST, Clustal Omega, ClustalW, HMMER, InterProScan, UniProtTools, CD-HIT, MMseqs2, and CLANS. In this implementation, the bioinformatics tool is CD-HIT. In this implementation, the sequence database is UniProt and the bioinformatics tool is CD-HIT.

[0160] In one embodiment, the sequence in step a) encodes a protein with a length between 150 and 800 amino acid residues. In another embodiment, the sequence in step a) encodes a protein with a length between 150 and 600 amino acid residues. In yet another embodiment, the sequence in step a) encodes a protein with a length between 600 and 700 amino acid residues.

[0161] In the implementation scheme, PPK is synthesized in step b) as having an N-terminal hexahistine tag and purified in step c) using nickel hyponitrotriacetate.

[0162] In one embodiment, step d) includes incubating the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, polyphosphate, and PPK at 25°C. In another embodiment, the incubation is carried out at 25°C for 24 hours. In yet another embodiment, the reaction is quenched after incubation at 25°C for 24 hours. In yet another embodiment, the reaction is quenched with EDTA at 80°C for 15 minutes. In yet another embodiment, the polyphosphate is sodium hexametaphosphate.

[0163] In this embodiment, the reaction mixture is centrifuged, and the presence of the corresponding modified nucleoside triphosphate in the supernatant is analyzed. In this embodiment, the reaction mixture is centrifuged at 4000 g for 15 minutes. In this embodiment, the supernatant is analyzed using HPLC or UPLC.

[0164] In the implementation scheme, the enzyme capable of producing the correspondingly modified nucleoside triphosphate is further engineered to increase the percentage (%) of conversion to the correspondingly modified nucleoside triphosphate. Methods for engineering the enzyme are known to those 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 methods that can be used include, among others, staggered extension (StEP), in vitro recombination (Zhao, et al., 1998, Nat. Biotechnol. 16:258–261), mutagenesis 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 that can be used for the purposes of this paper 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 artof creating genetic diversity: From directed evolution to synthetic biology”, Biotechnology Advances, 50:107762; Currin et al., 2015, “Synthetic biology forthe directed evolution of protein biocatalysts: navigating sequence spaceintelligently”, Chem Soc rev, 44:1172-1239;Dale, et al., 1996,“Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod,” Methods Mol. Biol. 57:369-74;Smith, 1985, “In vitro mutagenesis,”Ann. Rev. Genet. 19:423-462;Botstein, et al., 1985, “Strategies andapplications of in vitro mutagenesis,” Science 229:1193-1201; Carter, 1986,“Site-directed mutagenesis,” Biochem. J. 237:1-7;Kramer, et al., 1984, “PointMismatch 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 molecularbreeding,” Curr Opin Chem Biol 3:284-290;Christians, et al., 1999, “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling,” 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 arsenatedetoxification pathway by DNA shuffling," Nature Biotech 15:436-438; Zhang, etal., 1997, "Directed evolution of an effective fructosidase from agalactosidase 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.

[0165] In this disclosure, engineered PPKs with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 22 are provided. PPKs with 100% sequence identity to SEQ ID NO: 22 are not engineered PPKs.

[0166] The NTPs generated by the methods disclosed herein can be used to produce polynucleotides, including oligonucleotides.

[0167] Polynucleotides may consist entirely of nucleotide residues found in nature (i.e., all of them are “natural nucleotides” or “naturally occurring nucleotides”), or may contain at least one modified nucleotide residue, or at least one bond between modified nucleotide residues.

[0168] 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.

[0169] The modified nucleotide is not a naturally occurring nucleotide (i.e., it is a non-natural nucleotide). The modified nucleotide can be a naturally occurring nucleotide that has been modified (e.g., chemically modified). The modified nucleotide may contain a modified phosphate group, a modified sugar, and / or a modified nucleobase. It is known that certain modifications occur occasionally in nature, i.e., in naturally occurring nucleotides, such as 2'-OMe or C5 pyrimidine modifications; however, in this disclosure, these are considered modified nucleotides.

[0170] Modifications to the sugar moiety may include modifications at the 2'-position, modifications at the 3'-position, bicyclic sugars, or 4'-CH(CH3)-O-2' groups and combinations thereof. For example, modifications at the 2'-position of the sugar moiety may include 2'-F, 2'-OMe, or 2'-MOE.

[0171] Examples of modified nucleobases include modified cytosines, such as 5-methylcytosine, 5-methylpyrimidine, 7-deazoguanosine, and debased nucleotides. Further examples of modified nucleobases include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-Methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-hydroxy-n-valerylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxy-n-valerylcarbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); miI (1-methylinosine); m'Im (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2TO-methylcytidine); k2C (lysine); mlG (1-methylguanosine); m2G ​​(N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (peroxywybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wynoside); mimG (methylguanosine); Q (epoxyqueuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine);manQ (mannosyl-guanosine); preQo (7-cyano-7-deazoguanosine); preQi (7-aminomethyl-7-deazoguanosine); G (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-hydroxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); 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-carboxymethylaminomethyluridine); ncm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine) m4Cm(N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); £5Cm (5-formyl-2'-O-methylcytidine); mlGm (l,2'-O-dimethylguanosine); m'Am(1,2-O-dimethyladenosine)irinomethyluridine); tm5s2U (S-taurate methyl-2-thiouridine)); imG-14 (4-demethylguanosine); imG2 (isoguanosine);And ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C 2-C6)-Alynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-denitroguanine, 8-azaguanine, 7-denitro-7-substituted guanine, 7-denitro-7-(C2-C6)ynylguanine, 7-denitro-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-denitropurine, 7-aza-7-substituted purine, and 7-aza-8-substituted purine. For example, polynucleotides may include one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues.

[0172] Skeletal modifications include thiophosphates, phosphoramides, phosphoridamides, dithiophosphates, and / or methylphosphonates. Zero, one, several, or each nucleoside bond may be a modified nucleoside bond. The polynucleotides of this disclosure may consist only of phosphodiester bonds between nucleosides, but in other instances may contain thiophosphate, phosphoramide, phosphoridamide, dithiophosphate, and / or methylphosphonate bonds.

[0173] Polynucleotides, including oligonucleotides, can be single-stranded or double-stranded. The polynucleotides or oligonucleotides disclosed herein can be conjugated to another molecule, such as N-acetylgalactosamine (GalNAc) or its multimers (GalNAc clusters).

[0174] Modified nucleoside triphosphates known in the art include 2-amino-6-chloropurine ribonucleoside-5'-triphosphate, 2-aminopurine-ribonucleoside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, etc. Urate-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-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'- Triphosphate, 5-iodo-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-chloropurine ribonucleoside-5'-triphosphate, 7-deazoadenosine-5'-triphosphate 7-Denitroguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-ribonucleoside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, and flavin-5'-triphosphate.

[0175] Known base-modified nucleotides in this art include 5-methylcytidine-5'-triphosphate, 7-deazoguanidine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1 -Propynyl-pseudouridine, 5-Tauratemethyluridine, 1-Tauratemethyl-pseudouridine, 5-Tauratemethyl-2-thio-uridine, 1-Tauratemethyl-4-thio-uridine, 5-Methyl-uridine, 1-Methyl-pseudouridine, 4-Thio-1-methyl-pseudouridine, 2-Thio-1-methyl-pseudouridine, 1-Methyl-1-denitro-pseudouridine, 2-Thio-1-methyl-1 -Denitro-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-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-denitro-pseudoisocytidine, 1-methyl-1-denitro-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-pseudo-cytidine and 4-methoxy-1-methyl-pseudo-cytidine, 2-aminopurine, 2, 6-Diaminopurine, 7-Denitro-adenine, 7-Denitro-8-aza-adenine, 7-Denitro-2-aminopurine, 7-Denitro-8-aza-2-aminopurine, 7-Denitro-2,6-Diaminopurine, 7-Denitro-8-aza-2,6-Diaminopurine, 1-Methyladenosine, N6-Methyladenosine, N6-Isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-Methylthio-N6-cis-hydroxyisopentenyl)adenosine, N6-Glycineamidocarbonyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-methyladenosine, 2-methylthioadenosine and 2-methoxyadenosine, inosine, 1-methylinosine, woyoside, woyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-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 N2-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseuuridine, 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1 -Methyl-pseudouridine, 5,6-dihydrouridine, α-iodo-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deazo-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-methyl-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deazo-adenosine, pseudouridine, N1-methylpseudouridine, N1 -Ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseudouridine, 2-thio-1-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-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2'-O-methyluridine, pseudouridine (y), N1-methylpseudouridine, 5-methylcytosine, and 5-methoxyuridine.

[0176] In this implementation, the polynucleotide is a therapeutic polynucleotide. In this implementation, the therapeutic polynucleotide is therapeutic RNA. In this implementation, the therapeutic polynucleotide is therapeutic mRNA.

[0177] In this embodiment, the polynucleotide is an oligonucleotide. In this embodiment, the oligonucleotide is selected from the group consisting of: antisense oligonucleotides, siRNA, miRNA, aptamers, CRISPR guide RNA, and oligonucleotides for recruiting and guiding DNA and RNA editing enzymes. In this embodiment, the oligonucleotide for recruiting and guiding DNA and RNA editing enzymes is an A-to-I RNA base editing oligonucleotide (AIMer). In this embodiment, the oligonucleotide is a therapeutic oligonucleotide.

[0178] In this embodiment, the polynucleotide is prepared via an enzymatic method, i.e., via enzymatic synthesis, also referred to herein as enzymatically. In this embodiment, the enzyme is a polymerase. In this embodiment, the polymerase is a template-dependent polymerase. In this embodiment, the polymerase is a template-independent polymerase. In this embodiment, the enzyme is a transferase. In this embodiment, the enzyme is a terminal deoxynucleotidyl transferase (TdT).

[0179] In this disclosure, a method for preparing polynucleotides is provided, the method comprising incubating a nucleotide triphosphate, a suitable enzyme, and optionally a template under suitable conditions, wherein the nucleotide triphosphate is prepared by incubating a corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions.

[0180] In this disclosure, a method for preparing polynucleotides is provided, the method comprising the steps of: a) providing nucleoside triphosphates that have been prepared under suitable conditions using the corresponding nucleoside monophosphates and / or nucleoside diphosphates, PPK and polyphosphates; and b) using the nucleoside triphosphates and suitable enzymes under suitable conditions to produce polynucleotides.

[0181] In the implementation scheme, one or more or all of the NTPs are naturally occurring NTPs.

[0182] In some embodiments, one or more of the NTPs contain sugar, base, and / or α-phosphate modifications. In some embodiments, one or more of the NTPs contain base modifications. In some embodiments, one or more of the NTPs contain sugar modifications. In some embodiments, one or more of the NTPs contain 2' sugar modifications. In some embodiments, one or more of the NTPs contain 3' sugar modifications. In some embodiments, one or more of the NTPs contain α-phosphate modifications.

[0183] In the embodiments, the polynucleotide is mRNA. In the embodiments, one or more or all of the NTPs contain base modifications. In the embodiments, the provided NTPs comprise naturally occurring NTPs or consist of naturally occurring NTPs. In the embodiments, the NTPs are naturally occurring NTPs except that uridine triphosphate is replaced by pseudouridine triphosphate. In the embodiments, the provided NTPs are naturally occurring NTPs except that uridine triphosphate is replaced by N1-methylpseudouridine triphosphate.

