A mutant of terminal deoxynucleotidyl transferase and preparation method and application thereof

Abstract

Disclosed are a mutant of terminal deoxyribonucleotidyl transferase, a preparation method and application thereof. The wild-type terminal deoxyribonucleotidyl transferase is molecularly modified to improve the polymerization activity of the modified nucleotide monomers, improve the catalytic efficiency of the modified nucleotide monomers, realize high-efficiency single-base addition efficiency, and enable a plurality of 3'-O-blocked modified dNTPs substrates to be added to the 3'-OH end of an oligonucleotide single strand without a template, thereby providing a novel and effective tool enzyme for enzymatic de novo synthesis of nucleic acids.

Description

Terminal deoxyribonucleoside transferase mutant and its preparation method and application Technical Field

[0001] The present application belongs to the field of genetic engineering technology and relates to a terminal deoxyribonucleoside transferase mutant and a preparation method and application thereof. Background Art

[0002] Driven by high-throughput sequencing and gene editing technologies, synthetic biology has rapidly emerged, and DNA synthesis has become an emerging industry. The demand for DNA synthesis is increasing across many research and commercial sectors. If DNA synthesis can be provided on a large scale and at low cost, it will drive rapid development in technologies such as engineering biology, therapeutics, data storage, and nanotechnology. Following the discovery of the DNA structure, advances in solid-phase synthesis have enabled substantial milestones in de novo DNA synthesis. Currently, there are two main methods for de novo oligonucleotide synthesis: chemical synthesis (phosphoramidite synthesis) and biosynthesis (enzymatic synthesis). Phosphoramidite synthesis is the mainstream method for synthesizing short DNA chains, reliably providing short DNA chains of less than 200 nucleotides. Phosphoramidite synthesis involves a single base addition cycle using four steps: deprotection, coupling, capping, and oxidation. This cumbersome process is associated with long cycle times (6-8 minutes), high chemical reagent consumption, and high costs. Furthermore, the reaction process uses a large amount of toxic and flammable organic reagents, resulting in significant pollution. Currently, the synthesis of nucleotide sequences >200bp by phosphoramidite synthesis is still a heavy burden. To this end, researchers have tried to develop alternative technologies such as Gibbs assembly to produce longer DNA chains.

[0003] Enzymatic de novo DNA synthesis is a promising technology that has garnered considerable attention since the 1950s. Terminal deoxynucleotidyl transferase (TdT) is a widely used and advantageous DNA polymerase in de novo DNA synthesis. TdT can indiscriminately extend the four natural nucleotides (A, T, C, and G) to the 3' end of the starting strand without a template. Studies have shown that TdT can extend several nucleotides within a second and can synthesize DNA up to several kilobases. Its superior synthesis length and speed far exceed the capabilities of commercially available phosphoramidite synthesis techniques. However, TdT promiscuously extends oligonucleotides in the 5' to 3' direction of the starting strand. Because any of the four nucleotides participate in each step, the simultaneous formation of oligonucleotide sequences of varying lengths and coding sequences is crucial. An effective solution to this problem is to control nucleotide incorporation through a "reversible termination" mechanism: a reversibly removable protecting group (PG) is added to the 3-position of the pentose sugar of the nucleotide, ensuring that each reaction step terminates after the extension of a single nucleotide. In subsequent reactions, the PG can be cleaved and converted back to a hydroxyl group, allowing the next desired nucleotide to be extended (Figure 1). This TdT-catalyzed de novo DNA synthesis strategy requires only two steps: coupling and deprotection. This significantly improves the potential efficiency of a single cycle compared to phosphoramidite synthesis. Furthermore, the entire reaction is carried out in aqueous phase, resulting in mild reaction conditions and a greener environment. Achieving high single-base addition efficiency is crucial for TdT-based enzymatic de novo DNA synthesis. Because the scheme utilizes non-natural PG-modified nucleotide monomers, natural TdT has low activity against them, resulting in low synthesis efficiency. Therefore, molecular engineering is necessary to enhance the polymerization activity of TdT towards the modified nucleotide monomers. For example, CN114921436A discloses a terminal deoxynucleotidyl transferase mutant with improved thermal stability. The amino acid sequence of the wild-type terminal deoxynucleotidyl transferase shown in SEQ ID NO. 1 is mutated to obtain a mutant with six mutations: N135P, S138H, Q229D, K232D, L234R, and V366M.

