Terminal uracil nucleotidyl transferase mutants and uses thereof

Abstract

The application provides a terminal uracil nucleotidyl transferase mutant and application thereof. On the basis of wild-type terminal uracil nucleotidyl transferase (TUT) from Drosophila melanogaster, engineering terminal uracil nucleotidyl transferase variants containing single mutation and multi-site mutation are screened. The engineering terminal uracil nucleotidyl transferase variant designed in the application has a unique function of specifically catalyzing single base extension of 3'-O-modified nucleotide monomers, and can realize high-precision controllable synthesis of single-stranded DNA / RNA molecules under template-independent conditions. The application breaks the dependence of traditional enzyme synthesis on a template strand, and significantly improves the efficiency and sequence control precision of single-stranded oligonucleotide synthesis.

Description

Technical Field

[0001] This application belongs to the field of bioengineering technology, specifically relating to terminal uracil nucleotide transferase mutants and their applications. Background Technology

[0002] Currently, DNA / RNA synthesis mainly includes two methods: chemical synthesis and enzymatic synthesis. Chemical synthesis often employs the solid-phase phosphoramidite method, which involves a four-step cyclic reaction of deprotection, coupling, capping, and oxidation, progressively extending the target DNA / RNA molecule according to a predetermined sequence. While this method has matured over a long period, it still suffers from several insurmountable drawbacks due to its inherent reaction principles. These include: a cumbersome four-step cyclic process with long cycle times; the use of toxic and flammable chemical reagents, posing significant environmental hazards; exposure of the nucleic acid chain to a protic acid environment during synthesis, leading to depurination damage and acid hydrolysis; and limitations imposed by chemical reaction equilibrium in the deprotection step, hindering further efficiency improvements. These structural damages and reaction equilibrium limitations result in a high single-step error rate in chemical methods, which accumulates exponentially with chain length, leading to low fidelity of long fragment products and failing to meet the demands of high-quality nucleic acid synthesis.

[0003] Compared to chemical synthesis, enzymatic synthesis only requires a conventional aqueous biochemical reaction system and operates under mild conditions, significantly reducing nucleic acid chain damage and theoretically enabling the direct synthesis of longer sequences. Enzymatic nucleic acid synthesis uses polymerases as the core catalyst and can be achieved through both template-dependent and template-independent pathways, capable of preparing nucleic acid molecules up to tens of kb in length. For site-controllable template-independent sequence synthesis, polymerases with terminal nucleotide transferase (TENT) activity can be used in conjunction with nucleotide substrates that have 3'-O-reversible blocking modifications, thereby achieving ordered single-base extension.

[0004] Terminal deoxynucleotidyl transferases (TdTs) possess both template-dependent and template-independent TdT activities. When paired with 3'-O-reversibly blocking deoxyribonucleotide substrates, they have been used to explore enzymatic DNA or RNA synthesis. In natural cells, RNA undergoes various forms of terminal nucleotide modification during its life cycle, including poly-A addition, poly-U addition, and CCA addition. These enzymes involved in terminal modifications exhibit RNA-dependent TdT activity, and depending on their substrate preference, they are also known as poly-A polymerases, poly-U polymerases, or terminal uracil nucleotidyl transferases and CCA-adding enzymes. Similar to TdTs, RNA-dependent terminal uracil nucleotidyl transferases also have the potential for enzymatic DNA or RNA synthesis.

[0005] However, existing enzyme molecules still have significant shortcomings in terms of catalytic efficiency and substrate compatibility. Selecting suitable natural enzymes and making targeted modifications are issues that must be addressed before exploring practical applications. Summary of the Invention

[0006] This invention is based on the terminal uracil nucleotide transferase Tailor from Drosophila melanogaster. Through structural and sequence analysis, mutants with significantly enhanced catalytic activity were screened. These mutants can efficiently perform in vitro enzymatic DNA / RNA synthesis, greatly reducing the single-step synthesis time.

[0007] Specifically, the technical solution of the present invention is implemented in the following ways:

[0008] In a first aspect, the present invention provides a terminal uracil nucleotide transferase mutant, wherein the terminal deoxynucleotide transferase mutant is obtained by amino acid mutation based on the sequence shown in SEQ ID NO.1, and the amino acid mutation site is selected from at least one of the following positions: position 212, position 215, position 236, position 248, position 288, position 303, position 306, position 312, position 364, position 368, position 385, position 404, position 410, position 414, position 421, position 423, position 429, position 432, position 446, position 469, position 479, position 491, position 507, position 527, and position 540.

[0009] The terminal deoxynucleotidyl transferase mutant of the present invention, by adding or deleting some amino acids based on SEQ ID NO. 1, yields a mutant that still has the function of synthesizing nucleic acids using 3'-O-modified nucleotides.

[0010] In one or more embodiments, the amino acid mutation is A212V, M215F, V236E, C248L, T288R, K303F, A306Y, C312I, Q364L, Q368R, E385R / K, A404G, A410R, I414E, A421P, L423Y, V429C, A432S, A446P, H469P, A479P, H491P, Q507E, A527R / K, or C540L.

[0011] Preferably, the amino acid mutation is A306Y / M215F, A306Y / E385R, A306Y / E385K, A306Y / A410R, A306Y / H491P or A306Y / C540L.

[0012] More preferably, the amino acid mutation is A306Y / E385R / C248L, A306Y / E385R / K303F, A306Y / E385R / Q364L, A306Y / E385R / A432S, A306Y / E385R / H469P, A306Y / E385R / A527R, A306Y / E385R / A421P, A306Y / E385R / T288R, A306Y / E385R / C312I, A306Y / E385R / Q368R, A306Y / E385R / A404G, or A306Y / E385R / L423Y.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding a terminal uracil nucleotide transferase mutant as described in any of the above claims.

[0014] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0015] Fourthly, the present invention provides a host cell comprising the expression vector described above.

[0016] The terminal uracil nucleotide transferase mutant of the present invention can be obtained by the following method: the host cells described above are cultured under culture conditions that allow the expression of nucleic acids encoding the terminal uracil nucleotide transferase mutant, and the terminal deoxynucleotide transferase mutant is recovered.

[0017] Fifthly, the present invention provides a kit comprising the terminal uracil nucleotide transferase mutant of any one of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, or the host cell of the present invention.

[0018] In one or more embodiments, the kit of the present invention further includes one or more nucleotides, preferably one or more 3'-O-modified nucleotides.

[0019] In one or more embodiments, the kit of the present invention may optionally include at least one nucleic acid primer.

[0020] Preferably, the kit of the present invention comprises: 1) the terminal uracil nucleotide transferase mutant of any one of the present invention; 2) one or more nucleotides (preferably one or more 3'-O-modified nucleotides); 3) optionally at least one nucleic acid primer.

[0021] In a sixth aspect, the present invention provides the use of the terminal uracil nucleotide transferase mutant described in any of the above claims, the use being to catalyze the synthesis of single-stranded DNA / RNA from 3'-O-modified nucleotides in the absence of a template.

[0022] In some embodiments, such a method includes the steps of: (a) providing a starting fragment comprising an oligonucleotide having a free 3'-hydroxyl group; and (b) reacting the terminal uracil nucleotide transferase variant of the present invention with the starting fragment or the extended starting fragment under enzymatic extension conditions in the presence of a 3'-O-reversibly blocked nucleoside.

[0023] In some embodiments, such a method further includes step (c) deblocking the extended starting fragment to form an extended starting fragment having a free 3'-hydroxyl group, and (d) repeating steps (b) and (c) until a nucleic acid molecule with a predetermined sequence is synthesized.