[0184] In one embodiment, the enzyme used in step b) is a polymerase. In another embodiment, the polymerase is a template-dependent polymerase, and a suitable template is included in step a). In yet another embodiment, the polymerase is a template-independent polymerase. In another embodiment, the enzyme used in step b) is a transferase. In yet another embodiment, the enzyme used in step b) is a terminal deoxynucleotidyl transferase (TdT).

[0185] The enzymes disclosed herein, particularly PPK, can be prepared using any of many conventional techniques. For example, PPK can be purified from cells that naturally express them or produced in recombinant expression systems.

[0186] Many different expression systems and purification protocols are available for producing publicly disclosed PPKs. Typically, host cells are transformed with a recombinant expression vector encoding the desired PPK. The expression vector can 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 used, including prokaryotes (including Gram-negative or Gram-positive bacteria such as *Escherichia coli*, *Bacillus*, *Pseudomonas*, and *Corynebacterium*), eukaryotes including yeasts (e.g., *Saccharomyces cerevisiae*, *Pichia pastoris*), fungi (e.g., *Aspergillus*), or higher eukaryotes, including insect cells and mammalian-derived cell lines (e.g., CHO, NSO, PER.C6, HEK293, and HeLa). In this embodiment, the vector is the pET28a vector.

[0187] The host cell can be an isolated host cell. The host cell is typically not part of a multicellular organism (e.g., a plant or animal). The host cell can be a non-human host cell. In one embodiment, the host cell is a bacterial cell. In another embodiment, the host cell is a Gram-positive bacterial cell. In yet another embodiment, the host cell is *Escherichia coli*. In yet another embodiment, the host cell is *Escherichia coli* BL21(DE3).

[0188] Suitable cloning and expression vectors for use with bacteria, fungi, yeast and mammalian host cells are known in the art.

[0189] Cells can be cultured under conditions that promote PPK expression using various devices such as shake flasks, rotary flasks, and bioreactors. The peptide is recovered using conventional protein purification procedures. These procedures typically consist of a series of unit operations, including various filtration and chromatography processes developed for the selective concentration and separation of PPK. In a particular embodiment, a hexahistidine is fused to the N-terminus of PPK, and PPK is purified using immobilized metal affinity chromatography. In another particular embodiment, a hexahistidine is fused to the N-terminus of PPK, and PPK is purified using nickel hyponitrotriacetate (Ni-NTA). Example

[0190] Example 1: Selection of Polyphosphokinase (PPK)

[0191] INTERPRO (EMBL-EBI) provides functional analysis of proteins by classifying them into families and predicting domains and key sites (Paysan-Lafosse et al, Nucleic Acids Research, 2023, 51, D418-D427). INTERPRO entry IPR022488 corresponds to polyphosphate kinase-2-related proteins, i.e., the putative PPK2 compared to the characterized PPK2, and was used as a starting point (out of 28,000 proteins).

[0192] Sequences ranging from 150 to 600 amino acids in length were filtered using a standard Python script, and CD-HIT V4.6.8 was used to cluster sequences that shared at least 90% sequence identity with each other (11,779 clusters).

[0193] The characterized PPK2-III sequences (Nocek et al, ACS Catal. 2018, 8, 11, 10746-10760) and internal PPK2 sequences were inserted into the dataset without additional clustering to maintain sequence diversity.

[0194] Multiple sequence alignment was performed using MAFFT (Multiple Sequence Optimization) version 7.520 (March 2023), and phylogenetic tree files were generated. Visualization was performed using Christian Zmasek's Archaeopteryx software (https: / / sites.google.com / site / cmzmasek / christian-zmasek / software) V0.9928. Clusters of PPK2 belonging to different categories were manually examined by searching for relevant sequences in the generated tree (i.e., using characterized PPK2-III and known PPK2 I and II sequences). 4185 PPK2-III family members were identified for further research.

[0195] Sequences of PPK2-III family members were retrieved using UNIPROT. Column sorting and filtering removed fragments and poorly annotated sequences, leaving 4002 potentially interesting sequences. UNIPROT was then used to divide the sequences into three groups: sequences derived from archaea (34 sequences), metagenomic sequences (38 sequences), and bacterial sequences (3930 sequences).

[0196] Further clustering of bacterial sequences was performed using CD-HIT (a three-step process: 80%, 70%, and 60% sequence identity), resulting in 686 clusters. Representative sequences were aligned using Clustal Omega, generating phylogenetic tree files and visualizing them using Archaeopteryx software. Phylogenetic analysis divided the sequences into four main clades, and a further phylogenetic tree was constructed for each of these clades. The clades with the fewest populations from each of the four trees were removed, and, where available, sequences from clades of known extremophiles were selected as representative instances of the clades. After pruning the sequences to obtain maximum sequence diversity, 95 genes were finally selected. Sequences that failed to synthesize genes and were not produced in a sufficiently soluble form reduced the number of PPK sequences to 66.

[0197] Example 2: 5' phosphorylation of 2'-hydroxyadenosine 5'-monophosphate (AMP) by PPK to produce 2'-hydroxyadenosine 5'-triphosphate (ATP) (natural PPK reaction)

[0198] Wild-type PPKs, as shown in SEQ ID NO: 1-66, are each fused to a six-histidine tag (6xHis) at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using nickel hyponitrotriacetate (Ni-NTA) and used directly.

[0199] The reaction was established according to Table 1, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding ethylenediaminetetraacetic acid (EDTA) (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new polymerase chain reaction (PCR) plate. The presence of adenosine 5'-triphosphate (ATP) in the reaction mixture was analyzed by high-performance liquid chromatography (HPLC). Product peaks were characterized using liquid chromatography-mass spectrometry (LCMS).

[0200] Table 1: Reaction Setup

[0201]

[0202] Table 2: Reaction Results

[0203]

[0204]

[0205]

[0206]

[0207] *Product conversion (%) - Area of ​​5'-triphosphate and 5'-tetraphosphate peaks at 256 nm wavelength relative to 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and 5'-tetraphosphate peaks.

[0208] in conclusion

[0209] The experiments described and the resulting conversion data show that many tested wild-type PPKs (SEQ ID NO: 1-5, 7-9, 11-31, 33-41, 43-45, 47-57, and 59-66) can synthesize adenosine 5'-triphosphate from adenosine 5'-monophosphate via phosphorylation at 5'-phosphate. Among them, 13 wild-type PPKs (SEQ ID NO: 11, 12, 15, 21, 23, 26, 38, 41, 49-51, 54, and 65) convert about three-quarters or more of the substrate to the product under specific reaction conditions.

[0210] Example 3: 5' phosphorylation of 2'-methoxy-modified, 3'-phosphate-protected adenosine 5'-monophosphate by PPK to produce 2'-methoxy-adenosine 3'-phosphate-5'-triphosphate.

[0211] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0212] The reaction was established according to Table 3, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-adenosine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0213] Table 3: Reaction Setup

[0214]

[0215] Table 4: Reaction Results

[0216]

[0217]

[0218]

[0219]

[0220] *Product conversion (%) - Area of ​​5'-triphosphate and 5'-tetraphosphate peaks at 256 nm wavelength relative to 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and 5'-tetraphosphate peaks.

[0221] in conclusion

[0222] The described experiments and the obtained transformation data show that many wild-type PPKs (SEQ ID NO: 11, 14, 15, 19-23, 25-27, 29-31, 36-38, 40, 49-52, 65, and 66) can be synthesized from 2'-methoxy-modified, 3'-phosphate-protected adenosine 5'-monophosphate via phosphorylation at 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).

[0223] Example 4: 5' phosphorylation of 2'-fluoro,3'-phosphate-protected adenosine 5'-monophosphate by wild-type PPK to produce 2'-fluoro,3'-phosphate-protected adenosine 5'-triphosphate.

[0224] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0225] The reaction was established according to Table 5, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-fluoroadenosine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0226] Table 5: Reaction Setup

[0227]

[0228] Table 6: Reaction Results

[0229]

[0230]

[0231]

[0232]

[0233] *Product conversion (%) - Area of ​​5'-triphosphate and 5'-tetraphosphate peaks at 256 nm wavelength relative to 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and 5'-tetraphosphate peaks.

[0234] in conclusion

[0235] The described experiments and obtained transformation data show that many wild-type PPKs (SEQ ID NO: 11, 15, 19, 21, 22, 25, 27, 29, 31, 36, 38, 49, 52, 59, and 66) can be synthesized from 2'-methoxy-modified, 2'-fluoro, 3'-phosphate-protected adenosine nucleotide 5'-monophosphate via phosphorylation at the 5' phosphate site to 2'-fluoro, 3'-phosphate-protected adenosine nucleotide 5'-triphosphate. One wild-type PPK (SEQ ID NO: 22) works particularly well and converts 100% of the substrate to the product under specific reaction conditions.

[0236] Example 5: 5' phosphorylation of 2'-methoxy-modified, 3'-phosphate-protected guanosine 5'-monophosphate by PPK to produce 2'-methoxyguanosine 3'-phosphate-5'-triphosphate

[0237] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0238] The reaction was established according to Table 7, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-guanosine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0239] Table 7: Reaction Setup

[0240]

[0241] Results and Conclusions

[0242] The described experiments and obtained transformation data demonstrate that several wild-type PPKs (SEQ ID NO: 19, 22, 31, and 52) can be synthesized from 2'-methoxy-modified, 3'-phosphate-protected guanosine 5'-monophosphate via phosphorylation at the 5' phosphate position to 2'-methoxy-modified, 3'-phosphate-protected guanosine 5'-triphosphate (substrate to product conversion rates of 5.8%, 99%, 28.6%, and 4.0%, respectively). Representative chromatograms are shown below. Figure 2As shown. A wild-type PPK (SEQ ID NO: 22) works particularly well and converts 99% of the substrate to the product under specific reaction conditions.

[0243] Example 6: 5' phosphorylation of 2'-methoxy-modified, 3'-phosphate-protected uridine 5'-monophosphate by PPK to produce 2'-methoxyuridine 3'-phosphate-5'-triphosphate

[0244] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0245] The reaction was established according to Table 8, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-uridine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0246] Table 8: Reaction Setup

[0247]

[0248] Results and Conclusions

[0249] The described experiments and the obtained transformation data show that a single wild-type PPK (SEQ ID NO: 22) can synthesize 2'-methoxy-modified, 3'-phosphate-protected uridine 5'-triphosphate from 2'-methoxy-modified, 3'-phosphate-protected uridine 5'-monophosphate via phosphorylation at 5' phosphate (the conversion rate of substrate to 5'-triphosphate product was 39.6%, and the conversion rate to 5'-triphosphate and 5'-tetraphosphate products was 43.1%).

[0250] Example 7: 5' phosphorylation of 2'-fluoro-modified, 3'-phosphate-protected cytidine 5'-monophosphate by PPK to produce 2'-fluorocytidine 3'-phosphate-5'-triphosphate

[0251] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0252] The reaction was established according to Table 9, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-fluorocytidine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0253] Table 9: Reaction Setup

[0254]

[0255] Results and Conclusions

[0256] The described experiments and the obtained transformation data show that a single wild-type PPK (SEQ ID NO: 22) can be synthesized from 2'-fluoro-modified, 3'-phosphate-protected cytidine 5'-monophosphate via phosphorylation at 5' phosphate (substrate-to-product conversion rate of 22.6%).