[0004] In summary, the discovery and design of terminal deoxynucleotidyl transferases with high polymerization activity is of great significance to the field of nucleic acid synthesis.

[0005] Summary of the Invention

[0006] The present application provides a terminal deoxyribonucleoside transferase mutant, a preparation method, and an application thereof, in order to solve the problems of low efficiency, reaction energy consumption, and high pollution in synthesizing DNA by the traditional phosphoramidite synthesis method, as well as the low catalytic efficiency of wild-type terminal deoxyribonucleoside transferase.

[0007] In a first aspect, the present application provides a terminal deoxyribonucleoside transferase mutant, wherein the amino acid sequence of the terminal deoxyribonucleoside transferase mutant comprises any one of the following sequences:

[0008] (1) Based on the sequence of SEQ ID NO: 2, the following mutations occur: any one or at least two of the following: E459R, E459K, E459Q, E459V, R460Q, R460K, R460E, R460H, R460N, R460D, R460S, R460L, R460M, R460W, R460F, R460Y, R460C, or R460G; or

[0009] (2) a sequence obtained by substituting, deleting or adding one or at least two amino acid residues from the sequence described in (1), and having the same or similar functions as the sequence described in (1); or

[0010] (3) A sequence having at least 90% sequence homology with the sequence described in (1) or (2) and having the same or similar functions as the sequence described in (1).

[0011] In this application, the terminal deoxyribonucleoside transferase GeTdT from the gecko is molecularly modified. The polarity and spatial size of the active pocket are adjusted through molecular modification, so that it can well accommodate non-natural nucleotides, thereby improving its polymerization activity for modified nucleotide monomers, greatly improving the catalytic efficiency for modified nucleic acid monomers, achieving efficient single-base addition efficiency, and being able to add a variety of 3'-O-blocked modified dNTPs substrates to the 3'-OH end of oligonucleotide single chains in the absence of a template, providing a new and effective tool enzyme for enzymatic de novo synthesis of nucleic acids.

[0012] In the present application, the introduction of specific mutations into the wild-type terminal deoxyribonucleoside transferase can improve its polymerization activity for modified nucleotide monomers. It can be understood that based on the terminal deoxyribonucleoside transferase mutant, those skilled in the art can use the general technical means in the art to replace, delete or add one or at least two amino acid residues to obtain other sequences with the same or similar functions.

[0013] As used herein, the term "homology" can be assessed visually or using computer software, such as the software program described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The homology between two or more sequences can be expressed as a percentage (%), which can be used to assess the homology between related sequences. A polynucleotide sequence or amino acid sequence that has a certain percentage (e.g., 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when the sequences are aligned, that percentage of bases or amino acids are the same in the two sequences being compared.

[0014] In the present application, single-site mutations of E459 and combined mutations of E459 and R460 are involved. Preferably, (1) the mutations may include E459R and R460Q combined mutations (which can be written as E459R / R460Q), E459R and R460K combined mutations, E459R and R460E combined mutations, E459R and R460H combined mutations, E459R and R460N combined mutations, E459R and R460K combined mutations, E459R and R460E combined mutations, E459R and R460H combined mutations, E459R and R460N combined mutations, E459R and R460N combined mutations, E459R and R460K combined mutations, E459R and R460E combined mutations, E459R and R460H combined mutations, E459R and R460N combined mutations, E459R and R460N combined mutations, E459R and R460N combined mutations, E459R and R460N combined mutations, E459R and R460N combined mutations, E459R and R460K combined mutations, E459R and R460K combined mutations, E459R and R460E combined mutations, E459R and R460H ... Any one of the following: E459R and R460D combined mutations, E459R and R460S combined mutations, E459R and R460L combined mutations, E459R and R460M combined mutations, E459R and R460W combined mutations, E459R and R460F combined mutations, E459R and R460Y combined mutations, E459R and R460C combined mutations or E459R and R460G combined mutations.

[0015] In a second aspect, the present application provides a nucleic acid molecule encoding the glycosyltransferase mutant described in the first aspect.

[0016] In a third aspect, the present application provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0017] In a fourth aspect, the present application provides a recombinant cell, wherein the recombinant cell contains the recombinant vector described in the third aspect.

[0018] In a fifth aspect, the present application provides a method for preparing the terminal deoxyribonucleoside transferase mutant described in the first aspect, the preparation method comprising:

[0019] A nucleic acid molecule encoding the terminal deoxyribonucleoside transferase mutant described in the first aspect is inserted into an expression vector to obtain a recombinant vector, and the recombinant vector is introduced into a host cell, or the nucleic acid molecule is directly integrated into the genome of the host cell to obtain a genetically engineered bacterium, which is cultured and isolated and purified to obtain the terminal deoxyribonucleoside transferase mutant.