[0024] As one implementation, the removal of unreacted substrates and byproducts from the intermediate product, followed by the removal of the 3'-O-reversible blocking group to expose the 3' hydroxyl group, includes: removing the terminal uracil nucleotidyltransferase and unreacted nucleotides from the intermediate product; and using a cleavage agent to remove the 3'-O-reversible blocking group from the intermediate product to expose the 3' hydroxyl group.

[0025] In one embodiment, the cleavage agent is selected from one or more of tris(2-carboxyethyl)phosphine, palladium complex, sodium nitrite, or T4 polynucleotide kinase.

[0026] In one implementation, the initiating strand is DNA, RNA, or a mixture of DNA and RNA.

[0027] In one embodiment, the 3'-O-reversible blocking group is selected from -NH2, -CH2-CH=CH2, -CH2N3, -CH2OCH2CH=CH2, -CO-CH3, or -PO4.

[0028] In one embodiment, the 2' modifying group of the nucleotide is selected from -H, -OH, -O-CH3, -CH3, -F or -PO4.

[0029] In a seventh aspect, the present invention provides a method for the controlled synthesis of single-stranded DNA / RNA without a template, the method comprising the step of contacting a nucleic acid primer, at least one 3'-O-modified nucleotide, and a terminal deoxynucleotidyl transferase mutant according to any one of the present invention.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) This invention provides a variety of terminal uracil nucleotide transferase mutants, which can synthesize nucleic acid molecules efficiently and controllably without relying on templates, and surprisingly found that the efficiency of the various mutants is significantly higher than that of the wild-type terminal uracil nucleotide transferase before modification.

[0032] (2) The various terminal uracil nucleotide transferase mutants provided by this invention possess the unique function of specifically catalyzing the single-base extension of 3'-O-modified nucleotide monomers, enabling high-precision and controllable synthesis of single-stranded DNA / RNA molecules under template-free conditions. This invention overcomes the dependence of traditional enzymatic synthesis on the template strand, significantly improving the efficiency and sequence control precision of single-stranded oligonucleotide synthesis.

[0033] To provide a clearer understanding of this application, the specific embodiments of this application will be described below in conjunction with the accompanying drawings. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the terminal uracil nucleotide transferase and its variants catalyzing the reaction.

[0035] Figure 2 This is the real-time detection result of the activity of terminal uracil nucleotide transferase and its variant (single mutant) in catalyzing natural nucleotides in Example 2.

[0036] Figure 3 This is the real-time detection result of the activity of terminal uracil nucleotide transferase and its variant (double mutant) in catalyzing natural nucleotides in Example 2.

[0037] Figure 4 This is the real-time detection result of the activity of terminal uracil nucleotide transferase and its variants (multiple mutations) in catalyzing natural nucleotides in Example 2.

[0038] Figure 5 This is a gel imaging diagram showing the catalytic ability of the terminal uracil nucleotide transferase mutant in Example 3 to catalyze the 3' end reversible blocking group-initiated chain elongation of nucleotides.

[0039] Figure 6 This is a gel imaging image of 1 to 5 nucleotide synthesis cycles completed in Example 4. Detailed Implementation

[0040] The following will explain some of the terms and materials used in this invention so that those skilled in the art can understand them.

[0041] In this article, amino acids are represented by their single-letter or three-letter codes according to the following nomenclature: A: Alanine (Ala); C: Cysteine ​​(Cys); D: Aspartic acid (Asp); E: Glutamic acid (Glu); F: Phenylalanine (Phe); G: Glycine (Gly); H: Histidine (His); I: Isoleucine (Ile); K: Lysine (Lys); L: Leucine (Leu); M: Methionine (Met); N: Asparagine (Asn); P: Proline (Pro); Q: Glutamine (Gln); R: Arginine (Arg); S: Serine (Ser); T: Threonine (Thr); V: Valine (Val); W: Tryptophan (Trp); and Y: Tyrosine (Tyr).

[0042] In this document, the following terms are used to indicate substitutions: A212V indicates that the amino acid residue at position 212 of the parental sequence (alanine, A) is changed to valine (V); E385R / K indicates that the amino acid residue at position 385 of the parental sequence (glutamic acid, E) is changed to arginine (R) or lysine. A306Y / M215F indicates that the amino acid residue at position 306 of the parental sequence (alanine, A) is changed to tyrosine (Y), while the amino acid residue at position 215 of the parental sequence (methionine, M) is changed to phenylalanine (F). Other substitution forms can be deduced by analogy.

[0043] Unless otherwise stated, the positions disclosed in this application refer to the amino acid sequence numbers listed in SEQ ID NO. 1.

[0044] According to the present invention, terminal uracil nucleotide transferase and its variants can incorporate modified nucleotides, preferably modified 3'-O-nucleotides, and more preferably 3'-O-reversibly blocked nucleotides.

[0045] In the context of this invention, the term "modified nucleotide" refers to a molecule containing a nucleoside (i.e., a base attached to a deoxyribose or ribose molecule) bound to three phosphate groups, said nucleoside having at least one additional group at one end (2', 3', 5', or a base). This additional group blocks further addition of the nucleotide by preventing the formation of any phosphodiester bond (3-O-modification, 2', or 2-O-modification), or by spatially preventing polymerase from attaching to any nucleic acid fragment containing such a modified nucleotide (5' or base modification) at its 3' end. Furthermore, said additional group advantageously has a reversible property, allowing it to be removed by a specific cleavage reaction.

[0046] Nucleotides or nucleoside triphosphates include deoxyadenosine triphosphate (dTAP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), or deoxythymidine triphosphate (dTTP) as examples of nucleotides containing deoxyribose. Adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), or uridine triphosphate (UTP) are further examples of nucleoside triphosphates containing ribose. Other types of nucleosides, such as naturally occurring modified nucleosides and artificial nucleosides, can be combined with three phosphates to form nucleoside triphosphates. In a particular embodiment, the modified nucleotide is a 3'O-blocked nucleotide containing a group reversibly attached to the 3' end of the nucleoside triphosphate to prevent further nucleotide addition. The 3'-O-reversibly blocking group is selected from -NH2, -CH2-CH=CH2, -CH2N3, -CH2OCH2CH=CH2, -CO-CH3, or -PO4.

[0047] In some embodiments, the modified nucleotide comprises a modified nucleotide or nucleoside molecule containing a purine or pyrimidine base and a ribose or deoxyribose moiety having a removable 3'-OH blocking group covalently attached thereto, such that the 3' carbon atom has been attached to a group with a reversible structure (-OZ). Wherein -Z is any one of -C(R')2-OR", -C(R')2-N(R")2, -C(R')2-N(H)R", -C(R')2-SR", and -C(R')2-F, wherein each R" is a removable protecting group or part of a removable protecting group; each R' is independently a hydrogen atom, alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amide group, or a detectable marker attached by a linking group; provided that in some embodiments, such substituents have at most 10 carbon atoms and / or at most 5 oxygen or nitrogen heteroatoms; or (R')2 represents a pinalkylidene group of the formula =C(R"')2, wherein each R" The ' can be the same or different and is selected from the group consisting of hydrogen and halogen atoms and alkyl groups, provided that in some embodiments, the alkyl group of each R' has 1 to 3 carbon atoms; and wherein the molecule can react to produce an intermediate in which each R' is exchanged for H, or wherein Z is -(R')2-F, and F is exchanged for OH, SH, or NH2, preferably for OH, the intermediate being dissociated under aqueous conditions to provide a molecule having free 3'-OH; provided that when Z is -C(R')2-SR", neither of the two R' groups is H. In some embodiments, the R' of the modified nucleotide or nucleoside is an alkyl or substituted alkyl group, provided that such an alkyl or substituted alkyl group has 1 to 10 carbon atoms and 0 to 4 oxygen or nitrogen heteroatoms. In some embodiments, the -Z of the modified nucleotide or nucleoside is of the formula -C(R')2-N3. In some embodiments, Z is an azide methyl group.