[0257] Example 8: 5' phosphorylation of 2'-methoxy-ethyl modified methylcytidine 5'-monophosphate by PPK to produce methylcytidine 2'-methoxy-ethylmethylcytidine 3'-phosphate-5'-triphosphate

[0258] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0259] The reaction was established according to Table 10, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-ethyl-methylcytidine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0260] Table 10: Reaction Setup

[0261]

[0262] Results and Conclusions

[0263] The described experiments and the resulting transformation data show that several wild-type PPKs (SEQ ID NO: 22, 31, 49) can be synthesized from 2'-methoxy-ethyl-methylcytidine, 3'-phosphate-protected 5'-monophosphate via phosphorylation at 5' phosphate, although the substrate-to-product conversion percentages are low (5.4%, 2.4%, and 0.9%, respectively).

[0264] Example 9: 5' phosphorylation of 2'-methoxy-modified, 3'-phosphate-protected cytidine 5'-monophosphate by PPK to produce 2'-methoxy-modified, 3'-phosphate-protected cytidine 5'-triphosphate

[0265] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0266] The reaction was established according to Table 11, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-cytidine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0267] Table 11: Reaction Setup

[0268]

[0269] Results and Conclusions

[0270] The experiments described and the transformation data obtained show that several wild-type PPKs (SEQ ID NO: 22, 29 and 31) can be synthesized from 2'-methoxy-modified, 3'-phosphate-protected cytidine 5'-monophosphate via phosphorylation at 5' phosphate (substrate to product conversion rates of 10.4%, 0.3% and 38.1%, respectively).

[0271] Example 10: 5' phosphorylation of 2'-methoxy-modified, 3'-phosphate-protected adenosine 5'-thiomonophosphate by PPK to produce 2'-methoxy-adenosine 3'-phosphate-5' α-thiotriphosphate.

[0272] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0273] The reaction was established according to Table 12, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-adenosine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0274] Table 12: Reaction Setup

[0275]

[0276] Table 13: Reaction Results

[0277]

[0278]

[0279]

[0280]

[0281] *Product conversion (%) - Area of ​​5'-α-thio-triphosphate and 5'-α-thio-tetraphosphate peaks at 256 nm relative to the peaks of 5'-mono-, 5'-di-, 5'-tri-, and 5'-tetra-5'-α-thiophosphate (%)

[0282] in conclusion

[0283] The described experiments and the obtained transformation data show that many wild-type PPKs (SEQ ID NO: 1-4, 10-15, 18-23, 25-27, 29-31, 36-42, 47, 49-54, 56, 57, 59, 60, 65 and 66) can be synthesized from 2'-methoxy-modified, 3'-phosphate-protected adenosine 5'-α-thiotriphosphate by phosphorylation at 5'-thiophosphate.

[0284] Example 11: 5' phosphorylation of 2'-methoxy-modified, 3'-phosphate-protected cytidine 5'-thiomonophosphate by PPK to produce 2'-methoxycytidine 3'-phosphate-5'-α-thiotriphosphate.

[0285] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0286] The reaction was established according to Table 14, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-cytidine 3'-phosphate-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0287] Table 14: Reaction Setup

[0288]

[0289] Results and Conclusions

[0290] The described experiments and the obtained transformation data show that several wild-type PPKs (SEQ ID NO: 22, 31, 37 and 39) can be synthesized from 2'-methoxy-modified, 3'-phosphate-protected cytidine 5'-thiophosphate via phosphorylation at 5'-thiophosphate (substrate to product conversion percentages were 3.3%, 6.2%, 11.9% and 17.0%, respectively).

[0291] Example 12: 5' phosphorylation of adenosine 5'-thiomonophosphate by PPK to produce adenosine 5'-(α-thio)triphosphate.

[0292] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0293] The reaction was established according to Table 15, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of adenosine 5'-(α-thio)triphosphate in the reaction mixture was analyzed by HPLC. The product peaks were characterized using LCMS.

[0294] Table 15: Reaction Setup

[0295]

[0296] The formation of a phosphoric anhydride bond with α-thiophosphate results in a chiral center on the phosphorus of α-thiophosphate, producing two diastereomers. The diastereomers of adenosine 5'-(α-thio)diphosphate and adenosine 5'-(α-thio)triphosphate can be separated by ultra-high performance liquid chromatography (UPLC). The diastereomer ratios (first elution peak / second elution peak) and their percentage composition relative to monophosphate, diphosphate, and triphosphate are shown in Table 16 below.

[0297] Table 16: Results of diastereomers of the reaction

[0298]

[0299]

[0300]

[0301]

[0302]

[0303] in conclusion

[0304] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 1-9, 11, 12, 15-23, 25-41, 44-54, 56, 57, 59 and 61-66) can be synthesized from adenosine 5'-thiomonophosphate by phosphorylation at 5'-thiophosphate.

[0305] The ratio of the diastereomers of 5'-(α-thio)-triphosphate and 5'-(α-thio)-diphosphate generated can be 1 (indicating no stereopreferential treatment to phosphorylation), >1 (indicating a preference for generating early-eluting diastereomers), or <1 (indicating a preference for generating late-eluting diastereomers), depending on the PPK used (see [link to PPK]). Figure 3 ).

[0306] Example 13: 5' phosphorylation of 2'-deoxyadenosine 5'-thiomonophosphate by PPK to produce 2'-deoxyadenosine 5'-(α-thio)triphosphate.

[0307] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0308] The reaction was established according to Table 17, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-deoxyadenosine 5'-(α-thio)triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0309] Table 17: Reaction Setup

[0310]

[0311] Table 18: Reaction Results

[0312]

[0313]

[0314]

[0315]

[0316] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0317] in conclusion

[0318] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 2-4, 7-9, 11, 12, 14-23, 25-31, 33-40, 45, 47-54, 56, 57, 59, 61, 62, 64-66) can be synthesized from 2'-deoxyadenosine 5'-(α-thio)-triphosphate by phosphorylation at the terminal 5' thiophosphate.

[0319] Example 14: 5' phosphorylation of 2'-deoxyguanosine 5'-thiomonophosphate by PPK to produce 2'-deoxyguanosine 5'-(α-thio)triphosphate.

[0320] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0321] The reaction was established according to Table 19, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-deoxyguanosine 5'-(α-thio)triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0322] Table 19: Reaction Setup

[0323]

[0324] Table 20: Reaction Results

[0325]

[0326]

[0327]

[0328]

[0329] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0330] in conclusion

[0331] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 8, 9, 11, 15, 19-23, 25, 27, 29, 31, 32, 35, 37, 39, 45, 47, 49, 50, 54, 58, 61 and 64-66) can be synthesized from 2'-deoxyguanosine 5'-(α-thio)-triphosphate by phosphorylation at the terminal 5'-thiophosphate.

[0332] Example 15: 5' phosphorylation of 2'-deoxycytidine 5'-thiomonophosphate by polyphosphate kinase to produce 2'-deoxycytidine 5'-(α-thio)triphosphate.

[0333] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0334] The reaction was established according to Table 21, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-deoxycytidine 5'-(α-thio)triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0335] Table 21: Reaction Setup

[0336]

[0337] Table 22: Reaction Results

[0338]

[0339]

[0340]

[0341]

[0342] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0343] in conclusion

[0344] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 9, 14-16, 19, 20, 22, 23, 25, 27, 29, 31, 37, 39, 44, 47, 59 and 65) can be synthesized from 2'-deoxycytidine 5'-(α-thio)-triphosphate by phosphorylation at the terminal 5' thiophosphate.

[0345] Example 16: 5' phosphorylation of 2'-fluoro-cytidine-5'-monophosphate by PPK to produce 2'-fluoro-cytidine-5'-triphosphate

[0346] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0347] The reaction was established according to Table 23, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-fluoro-cytidine-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0348] Table 23: Reaction Setup

[0349]

[0350] Table 24: Reaction Results

[0351]

[0352]

[0353]

[0354]

[0355] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0356] in conclusion

[0357] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 10, 11, 15, 19, 21-23, 25-27, 29, 31, 32, 37, 39, 40, 49, 54, 59 and 66) can be synthesized from 2'-fluoro-cytidine-5'-monophosphate by phosphorylation at the terminal 5' thiophosphate.

[0358] Example 17: 5' phosphorylation of 2'-methoxy modified adenosine 5'-monophosphate by PPK to produce 2'-methoxy modified adenosine 5'-triphosphate.

[0359] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0360] The reaction was established according to Table 25, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 2'-methoxy-adenosine 5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0361] Table 25: Reaction Setup

[0362]

[0363] Table 26: Reaction Results

[0364]

[0365]

[0366]

[0367]

[0368] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0369] in conclusion

[0370] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 1, 2, 4, 5, 7, 9, 11, 12, 14-23, 25-40, 43-45, 47-54, 56, 57, 59-62 and 64-66) can be synthesized from 2'-methoxy-adenosine 5'-monophosphate by phosphorylation at the terminal 5' thiophosphate.

[0371] Example 18: 3'-(N'-methyl-o-aminobenzoyl)2'-deoxyadenosine-5'-monophosphate was 5'-phosphorylated by PPK to produce 3'-(N'-methyl-o-aminobenzoyl)2'-deoxyadenosine-5'-triphosphate.

[0372] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0373] The reaction was established according to Table 27, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 3'-(N'-methyl-o-aminobenzoyl)2'-deoxyadenosine-5'-triphosphate in the reaction mixture was analyzed by HPLC. The product peaks were characterized using LCMS.

[0374] Table 27: Reaction Setup

[0375]

[0376] Table 28: Reaction Results

[0377]

[0378]

[0379]

[0380]

[0381] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0382] in conclusion

[0383] The experiments described and the resulting transformation data show that many wild-type PPKs (SEQ ID NO: 2, 4, 6-9, 11, 12, 14-17, 19-23, 25-40, 42, 44, 45, 47-54, 56-62 and 64-66) can be synthesized from 3'-(N'-methyl-o-aminobenzoyl)2'deoxyadenosine-5'-monophosphate by phosphorylation at the terminal 5' phosphate.

[0384] Example 19: 3'-(N'-methyl-o-aminobenzoyl)2'-deoxyguanosine-5'-monophosphate was 5'-phosphorylated by PPK to produce 3'-(N'-methyl-o-aminobenzoyl)2'-deoxyguanosine-5'-triphosphate.

[0385] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0386] The reaction was established according to Table 29, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The presence of 3'-(N'-methyl-o-aminobenzoyl)2'-deoxyguanosine-5'-triphosphate in the reactants was analyzed by HPLC. The product peaks were characterized using LCMS.

[0387] Table 29: Reaction Setup

[0388]

[0389] Table 30: Reaction Results

[0390]

[0391]

[0392]

[0393]

[0394] *Product conversion (%) - Area of ​​the 5'-monophosphate and 5'-diphosphate peaks relative to the 5'-triphosphate peak at 256 nm wavelength.

[0395] in conclusion

[0396] The experiments described and the resulting transformation data show that many wild-type PPKs (SEQ ID NO: 2, 4-9, 11, 12, 14-40, 42, 44-47, 49-54, 56, 58, 59, 61 and 63-66) can be synthesized from 3'-(N'-methyl-o-aminobenzoyl)2'deoxyguanosine-5'-monophosphate by phosphorylation at the terminal 5' phosphate.