[0020] In a sixth aspect, the present application provides the use of the terminal deoxyribonucleoside transferase mutant described in the first aspect in the preparation of products for nucleic acid synthesis.

[0021] In a seventh aspect, the present application provides a product for nucleic acid synthesis, which comprises the terminal deoxyribonucleoside transferase mutant described in the first aspect.

[0022] In an eighth aspect, the present application provides the use of the terminal deoxyribonucleoside transferase mutant described in the first aspect in nucleic acid synthesis.

[0023] In a ninth aspect, the present application provides a method for synthesizing nucleic acid, the method comprising:

[0024] The terminal deoxyribonucleoside transferase mutant described in the first aspect is used to catalyze the ligation of dNTPs to the 3'-OH end of a single-stranded oligonucleotide.

[0025] Preferably, the dNTPs include 3'-O-blocking modified dNTPs, and the blocking modification includes, for example, a protecting group "Protecting Group, PG".

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] This application molecularly modifies the wild-type terminal deoxyribonucleoside transferase to improve its polymerization activity for modified nucleotide monomers, greatly improves the catalytic efficiency for modified nucleic acid monomers, achieves efficient single-base addition efficiency, and is able to add a variety of 3'-O-blocked modified dNTPs substrates to the 3'-OH end of oligonucleotide single chains in the absence of a template, providing a new and effective tool enzyme for enzymatic de novo synthesis of nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a flow chart of the terminal deoxyribonucleoside transferase-dominated enzymatic synthesis scheme.

[0029] FIG2 is an SDS-PAGE image of the purified protein of the GeTdT mutant.

[0030] Figure 3. Urea-PAGE image of terminal deoxyribonucleoside transferase products.

[0031] FIG4 is a capillary electrophoresis diagram of the transformation products of GeTdT wild type and its E459R and E459R / R460Q mutants.

[0032] FIG5 is a Urea-PAGE image of the terminal deoxyribonucleoside transferase product. DETAILED DESCRIPTION

[0033] To further illustrate the technical means and effects of this application, the following further describes this application in conjunction with examples and drawings. It should be understood that the specific implementation methods described herein are only used to explain this application, rather than to limit this application.

[0034] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] The overall scheme adopted in the specific embodiments of the present application is as follows: design primers for site-directed mutagenesis → obtain crude protein solution of TdT mutant through Escherichia coli expression system → obtain purified TdT mutant protein by nickel ion affinity chromatography → test the activity of protein → analyze the product by urea polyacrylamide gel (Urea-PAGE) and capillary electrophoresis (CE).

[0036] Example 1

[0037] Construction of terminal deoxyribonucleoside transferase GeTdT mutant.

[0038] The target of transformation is the terminal deoxyribonucleoside transferase GeTdT from Gecko, whose nucleotide sequence is shown in SEQ ID NO.1 and amino acid sequence is shown in SEQ ID As shown in NO.2, primers containing E459R, E459K, E459Q, E459V, E459R / (" / " indicates sum)R460Q, E459R / R460K, E459R / R460E, E459R / R460H, E459R / R460N, E459R / R460D, E459R / R460S, E459R / R460L, E459R / R460M, E459R / R460W, E459R / R460F, E459R / R460Y, E459R / R460C, and E459R / R460G mutations were designed and synthesized according to the sequence, and site-directed mutagenesis of GeTdT was performed. The recombinant expression plasmid pGS-21a / GeTdT carrying the gene encoding the partial peptide chain of GeTdT (amino acids G144 to A512 in SEQ ID NO. 2, i.e., the wild-type GeTdT described later) (G144—A512) As a template, the corresponding mutation primer pairs shown in Table 1 were used to introduce mutations at the characteristic sites by rapid PCR technology, and the coding gene of the GeTdT mutant was confirmed to be correct by Sanger sequencing. (G144—A512) The complete peptide chain sequence expressed is shown in SEQ ID NO.3.

[0039] Table 1 Corresponding primers for constructing GeTdT mutants

[0040] The PCR reaction system was as follows: 10 μL of 5× PS buffer, 4 μL of dNTPs Mix (2.5 mmol / L), 1 μL of forward primer (10 μmol / L), 1 μL of reverse primer (10 μmol / L), 1 μL of template DNA, 0.5 μL of Primer Star HS (Takara, 5 U / μL), and distilled water was added to 50 μL.