[0048] In some embodiments, Z is a cleavable organic moiety with a molecular weight of 200 or less, with or without heteroatoms. In other embodiments, Z is a cleavable organic moiety with a molecular weight of 100 or less, with or without heteroatoms. In still other embodiments, Z is a cleavable organic moiety with a molecular weight of 50 or less, with or without heteroatoms.

[0049] In another specific embodiment, a "3'-O-modified nucleotide" refers to a nucleoside triphosphate carrying a 3'-O-methyl, 3'-azido, 3'-O-azidomethyl, 3'-O-amino, 3'-aminooxy, or 3'-O-allyl group at its 3' end. In another embodiment, the 3'-O-blocked nucleoside triphosphate is blocked by a 3'-O-azidomethyl, 3'-aminooxy, or 3'-O-allyl group. In other embodiments, a "3'-O-modified nucleotide" refers to a nucleoside triphosphate carrying an ester, ether, nitrile, phosphate, carbonate, carbamate, hydroxylamine, borate, nitrate, sugar, phosphoramide, phosphoramidate, benzenesulfonate, sulfate, sulfone, or amino acid at its 3'-O end.

[0050] In other embodiments, the 3'-O-terminated group of the present invention includes methyl, 3'-O-(2-nitrobenzyl), allyl, amine, azidomethyl, tert-butoxyethoxy, or propargyl. In another specific embodiment, "3'-O-modified nucleotide" refers to a nucleoside triphosphate having a terminator effector modifying group.

[0051] According to a particular aspect, the present invention relates to variants of teluracil nucleotidyltransferases capable of working in a nuclease-enzymatic synthesis method with modified nucleotides, particularly with 3'-O-modified nucleotides (e.g., 3'-O-blocked nucleotides), and having the ability to produce long-length nucleic acid molecules or nucleic acid molecule derivatives.

[0052] According to the present invention, it is possible to implement a series of cycles including addition and deprotection by using variations of the invention. Therefore, this method will allow the controlled extension of the starting nucleic acid strand into a defined sequence by multiple cycles of adding reversibly modified nucleotides and further removing blocking groups.

[0053] The present invention envisions the use of the terminal uracil nucleotide transferase and variants thereof according to the invention in any enzymatic nucleic acid synthesis method.

[0054] Another object of the present invention is to provide a method for synthesizing nucleic acid molecules without a template, the method comprising the step of contacting a nucleic acid primer with at least one nucleotide, preferably at least one 3'-O-modified nucleotide and a variant of the present invention.

[0055] In the context of this invention, the term "cleavage reaction" refers to any action of substance or physical conditions capable of cleaving additional groups on previously described reversibly modified nucleotides. Those skilled in the art can determine the cleavage reaction of any of the previously listed groups. In one embodiment, the cleavage agent is a chemical cleavage agent. In an alternative embodiment, the cleavage agent is an enzymatic cleavage agent.

[0056] Those skilled in the art will understand that the choice of cleavage agent depends on the type of 3' nucleotide blocking group used. For example, tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave the 3'-O-azidomethyl group, a palladium complex can be used to cleave the 3'-O-allyl group, or sodium nitrite can be used to cleave the 3'-O-amino group. In a particular embodiment, the cleavage reaction includes TCEP, a palladium complex, or sodium nitrite.

[0057] In certain embodiments, the lysis reaction is carried out in the presence of additional components such as denaturing agents (e.g., urea, guanidine hydrochloride, formamide, or betaine). In other embodiments, the lysis reaction is carried out using one or more buffer solutions. Those skilled in the art will understand that the choice of buffer solution depends on the exact reaction mechanism.

[0058] This invention relates to variants of teluracil nucleotide transferases having the ability to quantitatively incorporate modified nucleotides. "Quantitatively" or "quantitatively reactant" means a reaction that proceeds completely, i.e., in which the reactants are completely converted into products. A polymerase that quantitatively incorporates reversibly modified nucleotides is a polymerase capable of extending each nucleic acid fragment with all available nucleotides, resulting in all starting fragments of length n being converted into fragments of length n+1.

[0059] As used herein, "starting fragment" refers to a short oligonucleotide sequence having a free 3' end that can be further extended. In one embodiment, the starting fragment is a DNA starting fragment. In an alternative embodiment, the starting fragment is an RNA starting fragment.

[0060] In one embodiment, the starting fragment has between 3 and 100 nucleotides, particularly between 3 and 20 nucleotides.

[0061] In one implementation, the starting fragment is single-stranded. In an alternative implementation, the starting fragment is double-stranded.

[0062] In one embodiment, the starting fragment is immobilized on a solid support. The starting fragment can be attached to the solid support by a variety of methods, resulting in a fragment that is stable under the various enzymatic or synthetic reaction conditions it will undergo.

[0063] In one embodiment, the starting fragment is immobilized on a solid support via a reversible interacting portion, such as a chemically cleavable linker, an antibody / immunogenic epitope, a biotin / biotin-binding protein, or a glutathione-GST tag. In another embodiment, the starting fragment is immobilized on a solid support via a chemically cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker.

[0064] In the starting fragment, the immobilized portion contains at least one restriction site. Prior art describes the selective hydrolysis of nucleic acid chains at specific sites using restriction enzymes and restriction sites. Any person skilled in the art can select an appropriate restriction enzyme that matches the sequence of the cleavage site in the starting fragment.

[0065] In an alternative implementation, the starting fragment contains at least one uridine. Treatment with uracil-DNA glycosylase (UDG) produces a base-free site. Treatment with a purine-free / pyrimidine-free (AP) site endonuclease on a suitable substrate will extract the nucleic acid strand.

[0066] Another object of the present invention is to provide nucleic acid molecules encoding variations of the invention. As used herein, "nucleic acid molecule" refers to a polymer of nucleosides. In one embodiment, the nucleic acid is DNA. In an alternative embodiment, the nucleic acid is RNA. In an alternative embodiment, the nucleic acid is XNA.

[0067] Those skilled in the art will understand that each of the previously listed nucleic acid molecules may carry modifications on the bases of the nucleotides that make up the polymer. Such modifications may be natural modifications, such as epigenetic modifications, or non-natural modifications, such as markers.

[0068] In one implementation, the nucleic acid molecule is DNA, RNA, or XNA carrying naturally occurring epigenetic modifications such as methylation, hydroxymethylation, formylation, or 5-carboxylation.

[0069] In one implementation, the nucleic acid molecule is DNA, RNA, or XNA carrying non-naturally occurring modifications such as fluorescent tags, fluorescent markers, or interacting groups.

[0070] The enzymatic synthesis method of the terminal uracil nucleotide transferase and polynucleotide of the present invention will be described in detail below.

[0071] First, let me describe the terminal uracil nucleotide transferase of the first aspect of the present invention.

[0072] Existing technologies, terminal uracil nucleotide transferases (TURBTs) suffer from significant deficiencies in catalytic efficiency, substrate adaptability, and enzyme stability, failing to meet the demands for efficient, precise, and long-fragment enzymatic polynucleotide synthesis. For example, traditional TURBTs primarily recognize native nucleotides (NTPs). When a reversible 3'-O-blocking group (such as -NH2, -CH2-CH=CH2, etc.) is introduced to achieve controllable single-base elongation, the modified group leads to severe steric hindrance and charge repulsion. Existing enzymes exhibit extremely low affinity for such blocking substrates, failing to effectively distinguish between the strand to be extended and the already blocked strand, resulting in ineffective cycles or premature termination, making template-independent stepwise synthesis difficult.