[0397] Example 21: 5' phosphorylation of 3'-neovaleryl 2'-methoxyguanosine-5'-monophosphate by PPK to produce 3'-neovaleryl 2'-methoxyguanosine-5'-triphosphate.

[0398] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0399] The reaction was established according to Table 31, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (2 μL, 100 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 20 μL of the supernatant was then transferred to a new PCR plate. The presence of 3'-neovaleryl-2'-methoxyguanosine-5'-triphosphate in the reaction mixture was then analyzed by HPLC.

[0400] Table 31: Reaction Setup

[0401]

[0402] Table 32: Reaction Results

[0403]

[0404]

[0405]

[0406]

[0407] *Product conversion (%) - Area of ​​the 5'-triphosphate peak at 256 nm relative to the peaks of 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate.

[0408] in conclusion

[0409] The experiments described and the transformation data obtained show that many wild-type PPKs (SEQ ID NO: 19, 20, 24, 32 and 36) can be converted from 3'-neovaleryl 2'-methoxyguanosine-5'-monophosphate to 3'-neovaleryl 2'-methoxyguanosine-5'-triphosphate by phosphorylation at 5' phosphate, with one wild-type PPK working particularly well (SEQ ID NO: 22, 72.5% conversion).

[0410] Example 22: 3'-neovaleryl 2'-methoxyuridine-5'-monophosphate was 5'-phosphorylated by PPK to produce 3'-neovaleryl 2'-methoxyuridine-5'-triphosphate.

[0411] Wild-type PPKs, as shown in SEQ ID NO: 1-66, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0412] The reaction was established according to Table 33, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (2 μL, 100 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 20 μL of the supernatant was then transferred to a new PCR plate. The presence of 3'-neovaleryl-2'-methoxyuridine-5'-triphosphate in the reaction mixture was then analyzed by HPLC.

[0413] Table 33: Reaction Setup

[0414]

[0415] Table 34: Reaction Results

[0416]

[0417]

[0418]

[0419]

[0420] *Product conversion (%) - Area of ​​the 5'-triphosphate peak relative to 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate at 256 nm wavelength.

[0421] in conclusion

[0422] The experiments described and the transformation data obtained show that only one wild-type PPK (SEQ ID NO: 24) can be converted from 3'-neovaleryl 2'-methoxyuridine-5'-monophosphate to 3'-neovaleryl 2'-methoxyuridine-5'-triphosphate (SEQ ID NO: 22, 28.5% conversion) by phosphorylation at 5' phosphate.

[0423] Example 23: 5' phosphorylation of deoxyadenosine 5'-thiomonophosphate by PPK to produce deoxyadenosine 5'-(α-thio)triphosphate.

[0424] Wild-type PPKs, as shown in SEQ ID NO: 22, 31, and 34, were each fused to a 6xHis fusion at the N-terminus. The gene was synthesized, cloned into pET28a, and the protein encoded by this gene was produced in *E. coli* BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0425] The reaction was established according to Table 35, with PPK added last. The reaction was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (2 μL, 100 mM), heating to 80°C for 15 minutes, and centrifuging at 4000 xg for 15 minutes. 20 μL of the supernatant was then transferred to a new PCR plate. The presence of deoxyadenosine 5'-(α-thio)triphosphate in the reaction mixture was then analyzed by HPLC.

[0426] Table 35: Reaction Setup

[0427]

[0428] The formation of a phosphoric anhydride bond with α-thiophosphate results in a chiral center on the phosphorus of α-thiophosphate, producing two diastereomers. The diastereomers of deoxyadenosine 5'-(α-thio)diphosphate and deoxyadenosine 5'-(α-thio)triphosphate can be separated by ultra-high performance liquid chromatography (UPLC). The diastereomer ratios (first elution peak / second elution peak) and their percentage composition relative to monophosphate, diphosphate, and triphosphate are shown in Table 36 below.

[0429] Table 36: Results of diastereomers of the reaction

[0430]

[0431] in conclusion

[0432] The experiments described and the transformation data obtained show that wild-type PPK (SEQ ID NO: 22, 31 and 34) can be synthesized from deoxyadenosine 5'-(α-thio)-triphosphate by phosphorylation at the terminal 5' thiophosphate.

[0433] The ratio of the diastereomers of 5'-(α-thio)-triphosphate and 5'-(α-thio)-diphosphate generated can be 1 (indicating no stereopreferentiality to phosphorylation), >1 (indicating a preference for generating early-eluting diastereomers), or <1 (indicating a preference for generating late-eluting diastereomers), depending on PPK.

[0434] sequence list

[0435]

[0436]

[0437]

[0438] SEQ ID NO:1

[0439] MRWIAALAEDDPIGKAVAGLVARYRVTDGEKFRIVDHATDDKAGLDKVTAKRFVKARRHHLAVLQENFAAARTRGIVVVLQGMDTAGKDSLIRHVMSGMNPQGTDVTSFKAPTPEEQAHDFLWRVHAAAPPRGKVGVFNRSHYEDVLVARVHAERLAMDGLPGDPSHPEFWDRRLEDIRHFEQYMTRQGFLFLKIFLHISFDEQRRRILRRLDRPEKRWKFDEGDLVERQFWPRYQVAYEEAIRATATEDAPWLVVPSDHKWLSRLVTMEALVGCLESLNPEPPPATSDLVRKIAEFRRALEKDTIVHPA

[0440] SEQ ID NO:2

[0441] MGSMDKTVYQYTGDQPVDLTQWATTTDTRIKTAQRNADLETLHDLQRRLYAQKQTGVVIILQGMDTAGKDGLIRHVFSGFNPAGTSVVSFKQPTSVDLQHDFLWRINRQLPARGEIRIFNRSQYEDVLISRVHPEILVGQHLPDVRSVADVTDKFFERRYHDLRHFETYLRHQGIMTYKFFLHISQKEQTARLERRLALPEKNWKFSTSDLKERQFWADYQTAYAQMLEHTATAKNPWYIIPADDKPTARRLVTAILTQELSQLDPQYPQVTADQQAHFQQLLQQLRKGQI

[0442] SEQ ID NO:3

[0443] MDAGGKDSAIKHLFTGINPQGCEVHSFKRPTFLELDHDFMWRSNIRTPSRGKIGVFNRSYYEEVLVVKVHEQILNSSKLPKELIHEDIWAERFEYIRAFERSMHQNGTNVLKFFLYLSPDEQKKRFISRMKNPEKNWKFDPNDLYERSFWGEYMQAYEEAIQSTACQDSPWYIIPADDKPYSRAVIADAVCQHLASLNLEYPTLSEEVQSNLQSYIEQLQNE

[0444] SEQ ID NO:4

[0445] MLRPDTTNQIRAGTDGGFSLSDHQPSWEGSGRVDGLTGAGLKKRAKKYVKAQRRLLAKAQEKLYADDRFSVLILLQAMDAAGKDSTIKQVMSGVNPQGCQVFSFKQPSAEELDHNFLWRYWRDVPERGRIGIFNRSYYEEVLVVRVHPELVQRQRLPAAPNGSDFWAARFEDINAFERHMTRNGTVILKFFLNVSKEEQRQRFVRRLTKPKKLWKFSANDLYERAHWDDYQHAYEELITATSTEWAPWYVIPADNKWLMRALVSHVIVEAVEGLGVTYPKVPPGREAEFRKALNALRAEGAASGKKGGIG

[0446] SEQ ID NO:5

[0447] MPHRHRLDHAPFTVQPGQRVRLKDYDSRETAGFKNKSEAKKALLEDVSLLAEMQQLLWAAKEYSLLIVFQALDAAGKDGAIRHVMSGVNPQGVDVYSFKAPTEEERLHHFLWRPAKVMPARGRIAIFNRSYYEEVLVVRVHPEWLERQWVPPRYRGGHYEELWPERFEEINAFERLMSENGVAILKFFLNVSKAEQRERFLERVTDPEKHWKYSAADLRERGYWDMYQAAYEDMLSHTSTKHAPWHVIPADRKWFTRACVADIIAAKIESLDLSYPTVSEEEHSAIEAARRQLEAESD

[0448] SEQ ID NO:6

[0449] MELKGPKVKDYIAKFIVEPGKQVRLKDFKTGWAENEQMKKLGEDKSKDLLVEMLEKDRSALAAVQDLLWSSRTHAVLIILQGMDTAGKDGTIRHVMSGVNPQGCSVHGFKVPSTQEHNHNFLWRYSNVLPERGEIGIFNRSYYEDVLVTRVHPNIISGFPKDIGPDADGFWKGRYYDINAFEEHLIRNGTLILKFFLHISKDEQKRRLLDRLEHKEKYWKFSVADLTERQFWDNYIKVYEKMLEATSTDIAPWFVIPGDYKWVARTAVAEAVVTAIKGLNLSYPSVSDDEIKRLMEAKKKLQAE

[0450] SEQ ID NO:7

[0451] MKINIKDFQVLQGDKVKLNKWPTQVGRVYSSKKNYHKLLKKQIKTLNELQQLQYAANTYSVLLVFQAMDAAGKDGVIRHVMSGINPQGCQVFSFKHPSATELDHDFLWRTNQCLPQRGRIGIFNRSYYEEVLIVRVHPEILHRQGLPDRLVDDETIWNGRYESIVDMENHLFRNGTRVIKFFLHLSKDEQRKRFVDRIDDPEKNWKFSEADIEERQFWDKYMGAYELCLSATSSKRAPWYVVPADDKKNARLIVSQVILDTFKSLNMQYPQTDEGRKQELLSIREQLIAEEE

[0452] SEQ ID NO:8

[0453] MKIKSGDYRVREGDKVRLKWPTLVKPVYSKEQYQEMLAEHVDELSKLQQLQYASDRYAFLLIFQAMDAAGKDGAIKHVMSGINPQGCQVFSFKHPSAQELHDFLWRTTQALPERGRIGIFNRSYEEVLIVRVHPEILRAQNIP DELLNTKTIWQERYRSIVDLEQHLHRNGTRIIKFFLHLSKEEQRKRFLARIDDPNKNWKFSPADIEERKYWDQYMLAYEACLSATSTKEAPWYVVPADDKENARLIISRIILDTFRLMLKMAYPKSGGEHQHELESIRRHLIKEGNAGK

[0454] SEQ ID NO:9

[0455] MTKNYLLARNGKVKLKAINPEHTGSFVNKEEVLEDFVILKEKLVDLQDKLIAGKKQSLLIIFQGMDCSGKDGVINKVLSAINPQGFQVTSFKKPTEEETSHDFLWRSHKASPAKGYIAAFNRSYYEDVLVTRVHGVIDDDEADRRFKHIRNFEKLLNDSDVRILKIFLHISKDFQIQKIKDRLENPNKRWKFDPSDLEERKYWDAYQSAYEDVFNHCNSKYAPWYAVPANHRWFRDYIVLKLVVKALEDMDLQYPDPDPELEKKISSLDLTKV

[0456] SEQ ID NO:10

[0457] MSKDNKQKSNAPKQSKVLSKKDLLQRARQFSEQYCVGDGKNFKLKDYKTNADFELGEEGKSLVKKTLKMGVNALSTMQDILYAQDKWSVLLIFQAMDAAGKDGAIKHVMSGINPQGCQVSSFKGPSSEELDHDFLWRCQKHLPERGRIGIFNRSYYEEVLVVRVHKAILEGQKIPKKLISDDIWENRFEDIRNFEKYLNRNGTIVIKFFLNVSKEEQKKRFIERIDNPDKNWKFSATDAKERGYWDDYMHAYEELIKNTSTEKSPWYVIPADNKSYARIAIASAIIHALDEMDLEYPKVSESKIVELNDVKQILLDEKE