[0041] The PCR amplification program was as follows: initial denaturation at 94°C for 5 min; 21 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C for 7 min 50 s; and a final extension at 72°C for 7 min, followed by incubation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.

[0042] Dpn I was added to the PCR product verified to be correct by gel electrophoresis, and the template was degraded by incubating in a 37°C water bath for 2 hours. The template was then transformed into Escherichia coli JM109 competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.). The transformation product was spread on LB solid medium containing 100 mg / L ampicillin and cultured at 37°C for 11 hours. The clones were picked and inoculated into LB liquid medium and cultured at 37°C for 9 hours. The bacterial solution was subjected to Sanger sequencing to verify whether the mutation site was correctly introduced. The plasmids of the clones with correct sequencing were extracted and transformed into the expression host Escherichia coli BL21 (DE3) competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.), obtaining 19 recombinant E. coli strains capable of expressing 18 mutants and GeTdT wild-type proteins.

[0043] It should be noted that due to the principle of codon degeneracy, the nucleotide sequence that translates the amino acid sequence is not a unique constant sequence. Any nucleotide sequence that can encode the same amino acid sequence is a nucleic acid sequence within the scope of this patent.

[0044] Example 2

[0045] Expression and purification of terminal deoxyribonucleotidyl transferase GeTdT and its mutants.

[0046] The 19 recombinant E. coli obtained in Example 1 were inoculated into LB medium, cultured at 37°C for 8 hours, and then transferred to 300 mL TB fermentation medium at an inoculum volume of 5% of the fermentation medium volume. First, culture at 37°C and 200 rpm for 3 hours. 600 When the pH value is 0.5-0.7, IPTG is added to a final concentration of 1 μM and the culture medium is incubated at 25°C and 200 rpm for 24 hours to induce fermentation. After the fermentation is completed, the fermentation broth is homogenized by high pressure (crushing conditions are 4°C and 80 MPa) and then centrifuged (12,000 rpm, 30 minutes, 4°C). The supernatant is the crude enzyme solution of the terminal deoxyribonucleoside transferase mutant produced by the recombinant bacteria.

[0047] The enzyme solution sample prepared in the previous step was subjected to protein purification as follows: Terminal deoxyribonucleoside transferase protein was purified using nickel affinity chromatography. The crude enzyme solution of the terminal deoxyribonucleoside transferase mutant was loaded onto a nickel affinity chromatography column (nickel column). The recombinant protein was purified using a gradient elution method: Purification Buffer A and Purification Buffer B were prepared into eluent 1 (containing 0% buffer B), eluent 2 (containing 5% buffer B), eluent 3 (containing 10% buffer B), eluent 4 (containing 20% ​​buffer B), eluent 5 (containing 50% buffer B), and eluent 6 (containing 100% buffer B). The protein bound to the nickel column was then eluted using these eluents in ascending order of imidazole concentration. All eluents were pre-cooled to 4°C during the purification process to ensure that the recombinant protein retained its biological activity. Eluent 4, containing the purified target protein, was concentrated by ultrafiltration using an ultrafiltration tube with a 30 kDa protein cutoff, and the purified eluent was exchanged with 2× enzyme storage buffer. Finally, an equal volume of glycerol was added to the concentrated protein, mixed, and stored at -20°C. The purity of the purified recombinant protein was analyzed by SDS-PAGE. In Figure 2, M represents protein standard; lanes 1-19 represent: 1, WT (wild type); 2, E459R; 3, E459K; 4, E459Q; 5, E459V; 6, E459R / R460Q; 7, E459R / R460K; 8, E459R / R460E; 9, E459R / R460H; 10, E459R / R460N; 11, E459R / R460D; 12, E459R / R460S; 13, E459R / R460L; 14, E459R / R460M; 15, E459R / R460W; 16, E459R / R460F; 17, E459R / R460Y; 18, E459R / R460C; 19, E459R / R460G, indicating that relatively pure protein was obtained.

[0048] Purification buffer A (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole.

[0049] Purification buffer B (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole.

[0050] 2× enzyme storage buffer (pH 7.2): 40 mM Tris-HCl, 400 mM NaCl, 5% glycerol.

[0051] Example 3

[0052] Semi-quantitative analysis of the activity of terminal deoxyribonucleoside transferase GeTdT and its mutants.