[0073] In view of this, the present invention provides a terminal uracil nucleotide transferase (derived from Drosophilamelanogaster) and its variants, the amino acid sequence of which is as follows:

[0074] (SEQ ID NO:1)

[0075] In this invention, the terminal uracil nucleotide transferase can synthesize nucleic acid molecules, such as DNA or RNA chains, without a template strand. This includes catalyzing the synthesis of oligonucleotides, or for other cyclic synthesis of functional DNA and / or RNA fragments, or for catalyzing the iterative synthesis of DNA / RNA in a DNA synthesizer. Specifically, the terminal uracil nucleotide transferase can efficiently couple with 3'-O-terminally modified nucleotides to synthesize nucleic acid molecules.

[0076] In this invention, "sequence similarity" refers to the numerical value represented by "similarity" given by the alignment software after sequence homology alignment. The sequence alignment software result "identity" is translated as "sequence identity" or "sequence sameness" in this invention. For example, it refers to the "similarity" or "identity" values ​​given by offline software such as SnapGene and DNAMAN, or online alignment software such as BLAST.

[0077] It is understood that the terminal uracil nucleotide transferase provided by this invention catalyzes the addition of a single base of a 3′-O-reversibly blocking modified nucleotide to the 3′-terminus of the initiating chain in a template-independent manner, thereby achieving enzymatically controlled polynucleotide synthesis. Specifically, the terminal uracil nucleotide transferase catalyzes the single-step controlled addition of a specified type of nucleotide to the 3′-terminus of the initiating chain, and through multiple extension-deprotection cycles, prepares a polynucleotide of a predetermined sequence (with excellent yield and / or higher yield). The terminal uracil nucleotide transferase and its mutants exhibit excellent catalytic activity, suggesting that the terminal uracil nucleotide transferase has a more significant application prospect in the synthesis of polynucleotides.

[0078] In particular, the terminal uracil nucleotide transferase provided by the present invention, in addition to SEQ ID NO.1 above, also includes a homologous sequence, mutant or functional fragment having at least 60% sequence similarity to the above sequence and retaining non-template-dependent nucleotide transfer activity.

[0079] In this invention, amino acid sequences with homology ≥ 60% and retaining the same function include, but are not limited to, sequences with homology of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 98%.

[0080] For example, taking SEQ ID NO:1 as an example, the terminal uracil nucleotide transferase of the present invention can undergo any one or more of the following mutations based on SEQ ID NO:1, i.e., using SEQ ID NO:1 as a template: A212V, M215F, V236E, C248L, T288R, K303F, A306Y, C312I, Q364L, Q368R, E385R / K, A404G, A410R, I414E, A421P, L423Y, V429C, A432S, A446P, H469P, A479P, H491P, Q507E, A527R / K, C540L; preferably, the terminal uracil nucleotide transferase mutant contains the following double mutations: A306Y / M215F, A306Y / E385R A306Y / E385K, A306Y / A410R, A306Y / H491P, A306Y / C540L; more preferably, the terminal uracil nucleotide transferase mutants claimed to contain the following multiple mutations: A306Y / E385R / C248L, A306Y / E385R / K303F, A306Y / E385R / Q364L, A306Y / E385R / A432S, A306Y / E 385R / H469P, A306Y / E385R / A527R, A306Y / E385R / A421P, A306Y / E385R / T288R, A30 6Y / E385R / C312I, A306Y / E385R / Q368R, A306Y / E385R / A404G, A306Y / E385R / L423Y.

[0081] It is understandable that the aforementioned mutants can be obtained by mutating the corresponding sites using existing site-directed mutagenesis techniques. Among them, mutants with multiple mutation sites can be obtained by superimposed mutations on the basis of a single mutant, that is, by mutating at another amino acid mutation site on the basis of a single mutant, thereby obtaining mutants with multiple mutation sites.

[0082] For example, using an expression vector containing the amino acid sequence shown in SEQ ID NO. 1 as a template, mutations are performed at the corresponding sites (site-directed mutagenesis) to obtain a recombinant plasmid encoding a terminal uracil nucleotide transferase mutant. Site-directed mutagenesis can be performed using existing mutagenesis techniques, such as polymerase chain reaction.

[0083] In summary, the terminal uracil nucleotide transferase and its variants provided by this invention can catalyze the generation of polynucleotides with target sequences from nucleotides modified with a reversibly blocking group of 3'-O-. The terminal uracil nucleotide transferase and its mutants have excellent catalytic activity, suggesting that the terminal uracil nucleotide transferase and its mutants have a more significant application prospect in the synthesis of polynucleotides.

[0084] Next, the nucleic acid molecule of the second aspect of the present invention will be described.

[0085] It is understood that the nucleic acid molecule provided by the present invention is a DNA molecule encoding the nucleotide sequence or complementary sequence of any of the above-mentioned terminal uracil nucleotide transferases and their mutants.

[0086] The recombinant vector (expression vector) of the third aspect of this embodiment will be described below.

[0087] The recombinant vector provided by this invention is a DNA molecule encoding the nucleotide sequence or complementary sequence of any of the above-mentioned terminal uracil nucleotide transferases and their mutants.

[0088] Exemplarily, the vector of the present invention may be a plasmid, bacteriophage, phage particle, granule, virus, YAC, BAC, Agrobacterium pTi plasmid, etc. The vector may preferably contain at least one element selected from: origin of replication, multiple cloning site, and optional gene. Preferably, the vector is a plasmid.

[0089] Examples, albeit non-exhaustive, of prokaryotic vectors include: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescriptSK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A; ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, pRIT5, and pET-28a. Preferably, the vector is an expression vector, and more preferably, pET-28a.

[0090] Next, the expression box of the fourth aspect of the present invention will be described.

[0091] In one specific embodiment, the expression cassette contains all elements for expressing the terminal uracil nucleotide transferase and its mutants, including elements essential for transcription and translation in the host cell.

[0092] For example, the expression cassette includes a promoter and a terminator. This embodiment does not specifically limit the promoter and terminator; they can be promoters and terminators known in the art capable of expressing the terminal uracil nucleotide transferase and its mutants. For example, the promoter can be prokaryotic or eukaryotic, and can be selected from, for example, Lacl, LacZ, pLacT, ptac, T3 or T7 phage RNA polymerase promoters, CMV promoters, HSV thymidine kinase promoters, SV40 promoters, mouse metallothionein-L promoters, etc.

[0093] In particular, the expression box described in this invention may optionally include an enhancer or other necessary elements.

[0094] In one specific embodiment, the host cell can be a prokaryote, such as *Escherichia coli*, or a eukaryote. The eukaryote can be a lower eukaryote, such as yeast (e.g., *Pichia pastoris* or *Kluyveromyces lactis*) or fungus (e.g., *Aspergillus*), or a higher eukaryote, such as insect cells (e.g., Sf9 or Sf21), mammalian cells, or plant cells. The cell can be a mammalian cell, such as COS (green monkey cell line), CHO (Chinese hamster ovary cell line), mouse cells, and human cells, etc.

[0095] Next, the enzymatic synthesis method of polynucleotides according to the fifth aspect of the present invention will be described.

[0096] This invention also proposes an enzymatic synthesis method for polynucleotides, such as... Figure 1 As shown, the enzymatic synthesis method includes the following steps:

[0097] (1) Provide an initiation chain, and under the catalysis of terminal uracil nucleotide transferase and its mutant, add 3'-O-nucleotides modified with reversible blocking groups to the 3' end of the initiation chain to obtain the final product.