[0458] SEQ ID NO:11

[0459] MPMVAAVEFAKSPAEVLRVGSGFSLAGVDPESTPGYTGVKADGKALLAAQDARLAELQEKLFAEGKFGNPKRLLLILQAMDTAGKGIVSHVVGAMDPQGVQLTAFKAPTDEEKSHDFLWRIEKQVPAAGMVGVFDRSQYEDV LIHRVHGWADAAELERRYAAINDFESRLTEQGTTIVKVMLNISKDEQKKRLIARLDDPSKHWKYSRGDLAERAYWDDYMDAYSVAFEKTSTEIAPWHVVPANKKWYARIAVQQLLLDALGGLQLDWPKADFDVAAERALVVES

[0460] SEQ ID NO:12

[0461] MGSMDKTVYQYTGDQPVDLTQWATTTDTRIKTAQRNADLETLHDLQRRLYAQKQTGVVIILQGMDTAGKDGLIRHVFSGFNPAGTSVVSFKQPTSVDLQHDFLWRINRQLPARGEIRIFNRSQYEDVLISRVHPEILVGQHLPDVRSVADVTDKFFERRYHDLRHFETYLRHQGIMTYKFFLHISQKEQTARLERRLALPEKNWKFSTSDLKERQFWADYQTAYAQMLEHTATAKNPWYIIPADDKPTARRLVTAILTQELSQLDPQYPQVTADQQAHFQQLLQQLRKGQI

[0462] SEQ ID NO:13

[0463] MTINRHDFLASPEKKLSEYATAHPDAASYSTEEVKEALFEEVISTLRDQQTRLFAEAKSGIVVILQAMDAAGKDEAVTTVFSNLSVQGLRESEPGEPSDKELKRDYLWRHHETLPKRGEIAILNRSYYEEVLGSRVHGSYQNEPMPDKWKEGPVWKRRYRHLNEYERYLTENGFVVLKFYLNVNKNVQKKRLLERMNNPNRNWEFSFSDVDDRDKWEDFYHAYDEAIKETSTGYAPWYVIPADDPWFTRLVIAEIFSQTLSELNPKLPVLSGEEATKLEEYKEKLKKQ

[0464] SEQ ID NO:14

[0465] MQGSQFRLEHGKKVRLANMDPNETGSLHDREEAEPNIERLKERLEALQNMLYAEKKQSVLFVIQGMDCSGKDGVVKRALGGLHPQGFQVHSFRTPTEEEEAHDFLWRAHRIVPAKGMIGAFIRSYYEDVLITRTHKWISEAEVKKRLKHINRFEKLLESSGVKIVKIFLHISKDFQLAKLRNRLTDETKRWKFDKNDLVERESWSAYEEAYEDVFKQCSTESAPWYVVPANHRWYRDLAVLQIAVDALESMRLAYPASDPELDQLLEQLQE

[0466] SEQ ID NO:15

[0467] MDVSRYRVPPGSGFDPEAWPTREDDFAGGKKEAKKELARLAVRLGELQARLYAEGRQALLIVLQGMDTAGKDGTIRHVFRAVNPQGVRVTSFKKPTALELAHDYLWRVHRHAPARGEIGIFNRSHYEDVLVVRVHELVPPEVWGRRYDHINAFERLLADEGTRIVKFFLHISKDEQKRRLEARLENPRKHWKFNPADLSERARWGDYAAAYAEALSRTSSDRAPWYAVPADRKWQRNRIVAQVLVDALEAMDPRFPRVDFDPASVRVE

[0468] SEQ ID NO:16

[0469] MATRTEDRADRIEELLDILRVPPGKKIDLRKDYDPGFTGKWMKKEEAEKTLAEGIQILAGMQDKLWAQNQYAVLMILQALDAAGKDGTIKHVMSGVNPQGVDVHSFKGPSSEELDHDYLWRNFKALPSRGRIGIFNRSYYEEVLVVRVHPEFLASQQLPPALKDERIWRRRFEEINNFEKYLVDNGIIVVKIFLYVSKEAQKERFLERAMMPEKNWKFSAADMKERAFWDDYIDAYEDMFNHTSTEWAPWHIVPADHKWFTRLAVAAVLYNTMENLNLAYPTISEQQKQALLVAKKELENEDGGGSKDKSVVKAKAKEAAYPEILDASEVTRDETKKRDRKKGKKNR

[0470] SEQ ID NO:17

[0471] MNIHWHGNSHAISQSPSRYPIVATPKVPSVAMPRNKGSVCEKNDCSKIRAVRAGGEIRVIDERYHKRLQVRPGIKVRLGEYNTGWAQNDALKEAGKDAVRERAMEILEENRAQLASAQELLWASNTYAVLIILQGMDTAGKDGTIKHVMSGVNPQGCKVHSFRVPTEEELDHTFLWRYWRALPNRGEIGIFNRSYYEDVLVVKVHPERLVNLPPGKRGKKFWNARYEDINAFERHLIRNGTVILKFYLHISKDEQKKRLLERLDDREKLWKFSLSDIEERKFWDRYQEVYEDALTMTSTDEAPWHIIPADHKWVARTLIADIITTKIQSLDLAFPTVSGEAIAQLKEARRHLEEE

[0472] SEQ ID NO:18

[0473] MKKTSAVDTSAYRVSNPETFVLSDAAADPPTGFDREQSKRVLKKNTKQLQSLQKLMWAERKQRLLIVLQAIDAGGKDGTIRSVFGPLNPQGVKVASFKRPTKTELAHDYLWRVHPHVPGNGEIVVFNRSHYEDVLVVKVHGYATADAIEKRYSHIRSFEQMLVDEGTTVVKIYLHISKDEQKKRFESRLNTPEKQWKFNKDDLKERAYWNQYQDAFETMLKETSTEDAPWYVVPANNKKYRNELISTLIRETLESLHMTWPKADEGIENVCLE

[0474] SEQ ID NO:19

[0475] MDKIKVDNYKANKNFKIADNETFEDFGKSDKELKKELSRIRRELGEFQDTIYAHGKYSILVCLQGMDTAGKDSLIREVFKDFNVSGVEVHSFKVPTELELSHNYLWRHYLVLPAKGKFGVFNRTHYENVLVTRVHPEYILGEHIPGIHSVDDIDQKFWDKRFDQINDFERHLAENGTLIFKFFLNLSKEEQRQRLLRRLAIKEKHWKFSPGDLKERKLWNVYQKCYEEAISKTTHDHAPWFAIPADNKKATRIIVASILLQSLKKYKDIKEPMLSDKIKSNLDSYEEQLQGEQ

[0476] SEQ ID NO:20

[0477] MKTINTDDFKITSPKKLSDLPTLVDLKATKQELKEALNETREALSDLQDKMYAHNRYGVLICLQGMDTSGKDSLIREVFKEFNARGVVVHSFKTPNSTELEHDYLWRHYLALPEKGKFSVFNRTHYENVLVTRVHPEYILNESLPGIEDVKDIPKDFWKKRMEQIVNFEKHIVENGVIVLKFFLHLSKEEQRQRLLRRLEKEKHNWKFSPSDLKERSFWKEYQHHYEEAINATSKEKAPWYVIPADDKNACRFLLAKAILDVLSQYKDIKFPELDEKVQENISFYKKELSNEK

[0478] SEQ ID NO:21

[0479] MMSAYPANLLEALRAEPGVKLSERATKDADWWPGDAPQDKKAAEKRMVQLAELLSDLQERLFAASVAGGQGPAVLLILQGMDTSGKGGTVRHVLGMLDPQGVKHHAFKAPTEEEAAHDFLWRITRQLPAGGLVGVFDRSHYEDVLVAKVKGFATPDAVESRYGKIVDFEKQLIEQGIHPIKVMLHISREEQFERLSARLERADKHWKYSPGDVDDRLLWDDFQSAYEVAINRTDNQGAPWHIVPADQKWKARLCVAELLLATLERVAPDWPVATFDVEQEKARLAAAK

[0480] SEQ ID NO:22

[0481] MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVTRVHPEYLLSEQTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSLEQKAELEKAKAELVAEKSSD

[0482] SEQ ID NO:23

[0483] MKIKKYFAGDDIKLKYFSTRCEEDLNKGEVKSILMPENLKKMTDYQSKLYAEGKKGLIVVFQAMDTAGKDGVIKHVMTSLNPQGMYVASFKAPSGVEMSHDYLWRIHKHAPSRGCVTVFNRSHYEDVIIARVHDLVKNQKLPDSMKHDGIWNDRYEQIRNYESYLHENGIHIVKFFLHLSKDEQRERLLSRIDEPEKNWKFSSADIHERKYWNDYQDAYEKVLQKTSTEKSPWYIIPADQKWFSRYLVSEILVEKFKDLNPEFPKLPEDELDNLAKWREELLND

[0484] SEQ ID NO:24

[0485] MSKIILANTTTKTYDRALKEKIKAENVKLVNQIVEFQRRMYSDGRHNLLIIFQGMDASGKDGATRKVFSGVNPLGIKVHAYKKPTDKEFAHDFLWRIHQNVPQQGMIQVFNRSHYEDILVPTIEGYFEESYIEKRYQHINDFEKLLIEGNNTTILKFYLHTSKEEQLERLTERIEIPEKYWKHNDKDWETRKKWDSYMSVYEQIFEKCNQPEWHIIPSDRNWEKENHIAKIVLKAFEEMDLKYPALDSELFKEKGM

[0486] SEQ ID NO:25

[0487] MGEAMERYFIKPGEKVRLKDWSPDPPKDFEGDKESTRAAVAELNRKLEVLQERLYAERKHKVLVILQGMDTSGKDGVIRSVFEGVNPQGVKVANFKVPTQEELDHDYLWRVHKVVPGKGEIVIFNRSHYEDVLVVRVHNLVPPEVWKKRYEQINQFERLLHETGTTILKFFLFISREEQKQRLLERLADPAKHWKFNPGDLKERALWEEYEKAYEDVLSRTSTEYAPWILVPADKKWYRDWVISRVLVETLEGLEIQLPPPLADAETYRRQLLEEDAPESR

[0488] SEQ ID NO:26

[0489] MIEEKYLERLLVKPGKKLKLTDFYTGWAQSNALREAGKEAVKEQAAEILEESRAALAEAQELLWADKQYSILVILQGMDTSGKDGTIKHVLSGVNPQGCRVMSFGVPTNDELHHTFLWRFWKAMPRRGEIGIFNRSYYEDVLVPKVHPVILEEQQLPPGYGSDAFWEARYEDIRAFERHHHANGTIILKFFLNISKEEQKERLLSRLKTEKKQWKFSLSDLTERQYWSHYQAAYEEAIFQTSTDDAPWYVIPADYKWVARTLVADIIVRAISRLDLRYPEISAEEMKEIEKARIHLESE