[0053] Use single-stranded Oligo(dT) with a polymerization degree of 18 18 (SEQ ID NO.40) was used as an oligonucleotide substrate, and four nucleotides with azidomethyl modifications at the 3' end (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, and 3'-O-Azidomethyl-dGTP) were used as nucleic acid monomer substrates (structures shown in Figure 3). The purified wild-type GeTdT and its mutants were tested for terminal transfer activity. Oligonucleotide substrate Oligo(dT) 18 Under the action of terminal deoxyribonucleoside transferase, one base is extended to obtain the product Oligo(dT) 18- dNTP-3'-O-Azidomethyl. The reaction system for the activity test of wild-type GeTdT enzyme and its mutants is shown in Table 2. After mixing, the reaction system was incubated at 37°C for 10 minutes. The reaction was terminated by heating at 95°C for 10 minutes. The reaction products of wild-type GeTdT and its mutants were semi-quantitatively analyzed by urea-polyacrylamide gel electrophoresis (20% denaturing gel), as shown in Figure 3. The 5× reaction buffer used was formulated as follows: 0.5M Na-Cacodylate, 5mM CoCl2, pH 7.2.

[0054] Table 2 Activity test reaction system of wild-type GeTdT enzyme and its mutants

[0055] In Figure 3, products were obtained by converting four nucleotide monomer substrates with azidomethyl modifications at their 3' ends (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, and 3'-O-Azidomethyl-dGTP) using GeTdT and its mutants; NC represents Oligo(dT). 18; PC stands for Oligo(dT) 19 Lanes 1-19 represent: 1, WT (wild type); 2, E459R; 3, E459K; 4, E459Q; 5, E459V; 6, E459R / R460Q; 7, E459R / R460K; 8, E459R / R460E; 9, E459R / R460H; 10, E459R / R460N; 11, E459R / R460D; 12, E459R / R460S; 13, E459R / R460L; 14, E459R / R460M; 15, E459R / R460W; 16, E459R / R460F; 17, E459R / R460Y; 18, E459R / R460C; 19, E459R / R460G. The activity of the GeTdT mutants shown in the figure towards the four nucleotides with azidomethyl modifications at the 3' end is better than that of the wild type. Among them, the conversion rates of the mutants E459R, E459R / R460Q, E459R / R460K, E459R / R460E, E459R / R460H, E459R / R460N and E459R / R460D are greatly improved compared with the wild type.

[0056] Example 4

[0057] Determination of the conversion rate of terminal deoxyribonucleoside transferase GeTdT and its mutants.

[0058] Use 5'-ROX-Oligo(dT) with a single-stranded 5' end modified with a ROX fluorescent group at a polymerization degree of 98 98 (SEQ ID NO.41) was used as an oligonucleotide substrate, and 3'-O-Azidomethyl-dTTP with an azidomethyl modification at the 3' end was used as a nucleic acid monomer substrate. The terminal transfer activity of the purified GeTdT wild type and its mutants was determined, and the conversion rate of the reaction product was quantitatively determined by capillary electrophoresis. Oligonucleotide substrate 5'-ROX-Oligo(dT) 98 The product 5'-ROX-Oligo(dT) is extended by one base under the action of terminal deoxyribonucleoside transferase. 99 The reaction system for determining the conversion rate of wild-type GeTdT and its mutants is shown in Table 3. The reaction system was mixed and reacted at 37°C for 10 minutes. After the reaction, the reaction was terminated by heating at 95°C for 10 minutes.

[0059] Table 3 Conversion rate determination reaction system of wild-type GeTdT enzyme and its mutants

[0060] The conversion rate of the terminal deoxyribonucleoside transferase reaction product was determined by capillary electrophoresis: the reaction product was diluted 200-fold with ddH2O, 1 μL of which was taken as a sample and mixed with 9 μL of HiDi formamide containing the internal standard. The resulting fragment length was analyzed using an Applied Biosystems 3730xl Genetic Analyzer. The conversion rate of the reaction product can be used to characterize the reaction activity. The calculation formula is: Conversion rate = Fragment 99 peak area / (Fragment 98 peak area + Fragment 99 peak area). The activity test results of the GeTdT wild-type and mutants are shown in Table 4 and Figure 4, where 98 represents the substrate 5'-ROX-Oligo(dT). 98 Peak 99 represents the product 5'-ROX-Oligo(dT) 99 -3'-O-Azidomethyl peak.