[0098] Typically, in step (1), a recombinant plasmid encoding terminal uracil nucleotide transferase and its mutant is first prepared. Then, the recombinant plasmid is transformed into a host cell (such as E. coli BL21(DE3)) to obtain a recombinant strain. The bacterial cells are then collected and purified by lysis (such as sonication, high-pressure disruption, or freeze-thaw) and purification operations to obtain the purified protein terminal uracil nucleotide transferase and its mutant. Based on the purified protein terminal uracil nucleotide transferase and its mutant, a nucleotide modified with a reversible blocking group is added to the 3' end of the initiating chain.

[0099] It is understandable that step (1) is a template-independent single-base controlled extension reaction of nucleic acid. The nucleotide modified with the 3'-O-reversibly blocking group is the reaction substrate. This step involves the directional addition of the 3'-O-reversibly blocking modified nucleotide to the 3' end of the initiating chain under the catalysis of terminal uracil nucleotide transferase, generating a single-base extension intermediate with the terminal reversibly blocked, which provides a basis for subsequent iterative nucleic acid synthesis.

[0100] The initiating chain is typically a synthetically produced DNA, RNA, or a mixture of DNA and RNA, with a free 3'-hydroxyl group (3'-OH) retained at its end. The initiating chain acts as an acceptor substrate for nucleotide addition, providing a nucleophilic reaction site and serving as the anchoring backbone for enzymatic elongation reactions.

[0101] In step (1), the chain extension reaction mediated by terminal uracil nucleotide transferase and its mutant is initiated to provide covalent linkage sites for the modified nucleotides and construct the starting structural unit of the target nucleic acid chain.

[0102] The terminal uracil nucleotide transferase and its mutant used in this invention are a type of polymerase that does not depend on nucleic acid templates. It is the core biocatalyst in step (1). The terminal uracil nucleotide transferase and its mutant can specifically recognize the free 3'-OH of the initiating chain, catalyze the formation of phosphodiester bonds between nucleotide monomers and the initiating chain, precisely mediate the single addition of mononucleotides, and inhibit the side reaction of continuous polymerization of polynucleotides.

[0103] Specifically, terminal uracil nucleotide transferase and its mutants can reduce the activation energy of phosphodiester bond formation, enabling directional, efficient, and controllable addition of modified nucleotides to the 3' end of the initiating chain, thus ensuring the enzyme-catalyzed specificity of the reaction and the precision of single-base extension.

[0104] It should be noted that the nucleotides modified with the 3'-O reversible blocking group are chemically modified nucleotide monomers. The 3'-hydroxyl group of the natural nucleotide is blocked by the reversible protecting group, while retaining the 5'-triphosphate structure.

[0105] In step (1), the nucleotide modified by the 3'-O-reversible blocking group serves as the monomeric structural unit for nucleic acid chain extension, providing bases and pentose backbones to form the nucleic acid backbone; the 3'-O-reversible blocking group blocks the terminal hydroxyl group, blocking the secondary extension after addition and forcing the reaction to stop at the single base addition stage; furthermore, the reversible blocking group can be gently removed in subsequent steps to restore the 3'-OH activity.

[0106] Based on this, step (1) can effectively achieve single-cycle single-base precise extension, avoid non-specific polymerization side reactions, provide controllable reaction units for iterative de novo nucleic acid synthesis, and ensure the editability and accuracy of nucleic acid sequences.

[0107] Typically, step (1) includes two processes: a non-covalent enzyme-substrate binding reaction and a covalent enzyme-catalyzed nucleoyl transfer reaction, with the covalent reaction being the key reaction.

[0108] In enzyme-substrate recognition and binding reactions (non-covalent reactions), terminal uracil nucleotide transferases and their mutants bind specifically to the initiating chain and the 3'-O-reversibly blocking modified nucleotide sequentially through intermolecular forces, forming an enzyme-initiating chain-modified nucleotide ternary complex. This reaction is a prerequisite for covalent catalysis; no new covalent bonds are formed, and only substrate localization and active site matching are achieved.

[0109] In the enzymatic phosphodiester bond formation reaction (covalent reaction), this reaction is a nucleophilic substitution reaction, namely a nucleoyl transfer reaction. The specific process is as follows: the free 3'-OH at the end of the initiating chain acts as a nucleophile to attack the α-phosphorus atom of the 5'-triphosphate of the modified nucleotide; under the catalysis of terminal uracil nucleotide transferase and its mutants, the phosphate anhydride bond of the 5'-triphosphate of the nucleotide breaks, accompanied by the departure of the pyrophosphate (PPi) group; a phosphodiester bond is formed between the 3' end of the initiating chain and the 5' end of the modified nucleotide, completing the mononucleotide covalent linkage.

[0110] In this process, since the 3'-OH of the modified nucleotide is blocked by a reversible blocking group, there is no free hydroxyl group at the end of the new chain after addition, and the reaction terminates automatically, generating only a single-base extension intermediate product without any continuous extension side reactions.

[0111] In one embodiment, the 3'-O-reversibly blocking group is selected from -NH2, -CH2-CH=CH2, -CH2N3, -CH2OCH2CH=CH2, -CO-CH3, or -PO4.

[0112] In one embodiment, the 2' modifying group of the nucleotide is selected from -H, -OH, -O-CH3, -CH3, -F or -PO4.

[0113] In summary, step (1) is a template-independent enzymatic single-base nucleic acid extension reaction. A pre-prepared oligonucleotide initiator chain is used as the reaction substrate backbone, which retains a free 3' hydroxyl group as a nucleophilic reaction site. Terminal uracil nucleotide transferase and its mutant are used as biocatalysts. Taking advantage of its template-independent polymerization characteristics, it recognizes and binds the initiator chain to the nucleotide substrate modified with the 3'-O-reversible blocking group. Under the action of enzyme catalysis, the 3' hydroxyl group of the initiator chain undergoes a nucleoyl transfer reaction with the 5'-triphosphate group of the modified nucleotide. A single molecule of 3'-O-reversible blocking modified nucleotide is covalently added to the 3' end of the initiator chain through a phosphodiester bond, accompanied by the generation of pyrophosphate byproduct. The reversible blocking group on the modified nucleotide blocks the 3' hydroxyl group at the newly generated chain end, so that the reaction terminates at the single-base addition stage, and finally obtains a nucleic acid intermediate with a reversible blocking modified group at the 3' end, which provides a basis for subsequent cyclic nucleic acid chain extension.

[0114] (2) Remove unreacted substrates and byproducts from intermediate products, and then remove the 3'-O-reversible blocking group to expose the 3' hydroxyl group.

[0115] Step (2) is the purification of intermediate products and the 3'-end deprotection and activation, which is the key step connecting the two rounds of single base extension. Step (2) first removes the unreacted substrate, enzyme, byproducts and buffer impurities (if any) remaining from the previous round of reaction by separation. Then, the high-purity intermediate product is subjected to the removal of the 3'-O-reversible blocking group, and the free 3'-hydroxyl group is exposed again, so that it has the reactivity to carry out enzymatic nucleotide addition again.

[0116] Step (2) can be divided into two stages: purification and deprotection activation. In step (1), the intermediate product (reaction mixture) is used as the starting material for step (2). The active ingredient is a monobasic extended oligonucleotide with a reversible blocking group at the 3' end, which is mixed with various impurities.

[0117] The aforementioned impurities include unreacted substrates and byproducts, specifically including unreacted initiation chains, remaining 3'-O-reversibly blocking nucleotides, terminal uracil nucleotide transferases and their mutants, etc. These impurities are all redundant components of the reaction and will inhibit subsequent deprotection and the next round of enzymatic extension. Therefore, they must be completely removed to avoid interfering with subsequent reactions.