[0490] SEQ ID NO:27

[0491] MGDNGARVLERIRVPAGKKVKLTEYPTSWKKAEEFKLNGLKLTKDNSKEILEKSKGELAAAQELLWAEDSYSMLIILQGMDAAGKDGIIEHVMSGVNPQGCEVTGFKTPSQIELDHDFMWRCYAAMPERGRIGIFNRSYYEEVLVVRVHPEILIGQRLPGKEKGEKKLWEERFESINDIERHLTRNGTVILKFFLHESKDEQRSRFLDRLSDTEKQWKFSPGDLAEREFWDQYTKAYEEMLSATSTEHAPWYIIPADQKWLARVMVAFVIVRTIESLDLGYPQLSEAQKKSLGEAKKKLQDE

[0492] SEQ ID NO:28

[0493] MIKEKNIRKLMVIPGKKVSLNRFETGWAQNDELKKVGKDALKDRAAALLEANRIVLAEAQELLWASNIYSVLIVLQGMDTSGKDGTIKHVMSGVNPQGCRVTSFKVPTSEETDHTFLWRYTRALPAKGEIGIFNRSYYEDVLVVRVHPEILDNQQVPPGKRGDTFWNARYEDINAFERHLVQNGTLILKFFLNISKEEQRRRLLERLENKDKYWKFSLLDLAERRYWDNYQGAYEIMFLKTSTEMAPWYIIPADYKWVARSLIAEIISQKIQMLDLRFPHVSAEGLKELEKAREELNSE

[0494] SEQ ID NO:29

[0495] MDIDNYRVKPGKRVKLSDWATNDDAGLSKEEGQAQTAKLAGELAEWQERLYAEGKQSLLLILQARDAAGKDGAVKKVIGAFNPAGVQITSFKQPSAEELSHDFLWRIHQKAPAKGYVGVFNRSQYEDVLVTRVYDMIDDKTAKRRLEHIRHFEELLTDNATRIVKVYLHISPEEQKERLQARLDNPGKHWKFNPGDLKDRSNWDKFNDVYEDALTTSTDDAPWYVVPADRKWYRDLVLSHILLGALKDMNPQFPAIDYDPSKVVIH

[0496] SEQ ID NO:30

[0497] MLRPDTTNQIRAGTDGGFSLSDHQPSWEGSGRVDGLTGAGLKKRAKKYVKAQRRLLAKAQEKLYADDRFSVLILLQAMDAAGKDSTIKQVMSGVNPQGCQVFSFKQPSAELDHNFLWRYWRDVPERGRIGIFNRSYYEEVLVVRVHPELVQRQRLPAAPNGSDFWAARFEDINAFERHMTRNGTVILKFFLNVSKEEQRQRFVRRLTKPKKLWKFSANDLYERAHWDDYQHAYEELITATSTEWAPWYVIPADNKWLMRALVSHVIVEAVEGLGVTYPKVPPGREAEFRKALNALRAEGAASGKKGGIG

[0498] SEQ ID NO:31

[0499] MINIYKIDKLNNNFNLNNHKTDDYSLCKDKDTALELTQKNIQKIYDYQQKLYAEKKEGLIIAFQAMDAAGKDGTIREVLKALAPQGVHEKPFSPSSTELAHDYLWRVHNAVPEKGEITIFNRSHYEDVLIGKVKELYKFQNKADRIDEN TVVDNRYEDIRNFEKYLYNNSVRIIKIFLNVSKKEQAERFLSRIEEPEKNWKFSDSDFEERWYWDKYQQAFEDAINATSTKDCPWYVVPADRKWYMRYVVSEIVVKTLEEMNPKYPTVTKETLERFEGYRTKLLEEYNYDLDTIRPIEK

[0500] SEQ ID NO:32

[0501] MRLPDAVLQQCHYQHASALNLDPNRDFGLPMRKADARLEDLRKWLDRQQQLLWANNGPAILVWLQGPDCSGKDGAIRNSLRGLNPQGIRVSDFQKPTEQQRSEHFLKRYREQLPTAGMITVFNRTPYEGLVSDLADGDIDSNQAAERLQQLLAFEEQLAEQNIHLLKIYLHISKSEQKKRLQQRFINPEKRWKISAADLIGHQNFEQIQANWNQAFAQSSSLSHPWHILPSHNKALRNLLICSLIAQKLEALDLQWPQPALPFAIEDLDLACSISG

[0502] SEQ ID NO:33

[0503] MAKTSFLQRFKVLPGKKIHLAEIDTDATPGYKNKQEAKADLADCLQRLRDLQYLLYAENKRSLLIVLQGLDASGKDGTIRHVLGYMNPQGCRVTSFKVPTEEEAAHDFLWRVHKAAPGRRQVAIFNRSHYEDVLVVRVHKLVPKSVWEKRYDQINEFERLLADNGTHILKFFLHISPEEQLRRFRKRLENPKKRWKISEADYSERKYWHDYQQAYEDALSRCSRDHAPWFVIPADKKWFRNLAISQIVVESLESLKMQWPNPQVDLDLIRLKYHEAVENS

[0504] SEQ ID NO:34

[0505] MTTIPEEMEDRIKAVMDIMRVPPDEKIDLEKDYDPGFTGKWMKKKEAKETLVRGIRLLAEMQNKLYAQDQYALLIVLQALDAAGKDSTIKHVMSGINPQGVDVHSFKTPSGEELDHDYLWRNFKALPARGRIGIFNRSYYEEVLVVRVHPEFLASQKLPPILKDKHIWKRRFEEINNFEKYLVDNGIIVLKFFLYVSKEKQRERFLERALLPEKNWKFSTADMKERARWDEYITAYEDMFNHTSTEWAPWYVVPADHKWFTRLAVAAVIYNTMDKLNLAYPVVSEQQKQALLVAEKELENENGGPDDKAVRKVKAKAVASRKNKKKDKKKK

[0506] SEQ ID NO:35

[0507] MIKKRYLKKIMVSPGSRVSLERYDTGWAQDDELKKVGKDAIKERAAEILEANRAALAESQELLWASNLYSFLIVLQGMDTSGKDGAIKHVMAGVNPQGCRVTSFKVPTSEETDHNYLWRYTRALPARGEIGIFNRSYYEDVLVVKVHPEILDHQQMPPGKRGDKFWNARYDDINAFERHLVQNGTFILKFFLHISREEQKKRLMERLEDKEKYWKFSLSDLAERRFWDDYQSAYEMMLSKTSTRIAPWYIIPADYKWIARSLIADIISTKIRALDLKYPEVSAETLKQLEGAREQLARE

[0508] SEQ ID NO:36

[0509] MKTNLIKDLVVRPNMKVKLNEWDPDYDGGIFKKEVESTLNKDLLDRMSDLQYKLFADKNQSLLIILQGIDTSGKDSTIRHVMGAFNPQSCKVISFKAPNDEELSHDYLWRIHKVTPAKGEIVIFNRSHYEDVIEAR VQRLTPEDMWFERYRQINEFERYLYENNIKTVKLLLHISKDEQKKRLEDRIKDPSKHWKFSEADIIKRKYWDQYITAYEEAISRCSTPWAPWYYIIPANTKWFRNFAIANIVVKSLEDMKLRFPAKIDLSKVVIDD

[0510] SEQ ID NO:37

[0511] MNIKTSEFKVPEGIAVDLSKLPTQVEPVYSKKEYKELLAEQVAALSAQQQLQYADNSHSLLIFQAMDAAGKDGVIKHVMSGVNPQGCQVFSFKHPSAQELDFLWRTTQCLPERGRIGIFNRSYEEVLIVRVHPEILKNQK LPDSLLNDDAIWPNRYKSIVDFEQHMHRNGTKVIKFFHLSKDEQRKRFIDRIDQPEKNWKFTVSDINERKHWTQYMKAYSCLTATSTKEAPWYVVPADDKKNARLIVSQIILDAYKDMDMKYPETSEERRQELLSIREQLIND

[0512] SEQ ID NO:38

[0513] MKEPILAVLPQGPVDLTAIPTDDTGDFDGNKADGEKALADLGPELSELQERLFAERTAGSTRRVLVVLQGMDTSGKGGVLRHTVGLVDPQGVRITSFKAPTDEERAHDFLWRIEKALPQPGTIGVFDRSHYEDVLIARVRKFAGDEEIERRYGAINDFEQRLVDDGVTIVKCMLHISAEEQKARLLARLDDPSKFYKFNPGDLDERALWPAYREAYEIALERTNTDAAPWHVIPSDKKWFRNLCVGQLLLEALRGLEQWPAADFDVEAQKARLRDEVSPE

[0514] SEQ ID NO:39

[0515] MGKSKSSENDPKKIKSPEKLKNFQKLSPKERIEHAKKFSEQFCVGDGNSFQLKDYKSDIDFDLGKGTKSLVADTLELGVDALATMQEILYAQDKWSLLLVFQAMDAAGKDGAIKHVLSGVNPQGCQVSSFKAPSSEELDHDFLWRCQKHLPERGRIGIFNRSYYEEVLVVRVHEHILKAQKLAEELITENIWEERFEDIKNFEKYLRRNGTVVVKFFLNVSKDEQKKRFIERIDDADKNWKFSTADAKERQHWDEYMHAYEDLIKNTSTKDSPWYVIPADNKAYARIAIASAVIHTLDEMGLEYPKVSDEKVAELNEIKKALLSEE

[0516] SEQ ID NO:40

[0517] MKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRWVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLVVRV KNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE

[0518] SEQ ID NO:41

[0519] MPHRHRLDHAPFTVQPGQRVRLKDYDSRETAGFKNKSEAKKALLEDVSLLAEMQQLLWAAKEYSLLIVFQALDAAGKDGAIRHVMSGVNPQGVDVYSFKAPTEEERLHHFLWRPAKVMPARGRIAIFNRSYYEEVLVVRVHPEWLERQWVPPRYRGGHYEELWPERFEEINAFERLMSENGVAILKFFLNVSKAEQRERFLERVTDPEKHWKYSAADLRERGYWDMYQAAYEDMLSHTSTKHAPWHVIPADRKWFTRACVADIIAAKIESLDLSYPTVSEEEHSAIEAARRQLEAESD

[0520] SEQ ID NO:42

[0521] MKTTTPKPTEGQRLQSTAPSFDGCPDWQALADAARVAPGTHVHLMRDHNPGRHTPGLARDGGEYALAEAKTLLLELQDRFFASSGHALVVVLQAIDAAGKDGTIKHVMSGLNPVGVDVYSFKEPTPIEAAHDYLWRYHRVLPERGRIAVFNRSHYEAVLTTRVHPEFLVPPVADADLHHLWHRRFQEIKEWERYLHDNGTVIVKLFLNLSKGEQKRRFLQRLENPQKNWKFSVNDLAERTHWDDYQFAFQEMLSHTSTTWAPWYVVPADHKWFSHLTTSAVLVQALRGMDPQYPALTDSARERLTAAKLRLDSEPC

[0522] SEQ ID NO:43

[0523] MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVT RVHPEYLLSEQTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSL

[0524] SEQ ID NO:44

[0525] MASRIEDMETRIEKLLDIMRVPPGKKIDLQKDYDPGFTGKWVTKEEAEETLAKGVQLLAEMQDKLWAQNQYALLMILQALDAAGKDGAIKHVMSGVNPQGVDVHSFKGPSSEELDHDYLWRNFKTLPSRGRIGIFNRSYYEEVLVVRVHPEFLASQQLPPALKDKRIWRRRF EEINNFEKYLVDNGIIVVKIFLYVSKEVQKERFLERAMMPEKNWKFSVADMKERAHWDDYIAAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYNTMKNLNLAYPTVSEQQKQALLAAKDELEREDGGLKDKAVVKAETKAATDPQAPDASSAVTKGKAKKKGKKKNK