[0118] This invention generates free 3'-hydroxyl groups (3'-OH) by breaking the connection between the 3'-oxygen atom and the blocking group, while releasing the blocking group fragments, and the product restores the reactivity required for enzymatic extension.

[0119] In summary, step (2) first purifies the enzyme-catalyzed reaction mixture (intermediate product) obtained in step (1) to achieve efficient separation of the target intermediate product from unreacted initiating chains, 3'-O-reversible blocking modified nucleotides, terminal uracil nucleotide transferase and its mutant catalyst, pyrophosphate byproducts and other impurities in the system. Then, in a suitable mild reaction system, the corresponding deprotection reagent is used to selectively remove the reversible blocking group at the 3' position of the intermediate product, breaking the covalent bond between the blocking group and the 3'-oxygen atom, thereby exposing and restoring the free 3'-hydroxyl group at the end of the nucleic acid chain. If necessary, a quenching reagent is added to terminate the deprotection reaction, and finally, an activated oligonucleotide product that can directly enter the next round of terminal uracil nucleotide transferase and its mutant catalyze single-base extension is obtained.

[0120] (3) Repeat the above steps until the target sequence of polynucleotide is obtained.

[0121] Step (3) is an iterative and cyclical controlled synthesis stage. Its core is to repeatedly perform the single-base addition in step (1) and the purification and deprotection in step (2) according to the preset base sequence, gradually increasing the nucleic acid chain by extending one base per cycle until the target polynucleotide with the required sequence and length is obtained. It can be understood that step (3) is a completely new reaction type, which is entirely a repetition and iteration of the core reactions in the first two steps.

[0122] Specifically, after the previous round of deprotection in step (2), the oligonucleotide chain with free 3'-hydroxyl groups serves as the new initiating chain; under the catalysis of terminal uracil nucleotide transferase and its mutants, the 3'-OH group of the initiating chain attacks the 5'-triphosphate α-phosphoatom of the 3'-O-reversible blocking modified nucleotide; wherein, the phosphate anhydride bond breaks and releases pyrophosphate (PPi), forming a new phosphodiester bond at the chain end, completing the precise addition of a single base; the reversible blocking group on the modified nucleotide forces the chain extension to terminate, ensuring that only one base is added in each round.

[0123] Once the chain length and base sequence meet the target requirements, the cycle is terminated. Alternatively, the terminal reversible blocking group can be retained, or the 3′-hydroxyl group can be permanently blocked to completely terminate chain extension and obtain a structurally stable target polynucleotide without subsequent side reactions.

[0124] In summary, step (3) adopts an iterative cyclic synthesis strategy, integrating the terminal uracil nucleotide transferase and its mutant catalyzed 3'-O-reversible blocking modified nucleotide single-base addition reaction described in step (1), the removal of unreacted substrate and byproducts in step (2), and the deprotection activation reaction of the 3'-O-reversible blocking group into a single cyclic unit. According to the preset target polynucleotide base sequence, the corresponding base of the 3'-O-reversible blocking modified nucleotide substrate is replaced sequentially, and the above cyclic unit is repeated. In each cycle, a single base with reversible blocking modification is covalently linked to the 3' end of the activated oligonucleotide chain. After physical separation and purification to remove impurities from the system, the 3'-end reversible blocking group is removed to re-expose the free hydroxyl group, so that the product has the reactivity for the next round of extension. Through iterative extension, the nucleic acid chain gradually grows according to the preset sequence. When the chain length and base composition of the synthesized product meet the requirements of the target polynucleotide, the cycle is terminated, and finally the target polynucleotide with accurate sequence and complete structure is obtained.

[0125] In one embodiment, step (2), the removal of unreacted substrates and byproducts of the intermediate product, and the removal of the 3'-O-reversible blocking group to expose the 3' hydroxyl group, includes: step (2.1) removing the terminal uracil nucleotide transferase of the intermediate product and its mutants and unreacted nucleotides; and step (2.2) using a cleavage agent to remove the 3'-O-reversible blocking group of the intermediate product to expose the 3' hydroxyl group.

[0126] Step (2.1) First, the reaction system of step (1) is purified by physical separation methods such as ultrafiltration and chromatographic separation. Relying on the differences in molecular weight, charge and hydrophobicity, the terminal uracil nucleotide transferase protein, unreacted 3'-O-reversible blocking modified nucleotides and by-products and impurities such as pyrophosphate are removed from the system to obtain a high-purity oligonucleotide intermediate. Then, in step (2.2), a corresponding type of chemical cleavage agent is added to the purified product. Based on the chemical characteristics of the 3'-O-reversible blocking group, the connection between the blocking group and the 3'-oxygen atom is broken through the selective covalent bond breaking reaction of the cleavage agent. The reversible blocking modification is removed without destroying the nucleic acid backbone structure, thereby exposing and restoring the free hydroxyl group at the 3' end of the oligonucleotide, so that it has the activity to carry out the terminal uracil nucleotide transferase-catalyzed single base extension reaction again.

[0127] In one embodiment, the cleavage agent is selected from one or more of tris(2-carboxyethyl)phosphine, palladium complex, sodium nitrite, or T4 polynucleotide kinase.

[0128] In one implementation, the initiating strand is DNA, RNA, or a mixture of DNA and RNA.

[0129] In summary, the terminal uracil nucleotide transferase and its mutants provided by this invention catalyze the addition of a single base of a modified nucleotide to the 3'-terminus of the initiating chain in a template-independent manner, achieving enzymatically controlled polynucleotide synthesis. Specifically, the terminal uracil nucleotide transferase and its mutants catalyze the single-step controlled addition of a specified type of nucleotide to the 3'-terminus of the initiating chain, followed by multiple extension-deprotection cycles to prepare polynucleotides of a predetermined sequence. The terminal uracil nucleotide transferase and its mutants exhibit excellent catalytic activity, suggesting that they have significant application prospects in the synthesis of polynucleotides.

[0130] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0131] It should be understood that the described embodiments are merely some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of the embodiments of this application.

[0132] Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the raw material supplier; unless otherwise specified, the materials, reagents and instruments used in the following examples can be purchased commercially.

[0133] The protein buffer used in the following examples is: 50mM Tris-HCl, 150mM NaCl, pH 8.0.

[0134] Example 1: Obtaining terminal uracil nucleotide transferase and its mutant protein

[0135] Through literature review and database search, the following terminal uracil nucleotide transferase from the species was obtained, with the amino acid sequence as follows: SEQ ID NO:1 (Drosophila melanogaster).

[0136] 1.1 Construction of expression vector for terminal uracil nucleotide transferase.

[0137] In this embodiment, the gene sequences encoding terminal uracil nucleotide transferase protein from the above-mentioned species were constructed into the expression vector pET-28a(Novagen, Kan+), with BamHI and EcoRI restriction sites at both ends, respectively, to obtain the recombinant plasmid encoding SEQ ID NO:1.

[0138] 1.2 Construction of mutant expression vector.

[0139] Using the recombinant plasmid encoding SEQ ID NO:1 as a template, mutation primers were designed, with the bases to be mutated placed in the middle of the primers.

[0140] The PCR amplification system includes: 25uL Q5 Quick-Load™ Ultra Fidelity 2X premix, 2uL primer F, 2uL primer R, 0.5uL template plasmid, and 20.5uL sterile water.

[0141] Reaction procedure: denaturation at 95℃ for 20 seconds; annealing at 53℃ for 20 seconds; extension at 72℃ for 4 minutes, repeated 30 times.

[0142] The sequences of the mutant and upstream and downstream mutant primers in this embodiment are shown in Table 1 below.

[0143] Table 1: Mutants and upstream and downstream mutant primers

[0144]

[0145] Next, after the reaction, the template plasmid was digested with DpnI, and the PCR product length was checked by electrophoresis. The PCR product with the expected length was purified, chemically transformed into DH5α competent cells, and positive clones were screened on kanamycin-resistant plates (Kan+, 50 mg / L). For positive clones, single clones were picked and placed in 5 mL of kanamycin-resistant LB liquid medium (10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, Kan+, 50 mg / L), and cultured at 37℃ and 220 rpm. Plasmids were extracted, and sequencing was performed to obtain the plasmid vectors corresponding to mutants m1-m26 with SEQ ID NO:1 as the parent.

[0146] 1.3 Gene expression.

[0147] The recombinant plasmids were transformed into *E. coli* BL21(DE3) to obtain recombinant bacteria. Positive clones were screened using kanamycin-resistant plates (Kan+, 50 mg / L), and single clones were picked and transferred to 5 mL of kanamycin-resistant LB broth (10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, Kan+, 50 mg / L), and cultured at 37℃ and 220 rpm until the OD600 reached 0.6-0.8. The bacterial culture from 5 mL of LB broth was then transferred to 1000 mL of kanamycin-resistant LB broth (10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, Kan+, 50 mg / L), and cultured at 37℃ and 220 rpm until the OD600 reached 0.6-0.8. The temperature was then lowered to 22℃, and IPTG was added to a final concentration of 0.5 mM for 22 h to induce expression.

[0148] Collect the above-mentioned bacterial culture into a collection bottle and centrifuge at 3000g for 15 min. Discard the supernatant, suspend the obtained bacterial pellet with 35 mL of protein buffer, pour it into a 50 mL centrifuge tube, and store at -80℃.

[0149] 1.4 Protein purification.

[0150] Sterilization: Thaw the bacterial cells suspended in the buffer solution, add lysozyme to a concentration of 0.05 mg / ml, and lyse twice using a high-pressure cryo-lysis device at 600 bar and 4°C. Centrifuge at 20,000 g for 30 min at 4°C and obtain the supernatant.

[0151] First, wash with ddH2O for 2 column volumes, then equilibrate the Ni affinity chromatography column (Tiandi Renhe, Ni SmartBeads 6FF, 5 mL) with protein buffer for 1 column volume. Slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5 mL / min, repeating once. Wash with protein buffer for 1 column volume, then elute weakly bound proteins with 5 column volumes of protein buffer containing 20 mM imidazole. Elute the target protein with 3 column volumes of protein buffer containing 100 mM, 200 mM, and 300 mM imidazole, respectively, collecting the 200 mM imidazole eluent for subsequent experiments. Concentrate the collected target protein using a 50 mL Amicon ultrafiltration tube (30 kDa, Millipore) at 4°C and 3400 rpm to 1 mL. Add 10 mL of protein buffer and concentrate to 1 mL. Repeat this process twice to obtain purified terminal uracil nucleotide transferase.

[0152] The concentration of the concentrated protein was determined to be 5 mg / mL using a NanoDrop 2000 micro-spectrophotometer. This yielded purified and concentrated terminal uracil nucleotide transferase and mutant protein, which were used for subsequent enzyme catalytic activity assays.

[0153] Example 2: Detection of the catalytic activity of terminal uracil nucleotide transferase and its mutants on native nucleotides

[0154] In this embodiment, DNA-RNA chimeric oligonucleotides were used as the initiation chain. The substrates included adenine ribonucleotide (ATP), guanine ribonucleotide (GTP), cytosine ribonucleotide (CTP), and uracil ribonucleotide (UTP). The initiation chain start sequence was: ATGACGTGCTACG(rA)(rA)(rC). The catalytic activities of the above-mentioned terminal uracil nucleotide transferase and mutants (wt, m1~m26) were measured.

[0155] In vitro purified enzyme reaction: The total volume of the reaction system was 50 μL, and the final concentrations of each component in the system were as follows: 50 mM Tris-HCl (pH 8.5), 1 U / μL RNase inhibitor, 5 mM MnCl2, 0.05 μM initiation sequence (initiator strand, 16 bp), 200 μM ribonucleotides (ATP, GTP, CTP, UTP, 50 μM each), 0.2x Helixyte Green ssDNA dye (Xi'an Baiying Biotechnology), enzyme: 0.1 mg / mL. Example 1: Preparation of terminal uracil nucleotide transferase and mutants, with nuclease-free water added to a final volume of 50 μL.

[0156] Real-time detection reaction conditions: The above system was placed in a black ELISA plate and tested using a TECAN SPARK ELISA reader at 37°C with 488nm excitation. Emission at 535nm was detected every two minutes. Helixyte Green ssDNA specifically binds to the bases and phosphate backbone of single-stranded DNA or RNA, resulting in a significant increase in fluorescence intensity. The fluorescence intensity is positively correlated with the amount of single-stranded DNA or RNA. Using the TECAN SPARK ELISA reader, mutants with enhanced RNA catalytic efficiency can be rapidly screened.

[0157] Figures 2 to 4 The figures show the changes in catalytic efficiency of each mutant relative to the wild type and relative to the previous generation parent, respectively. Compared to the wild type, mutant m3 (A306Y) significantly improves the catalytic activity for natural NTPs. Using m3 as the parent and adding additional mutations, a second round of optimization was performed to obtain mutant m10 (A306Y / E385R) with improved activity. In the third round of optimization, using m10 as the parent and adding additional mutations again, optimized mutants m16 (A306Y / E385R / K303F) and m20 (A306Y / E385R / A527R) were obtained.

[0158] Example 3: Detection of the catalytic ability of terminal uracil nucleotidyltransferase and its mutant to catalyze the chain elongation of nucleotides with reversible blocking groups at the 3' end.

[0159] In this embodiment, a DNA-RNA chimeric oligonucleotide was used as the initiation chain, with the starting sequence being: ATGACGTGCTACG(rA)(rA)(rC). The substrate was uracil ribonucleotide (UTP), and the 3'-OH end of the UTP substrate was replaced with a reversible blocking group -CH2N3. The catalytic activities of the terminal uracil nucleotide transferase of SEQ ID NO:1 and its mutants m3, m10, and m16 were measured.

[0160] In vitro purified enzyme reaction: The total volume of the reaction system was 20 μL, and the final concentrations of each component in the system were as follows: 50 mM Tris-HCl (pH 8.5), 1 U / μL RNase inhibitor, 5 mM MnCl2, 1 μM initiator sequence (initiator chain, 16 bp), 40 μM 3'-O-ribonucleotide with reversible repressor group, enzyme: 0.025 mg / mL SEQ ID NO:1 terminal uracil nucleotide transferase and its mutants m3, m10 and m16 prepared in Example 1 (parallel experimental groups were set up and the catalytic activity of each recombinant enzyme was measured separately), and the volume was made up to 20 μL with nuclease-free water.

[0161] Reaction conditions: React at 37℃ for 5 min, terminate the reaction by mixing an equal volume of 2x Urea-PAGE loading buffer, denature at 95℃ for 5 min, perform 20% Urea-PAGE at 300V for 40 min, and stain with Gel-Red. The electrophoretically separated RNA synthesis products were detected using the Bio-Rad GelDoc Go automated nucleic acid analysis system. Under the same reaction and electrophoresis conditions, the intensity of the RNA band of the synthesis product directly reflects the amount of product generated, thus enabling a comparison of the catalytic activity of terminal uracil nucleotide transferase for different ribonucleotides (by comparing the intensity of the synthesized RNA). Specifically, the intensity of the target product was compared with the starting sequence, increasing the product intensity by one nucleotide unit to assess the catalytic activity of terminal uracil nucleotide transferase for ribonucleotides with a reversible blocking group at the 3' end.