[0526] SEQ ID NO:45

[0527] MANRIEELLDVLRVPSGKKINLKKDYDPDFTGKWVKKEEAKEALTEGIQILAEMQDKLWAQDQYALLIILQALDAAGKDSTIKHVMSGVNPQGVDVHSFKVPSGEELHDYLWRNFKALPARGRIGIFNRSYEEVLAVRVHPEFLAGQKLPPSLKDKNIWNRRFEEIN NFEKYLVDNGIIVVKLFLYVSKETQKERFLERTLEPEKNWKFSAADMKERAFWDDYMAAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYNTMKSLNLAYPTVSEEQKQALLVAKEELEHEDGGLKDKAVTRAEAKTAADPKTLAARSKVTKEEVKKNSTKGKKNK

[0528] SEQ ID NO:46

[0529] MDVSGPRVRKYLRQFVADPTKKIRLKYDTARVPRKELSAESDDEAKALLKDLLDKNRQELARAQDLLAASAQYSILIVLQGMDTSGKDGTIRHVMSGINPQGCAVHSFKVPGAEEHAHDFLWRYEKVLPERGMIGIFNRSYYEDVLVVHVH PDRMESLPAAIGPENAGFWEGRYKDINAFEKHLVRNGTVVLKFFLHISRDEQKRRLLDRLSQEKFWKFSFTDIAERQYWDQYQVAYEEMLSATSTEYAPWFIVPADHKWVARTVVAEVIASAIEGLHLCYVPDFDEKIRQLEKAKAALERE

[0530] SEQ ID NO:47

[0531] MDTLKDLRSLIVEPGRKFDLADRATALDPLPKEEIEAELATLREQIGDIQDRMFTRHEQALLVVLQGMDSAGKDSTTKAVFTGVNPLGIRAASFSVPTPLERRHDYLWRIHAQVPQLGCIGVFNRSHYEDPVI RRVHGEIKNGAMKQYLQQIAEFERLLTETGTTIVKCFLHIGFDEQRERLQERIDNPAKNWKFDPSDLQERRYWKAYQSAYSEALTSTSTEQAPWYVVPADRKPARNLLVARLVLQALKGLSAPEPAPKPELKGLKVR

[0532] SEQ ID NO:48

[0533] MARSTAYLKTYRVGRKLRLKDIDPGARPAASSSREADDARLAELAIEIDRLQDLLYANGSAGRPPKLLLVLQGMDTSGKDGTARVFRQCSPLGVRVAAFKAPTEVERAHDFLWRVHAVAPRAGEVVVFNRSHYEDVLVPFVEGWIDAAERQRRLAHINAFERLLHDSGTTIVKCFLHISKDEQRERLQARLDDPAKRWKFQVGDLETREKWKAYLAAYETALAATSTACAPWHVVPADSKSNRNLMIATLVAQALAGMKLKPPKPDFDPAAVRVV

[0534] SEQ ID NO:49

[0535] MLFKPERYLWRRQSAPDLRQCATRLPYEDKDSRDQLKREAKDLLEESQKELIESQHRLNVIDTHAVLLILQGMDAAGKDGIIKHVLSGLNPLYCQITGFRAPSSIELEYPFLSRYVAHLPQRGHIGLFNRSWYEETVTVKIFPEFLHKQRI ALEAINDRFWQHRFEDINHFEQHLTRNGIAIIKCFLHVSREEQRQRLLDRVVKPEKFWKDFPFDLETRKRWDEFMQAWNETLTATDSEHAPWHVIPADDKPMARALVARLLASTIDALPLKEKSFSETQLERIKEARMILENEKDDSAEHD

[0536] SEQ ID NO:50

[0537] MNLKDYCFFGNKPFVIEQFDPAQTGKFQLKNEVEELHHTNLLKMQELQDRLYAENKEALLIIFQGMDASGKDSAVKHVMGGVNPQGINVHNFKKPSSEELDHDYMWRSMRVIPERGKIGVFNRSYYEDVLVGKVHKLYQESYLPDRCKTDKIFEQRYQQIKNYERYLYENGIRVIKFFLHISKEEQKKRFLERIEDDQKNWKFSDSDMIERDYWEEYQRAYHDAINATATRIAPWYVVPSDKKWFGHFIISETIIDTLEKINPQYPAVTRERKERLLEFRSKLLREEEQSSF

[0538] SEQ ID NO:51

[0539] MQLDRYRVPPGQRVRLSNWPTDDDGGLSKAEGEALLPDLQQRLANLQERLYAESQQALLIVLQARDAGGKDGTVKHVIGAFNPSGVQVSNFKVPTEEERAHDFLWRIHRQTPRLGMIGVFNRSQYEDVLVTRVHHLIDDQTAQRRLKHICAFESLLTDSGTRIVKFYLHISPEEQKKRLEARLADPSKHWKFNPGDLQERAHWDAYTAVYEDVLTTSTPAAPWYVVPADRKWFRNLLVSQILVQTLEEMNPQFPAPAFNAADLRIV

[0540] SEQ ID NO:52

[0541] MDTSQFQVSNPDSYVLADAPTVAPSDFDREESKRRVKQLTKQIQALQEKMWAGRKHRLLVVLQAIDAGGKDGTVRSVFGRTNPQGVKVHSFKRPTEQELAHDYLWRVHPHVPGNGEITVFNRSHYEDVLVVKVHGYAPLDVVEKRYQHIRNFEQMLVDEGTTXIKIFLHISKDEQKKRFEARLNRPEKNWKFKKGDIKERAFWEQYHEVFEQMLKKTTTDDSPWYVVPADNKKYRNEIISSLILETLESFKMQWPQPEEDLSDIVIE

[0542] SEQ ID NO:53

[0543] MIFNGENSVLAEELIAKFRIDEGQKVKLRDYDTGWKVSDEIAYFQKEEVKNKAKDILEKNREDLARGQSLLWANNTYSLLVIIQGMDTAGKDSLIKHVMSGVNPQGCRVSSFDVPSKEELDHDYFWRCNLKLPKRGEIGIFNRSYYEEVLITKVHPEILEGRHLPQKEFHDEFWKMRYEDINSYERHLSNSGTVVLKFFLHISKEKQKERLLARFENESKYWKISPSDIAERKFWDNYIRAYEDMLNNTSTKSAPWYIIPADYKWVARTIVSDIIASKLISLHDSYPAVSEETLEKIKKAADMLKNE

[0544] SEQ ID NO:54

[0545] MKNEFKGLIVQGDKKFSLKNHKTDFTGGYNKEKAKDALVNSKIELSHLQEKLYASGKHSVLIIFQAMDAAGKDSAIEHVMSGLNPQGCQVYSFKVPTSEEYQHDFLWRHYKALPERGRIGIHNRSHYENVLVCKVHPEYILSEDIPGITDVKKISKDFWKQRYQSIRNFEQHLTANGTVILKFFLNVSKDEQKQRFLDRIEDPSKNWKFSSGDIKERALWDKYMEAYQDAINETSTAESPWHIIPADKKWFARLAISEIIEDRLKNLELKFPVLGAEETLKLSETKNALLAE

[0546] SEQ ID NO:55

[0547] MIPQDFLDEINPDRYIVPAGGNFHWKDYDPGDTAGLKSKVEAQELLAAGIKKLAAYQDVLYAQNIYGLLIIFQAMDAAGKDSTIKHVMSGLNPQACRVYSFKAPSAEELDHDFLWRANRALPERGCIGIFNRSYYEEVLVVRVHPDLLNRQQLPPETKTKHIWKERFEDINHYERYLTRNGILILKFFLHISKAEQKKRFLERISRPEKNWKFSIEDVRDRAHWDDYQQAYADVFRHTSTKWAPWHIIPANHKWFARLMVAHFIYQKLASLNLHYPMLSEAHREQLLEAKALLENEPDED

[0548] SEQ ID NO:56

[0549] MVGKYRVDGKKSIKLKDFPTADKGIFKNKEEGLLKLGENIEIISELQNKLYAEDTYSLLIIFQAMDAAGKDGTIKHVFSGINPQGFQIFNFKQPSREELDHTYMWRTSKSMPERGRIGVFNRSYYEDVLVVRVHNLLSTQHLPKEKQYKDIWKRRFHNIREQERYLFENGVIPIKFFLNVSKEVQKERFLERIEDPSKNWKFSSADIEERKYWDDYQDAYEDAINHTSTSYAPWYVIPADKKWFAKFAVSEIIKETLESLNLKYPELNKTQKSELKNYKEILLKEK

[0550] SEQ ID NO:57

[0551] MGFCSIEFLMGAQMKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRWVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYED VLVVRVKNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVHILVETLEGLAMQYPQPETESAKIVIE

[0552] SEQ ID NO:58

[0553] MKINSEDFIVSTAGRKKVNLKHRPTIVKPLYGSKKQYKEILNEHIEELSSLQELLFASNRYSLLVIFEGMDAAGKDSAIKHVMSGVNPQGCQVFSFKRPTPIELQHDFLWRYVVCLPERGRIGIFNRSYYEGVLAARVHPEVDLRSEGFLDQDDGPAADEEKKKEEVWEQRYESIVNLEKHLYHNGTRIIKIFLHLSEEEQRKRFLERIDDPKRNWKFSLADVEERKFWHDYMKAYEDCLSATSTTTYAPWYVVPADNKENARLIISEIILETLNGLEMSYPKLDPKRLDELQSTRKKLLLNK

[0554] SEQ ID NO:59

[0555] MDTSEFLISNAEDFALSNWQVDPPAEFDEKQSKRTLKQNTKVLQSLQKLMWAERKQRVLVVLQAIDAGGKDGTIRSVFGGLNPQGVKVQSFKRPTKQELAHDYLWRVHKHVPGGNGEIMVFNRSHYEDVLVVKV HEYAPLDLVEKRYEHIRNFEQLLADEGTTIIKIFLHISKDEQKRRFEDRLNEPDKNWKFKKEDLKERAFWDQYQDAFETMIEQTSTKDAPWYVVPSNNKKYRNEIISNLLIETLSGLNMEFPPAADGLDNIVID

[0556] SEQ ID NO:60

[0557] MSAIRLTDIPTRAPGNMDKKKTRKATHKLKKAIADLQNILYAEAKHSLLVILQGMDASGKDGAIRHVFNRINPQGIQVTSFKVPTPEEAAHDFLWRIHAHAPARGMMHIFNRSHYEDVLVTRVHGWIDDDTAHRRMQHINHFEQLLLDHQTTILKFYLHISREEQAKRLQERLKNPKKRWKYNPSDLEEAQKWHDYLHYYEEVFATCSPEIPWIIVPADQNWYKEFVIAEAVHQALKNMQLRYPRRASLASAGHAHNRKK

[0558] SEQ ID NO:61

[0559] MSNGTRQPIVYPPHTRVDLRKFDPAATNGMSREEAEAELVGLRLRLNELQNMLYADARYALLVVLQGIDAAGKDGTINSVFEQVGPIGCSVVSFKAPTPEELAHDYLWRYHRVMPERGHITIFNRSYYEAVLVERVKGIVPKATWEQRYEDINRLEEYLLRNGTVVMKFFLAISKEEQRERLQERIDNPKKQWKFRLGDLEERKLWDEYLEAFEDMLERCNTKHAPWHVVPADRKWYRDVVVARTLVEKLASLGLRYPPADPAVLGLKVD

[0560] SEQ ID NO:62

[0561] MANRTEDMETRIEDVEARIEALMEFMRVPPGRKINLRKDYDPGFTGRWLKKAEAKETLARGIQMLAEMQDKLWAQNQYALLMVLQALDAAGKDGTIKHVMSGVNPQGVDVYSFKAPSGEERDHDYLWRNFRALPARGDIGIFNRSYYEEVLVVRVHPEILAGQQLPPALKDEGIWKRRFEEINNFEKYLVDNGIIVVKFFLYVSKEAQKKRFLKRTMLPEKNWKFSAADIKERAHWDDYLDAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYSTMKKLNLAYPTVSEQKKQALLVAKEELENEDGGKKDKAVVKAKAKAAANKKVPASGREGTRNESKKKGKKNR

[0562] SEQ ID NO:63

[0563] MATPLEKLADIFRVPPGRNIDLRKEYDPGFTSDSVSKENAQKYLETGIQKLAEQQDKLYAQNTLALLINFQAMDAAGKDSTIKQVMSGVNPQGVQVKSFKVPSAEELDHDYLWRTFLELPNRGHIGIFNRSYYEEVLVVRVHPEFLERQQLPPKQKGEDIWKRRYGEINNFEKYLTDNGIVVLKFFLNVSKEEQKKRFLERIEEPEKNWKFSANDVLERQYWDRYMDAYEEMFNNTSTPWAPWYIIPADHKWFTRFAVVSVINYTIDGMNLAYPSVSAEQKEALQKAKEAMEQEETHPEKTTGTKKKRKR

[0564] SEQ ID NO:64

[0565] MKIRSKDFRVPPGEKLDEWPTLIKPAYKSKKAYAELLQEHVAELSTLQQLHYASGRYALLLIFQAMDAAGKDGAIKHVMSGVNPQGCQVFSFKHPSATELEHDFLWRTNQCLPERGRIGIFNRSYEEVLIVRVHPEILRSQGLPDG AVDEKTVWKERYRSIVDLENHLYRNGTRIIKFFLHLSAEEQRKRFLERIDEPEKNWKFSLADIQERKFWPQYMQAYAECLGATSTKIAPWHVVPADDKENARLIVSQIILDAFKALKMRYPETDAKRRQELLSIRHGLSEEGRRNFSPQ

[0566] SEQ ID NO:65

[0567] MIKLIKDDRITITSGKNFKLKNFDTDFSLDFVSKESAGVIMQEHIQRIAALQERLYASDNHAILMIFQARDAAGKDSTIKHVMSGINPSGCQVVSFKVPTSTEIEHDFLWRTAVQLPERGRIGIFNRSYEEVLVTKVHPEIILNQRL PSVLTEKDLDKKFWEHRYESIRDHEKHLSRNGFVILKFFNLNVSKKEQKKRFLERIDQPDKNWKFSYGDIKERQYWDQYEDAFEQMIETAEEHAPWFVIPSDKKWFAQLAVARRILETLESLDLKFPELGDEEKAVLEKAKMELESEKD

[0568] SEQ ID NO:66

[0569] MKAINTEDFKVTGKFDVKGVPTFIDVETSDKKIANSLEEVSIKLGKLQDKMYANNRYSVLICLQGMDTAGKDSLIREVFKNFNARGVNVYSFKKPSSSELEHDYLWRHYIALPEKGKFAVFNRTHYENVLVTRVHPEFLMYENLPGIETVEEIPADLWEQRYEQINNFEKHISANGTIIFKFFLHLGKDEQKKRLLRLEKQSHNWKFSPGDLTERDLWDKYQQYYEEAINKTSKEHAPWYVIPADKKETARYLVAKTILDTLKKYDDIKEPEVSEDVLANISLYRDQLMNEE.

Claims

1. Use of polyphosphate kinase (PPK) in the production of sugar-modified and / or α-phosphate-modified nucleoside triphosphates, wherein the sugar-modified and / or α-phosphate-modified nucleoside triphosphates are produced from the corresponding modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate.

2. The use as claimed in claim 1, wherein the PPK is PPK2.

3. The use as described in claim 1 or claim 2, wherein the PPK is PPK2-III.

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

22.

6. A method for screening a set of putative and known PPKs to identify enzymes capable of producing correspondingly modified nucleoside triphosphates using sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates as substrates, said method comprising: a) Identify the sequence encoding the set of presumed and known PPKs; b) Synthesize the PPK; c) Purify the PPK; d) Each of the purified PPKs is mixed with the sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates to form a reaction mixture; and e) Analyze the presence of the corresponding modified nucleoside triphosphate in each of the said reaction mixtures to determine which enzyme(s) produce the corresponding modified nucleoside triphosphate.

7. The method of claim 6, wherein the sequence in step a) is identified using a sequence database and bioinformatics tools.

8. The method of claim 6 or claim 7, wherein the sequence in step a) encodes a protein with a length between 150 and 600 amino acid residues.

9. The method of any one of claims 6 to 8, wherein the PPK is synthesized in step b) having an N-terminal hexahistine tag and purified in step c) using nickel hyponitrotriacetate.

10. The method of any one of claims 6 to 9, wherein step d) comprises incubating the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, 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 presence of the corresponding modified nucleoside triphosphate in the supernatant is analyzed.

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

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

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

16. An enzymatic method for producing sugar-modified and / or α-phosphate-modified nucleoside triphosphates, said method comprising: PPK was incubated with correspondingly modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates 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 Mg 2+ Mn 2+ or Co 2+ .

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 the modified nucleoside triphosphate obtained by the method of any one of claims 16 to 20 in the preparation of polynucleotides.

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

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

24. The use as described in claim 21 or claim 22, wherein the polynucleotide is an oligonucleotide.

25. The use of claim 24, wherein the oligonucleotide is selected from the group consisting of: antisense oligonucleotides, siRNA, miRNA, aptamers, CRISPR guide RNA, and oligonucleotides for recruiting and guiding DNA and RNA editing enzymes.

26. The use of claim 25, wherein the oligonucleotide for recruiting and directing DNA and RNA editing enzymes is an A to I RNA base editing oligonucleotide (AIMer).

27. The use according to any one of claims 21 to 26, wherein the preparation of the polynucleotide is carried out 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 according to claim 28, wherein the polymerase is a template-independent polymerase.

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

32. The use or method of any 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, an O-methylene bridge with a 4' carbon, and an O-restricted ethylene bridge with a 4' carbon.

34. The use or method of claim 32, wherein the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate comprise locked nucleic acids (LNA).

35. The use or method of claim 32, wherein the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate comprise a restricted ethyl group (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 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 ester (PO), thiophosphate (PS), phosphoramide (PN), dithiophosphate, methylphosphonate, borophosphate, selenophosphate, N-methyl-o-aminobenzoyl (MANT), acetyl (Ac), benzyl (Bn), benzoyl (Bz), allyl (All), 2-cyanoethyl (CE), azidomethyl (AzM), cyanovinyl (Cv), methoxymethyl (MOM), methoxyethoxymethyl (MEM), methoxymethylphenylmethyl (MPM), diphenylmethyl (DPM). Tetrahydropyranyl (THP), dimethoxytriphenylmethyl (DMTr), methoxycarbonylmethyl (MOMcl), ethoxycarbonyl (EOC), benzyloxycarbonyl (Cbz), trifluoromethanesulfonate (triflate), mesylate, tosylate, trimethylacetyl (Piv), o-nitrobenzyl (o-NB), bromomethylcoumarinylmethyl (Bhc), (7-(diethylamino)coumarin-4-yl)methyl (DEACM), 6-nitroveratroloxycarbonyl (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 of the preceding claims, wherein the α-phosphate modification is selected from the group consisting of: PS, PN, methylphosphonate, borophosphate and selenophosphate.

44. The use or method according to any of the preceding claims, wherein the α-phosphate modification is stereospecific.

45. The use or method according to claim 43 or claim 44, wherein the α-phosphoric acid modification is PS.

46. ​​The use or method according to claim 43 or claim 44, wherein the α-phosphoric acid modification is PN.

47. The use or method of any of the preceding claims, wherein the bases in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate are 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 (Ψ), N1-methylpseudorazine (m1Ψ), 1-ethylpseudorazine, 2-thio-1-methyl-1-deazo-pseudorazine, 2-thio-1-methylpseudorazine, 2-thio-5-azauracil, 2-thio-dihydropseudorazine, 2-thio-dihydrouracil, 2-thio-pseudorazine, 4-methoxy-2-thio-pseudorazine, 4-methoxy 4-Thio-1-methyl-pseudouracil, 4-Thio-pseudouracil, 5-aza-uracil, dihydropseudouracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyluracil (2moU), 7-denitroguanine (7-denitro-G), 8-oxoguanine (8-oxo-G), 5-propynyluracil (pU), 5-propynylcytosine (pC), N6-methyluracil Adenine (m6A), N4-acetylcytosine (ac4C), O6-methylguanine (O6meG), hypoxanthine (H), xanthine (X), isocytosine (iC), isoguanine (iG), queuine (Q), 2-thionuracil (s2U), 4-thionuracil (s4U), 5-iodouracil (5Iu), 2-thionuracil (s2C), 5-chlorouracil (5ClU), and isouriacil.

48. The use or method of claim 47, wherein the bases in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate are selected from the group consisting of: A, U, C, G, T, m5C, I, Ψ and m1Ψ.

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

50. The use or method according to claim 47 or claim 48, wherein the bases in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate are m5C.

51. The use or method according to claim 47 or claim 48, wherein the bases in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate are m1Ψ.

52. Applications of PPK in generating stereospecific α-phosphate-modified nucleoside triphosphates from α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

53. The use according to claim 52, wherein the stereospecific α-phosphate modified nucleoside triphosphate is a stereospecific PS modified nucleoside triphosphate.

54. The use as described in claim 53, wherein S p diastereomers and R p The ratio of diastereomers is approximately 50:50, and the S p diastereomers and the R p The ratio of diastereomers is approximately 55:45, or the R... p diastereomers and the S p The ratio of diastereomers is approximately 55:

45.

55. The use as described in claim 53, wherein the S P :R p The 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.

56. The use as claimed in claim 53, wherein the R p :S P The 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, wherein the engineered PPK has at least 90% sequence identity with SEQ ID NO:

22.

58. A method for preparing polynucleotides, the method comprising incubating a nucleoside triphosphate, a suitable enzyme, and optionally a template under suitable conditions, wherein the nucleoside triphosphate is prepared by incubating a corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions.

59. The method of claim 58, wherein the nucleoside triphosphate comprises or is composed of naturally occurring nucleoside triphosphates.

60. The method of claim 58, wherein the nucleoside triphosphate comprises or is composed of modified nucleoside triphosphates.

61. The method of claim 59, wherein the nucleoside triphosphate is composed of naturally occurring nucleoside triphosphates, except that uridine triphosphate is replaced by N1-methylpseudouridine triphosphate.

62. The method of claim 60, wherein one or more or all of the nucleoside triphosphates are modified nucleoside triphosphates comprising sugar, α-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 terminal deoxynucleotidyl transferase (TdT).

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