[0162] Figure 5 This is a schematic diagram showing the catalytic ability of the terminal uracil nucleotide transferase mutant in this embodiment to catalyze the catalytic chain elongation of nucleotides with reversible blocking groups at the 3' end.

[0163] in, Figure 5 The image on the left is a gel imaging diagram of nucleotides with reversible blocking groups at the 3' end, output by a fully automated nucleic acid analysis system. Figure 5 The right side shows a schematic diagram of the catalytic ratio of nucleotides with reversible blocking groups at the 3' end catalyzed by different terminal uracil nucleotransferase mutants. WT is the terminal uracil nucleotransferase shown in SEQ ID NO:1.

[0164] according to Figure 5 The test results showed that the terminal uracil nucleotide transferase shown in SEQ ID NO:1 and its mutants have high catalytic efficiency in catalyzing the addition of thymine ribonucleotides with reversible blocking groups to the starting sequence; among them, mutants m3, m10 and m16 all showed significantly higher catalytic efficiency than the wild type.

[0165] Example 4: Detection of the ability of terminal uracil nucleotide transferase mutant to catalyze multi-round nucleotide synthesis

[0166] In this embodiment, a DNA-RNA chimeric oligonucleotide was used as the initiation chain. The starting sequence of the initiation chain was: ATGACGTGCTACG(rA)(rA)(rC) [5' biotinylated and immobilized on streptavidin magnetic beads]. The substrates included adenine ribonucleotide (ATP), guanine ribonucleotide (GTP), cytosine ribonucleotide (CTP), or uracil ribonucleotide (UTP). The 3'-OH end of the above nucleotides was changed to a reversible blocking group -CH2N3. The ability of the terminal uracil nucleotide transferase mutant m10 of SEQ ID NO:1 to catalyze multiple rounds of nucleotide synthesis was measured.

[0167] (1) Synthesis: The total volume of the reaction system was 20 μL, and the final concentrations of each component in the system were as follows: 50 mM Tris-HCl (pH 8.5), 1 U / μL RNase inhibitor, 5 mM MnCl2, 0.5 μM initiating sequence (ATGACGTGCTACG(rA)(rA)(rC), 5' biotinylated, immobilized on streptavidin magnetic beads), 40 μM 3'-O-ribonucleotide with reversible repressor group, enzyme: 0.1 mg / mL SEQ ID NO:1 terminal uracil nucleotide transferase mutant m10 prepared in Example 1. Reaction conditions: 37℃ for 5 min. Washing: The initiating chain on the magnetic beads was separated from the solution system using a magnetic rack, and the washing solution (50 mM Tris-HCl, pH 8.5, 500 mM NaCl, 0.1% Tween 20) was used for washing three times.

[0168] (2) Deprotection: Deprotection solution (50mM Tris-HCl, pH 8.5, 500mM NaCl, 0.1% Tween 20, 20mM TECP), reaction at 45℃ for 10 min. Cleaning: Cleaning: Separate the initiation chain and solution system on the magnetic beads using a magnetic rack, and clean three times with cleaning solution (50mM Tris-HCl, pH 8.5, 500mM NaCl, 0.1% Tween 20).

[0169] Repeat steps (1) and (2), cycling each of the five samples 1 to 5 times.

[0170] After each sample completed the corresponding number of cycles, the washing solution was removed, and 40 μL of 1x Urea-PAGE loading buffer was added to the magnetic beads to terminate the reaction. The mixture was then denatured at 95°C for 5 min, followed by 15% Urea-PAGE at 300V for 40 min, and then stained with Gel-Red. The RNA synthesis products separated by electrophoresis were detected using the Bio-Rad GelDoc Go automated nucleic acid analysis system. Under the same reaction and electrophoresis conditions, with each addition cycle, the nucleic acid molecule lengthened by one nucleotide unit, slowing down the electrophoretic velocity. This reaction is reflected on the electrophoresis pattern as the band shifting upwards.

[0171] Figure 6 This is a gel imaging diagram showing the completion of 1 to 5 nucleotide synthesis cycles in this embodiment. Cycle 0 is the starting sequence of the notebook. Cycles 1 to 5 are ribonucleotides with 3'-O-reversible repressor groups added sequentially according to the ACUGA sequence. Due to the presence of the repressor group, only one nucleotide unit can be added at a time. Other nucleotide units can only be added after TCEP deprotection.

[0172] according to Figure 6 According to the test results, the terminal uracil nucleotide transferase mutant shown in SEQ ID NO:1 can control the addition of ribonucleotides with reversible repressor groups in the synthesis stage, and cycle the deprotection step to synthesize the target sequence.

[0173] In summary, the terminal uracil nucleotide transferase and its mutants provided by this invention possess excellent catalytic activity, capable of catalyzing the generation of polynucleotides with target sequences from nucleotides modified with a reversibly blocking group of 3'-O-, suggesting that this terminal uracil nucleotide transferase has a more significant application prospect in the synthesis of polynucleotides.

[0174] The above are merely embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A terminal uracil nucleotide transferase mutant, characterized in that, The terminal deoxynucleotidyl transferase mutant is obtained by mutating amino acids based on the sequence shown in SEQ ID NO.

1. The mutated amino acid sites are selected from at least one of the following positions: position 212, position 215, position 236, position 248, position 288, position 303, position 306, position 312, position 364, position 368, position 385, position 404, position 410, position 414, position 421, position 423, position 429, position 432, position 446, position 469, position 479, position 491, position 507, position 527, and position 540.

2. The terminal uracil nucleotide transferase mutant according to claim 1, characterized in that, The amino acid mutation is A212V, M215F, V236E, C248L, T288R, K303F, A306Y, C312I, Q364L, Q368R, E385R / K, A404G, A410R, I414E, A421P, L423Y, V429C, A432S, A446P, H469P, A479P, H491P, Q507E, A527R / K, or C540L.

3. The terminal uracil nucleotide transferase mutant according to claim 1, characterized in that, The amino acid mutation is A306Y / M215F, A306Y / E385R, A306Y / E385K, A306Y / A410R, A306Y / H491P or A306Y / C540L.

4. The terminal uracil nucleotide transferase mutant according to claim 1, characterized in that, The amino acid mutation is A306Y / E385R / C248L, A306Y / E385R / K303F, A306Y / E385R / Q364L, A306Y / E385R / A432S, A306Y / E385R / H469P, A306Y / E385R / A527R, A306Y / E385R / A421P, A306Y / E385R / T288R, A306Y / E385R / C312I, A306Y / E385R / Q368R, A306Y / E385R / A404G, or A306Y / E385R / L423Y.

5. A nucleic acid molecule encoding the terminal uracil nucleotide transferase mutant according to any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the expression vector of claim 6.

8. A reagent kit, characterized in that, It comprises the terminal uracil nucleotide transferase mutant according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, or the host cell according to claim 7.

9. The use of the terminal uracil nucleotide transferase mutant according to any one of claims 1 to 4, wherein the use is to catalyze the synthesis of single-stranded DNA / RNA from 3'-O-modified nucleotides in the absence of a template.

10. A method for the controlled synthesis of single-stranded DNA / RNA without a template, characterized in that, The method includes the step of contacting a nucleic acid primer, at least one 3'-O-modified nucleotide, and the terminal deoxynucleotidyl transferase mutant as described in any one of claims 1 to 4.

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