[0061] Table 4 Activity test results of wild-type GeTdT enzyme and its mutants

[0062] The results show that the activities of the GeTdT mutants listed in the table towards 3'-O-Azidomethyl-dTTP are all better than those of the wild type, and the conversion rates are all increased from 45% of the wild type to more than 50%. Among them, mutants E459R, E459R / R460Q, E459R / R460K, E459R / R460E, E459R / R460H, E459R / R460N and E459R / R460D are the better mutants, with conversion rates greater than 90%.

[0063] Example 5

[0064] Activity testing of terminal deoxyribonucleotidyl transferase GeTdT and its mutants towards different 3'-O-blocked modified dTTPs.

[0065] Use single-stranded Oligo(dT) with a polymerization degree of 18 18 (SEQ ID NO.40) was used as an oligonucleotide substrate, and four nucleotides (3'-O-Amide-dTTP, 3'-O-Allyl-dTTP, 3'-O-Methy-dTTP, and 3'-O-Oxime-dTTP) with different 3'-end protective groups (PG) were used as nucleic acid monomer substrates (structures shown in Figure 5). The purified wild-type GeTdT and its mutants were tested for terminal transfer activity. Oligonucleotide substrate Oligo(dT) 18 Under the action of terminal deoxyribonucleoside transferase, one base is extended to obtain the product Oligo(dT) 19-3'-O-PG. The reaction system for the terminal transfer activity test of the wild-type GeTdT enzyme and its mutants is shown in Table 5. After the reaction system is mixed, the reaction is incubated at 37°C for 10 minutes. After the reaction is completed, the reaction is terminated by heating at 95°C for 10 minutes. The reaction products of the wild-type GeTdT and its mutants were semi-quantitatively analyzed by urea polyacrylamide gel electrophoresis (20% denaturing gel). The results are shown in Figure 5. The products were obtained by converting GeTdT and its mutants into four nucleotide monomer substrates modified with different 3'-end protecting groups (Amide, Allyl, Methy, and Oxime). NC represents Oligo(dT) 18 Lanes 1-3 represent: 1, WT; 2, E459R; 3, E459R / R460E. The 5× reaction buffer used in this application has the following formula: 0.5 M Na-Cacodylate, 5 mM CoCl2, pH 7.2.

[0066] Table 5. Terminal transfer activity test reaction system of wild-type GeTdT enzyme and its mutants

[0067] The results show that the activities of the GeTdT mutant shown in FIG5 toward the four nucleotides modified with different protecting groups at the 3' end are better than those of the wild type.

[0068] In summary, the present application obtains advantageous TdT mutants by molecularly modifying natural TdT, which greatly improves the catalytic efficiency of modifying nucleic acid monomers and achieves efficient single-base addition efficiency.

[0069] The applicant declares that while the above-mentioned embodiments are used to illustrate the detailed methods of the present application, the present application is not limited to the above-mentioned detailed methods, which does not mean that the present application must rely on the above-mentioned detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials of the present application's products, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A terminal deoxynucleotidyl transferase mutant, the amino acid sequence of which comprises any one of the following sequences: (1) The following mutations occur on the basis of the sequence shown in SEQ ID NO:2: any one or at least two of E459R, E459K, E459Q, E459V, R460Q, R460K, R460E, R460H, R460N, R460D, R460S, R460L, R460M, R460W, R460F, R460Y, R460C or R460G; or, (2) A sequence obtained by substituting, deleting or adding one or at least two amino acid residues to the sequence as described in (1), and having the same or similar function as the sequence described in (1); or, (3) A sequence having at least 90% sequence homology with the sequence described in (1) or (2), and having the same or similar function as the sequence described in (1).

2. The terminal deoxynucleotidyl transferase mutant according to claim 1, wherein, (1) The mutations described above include any one of the combined mutations of E459R and R460Q, E459R and R460K, E459R and R460E, E459R and R460H, E459R and R460N, E459R and R460D, E459R and R460S, E459R and R460L, E459R and R460M, E459R and R460W, E459R and R460F, E459R and R460Y, E459R and R460C or E459R and R460G.

3. A nucleic acid molecule encoding the glycosyltransferase mutant according to claim 1.

4. A recombinant vector containing the nucleic acid molecule according to claim 3.

5. A recombinant cell containing the recombinant vector according to claim 3.

6. A method for preparing the terminal deoxynucleotidyl transferase mutant according to claim 1 or 2, comprising: Inserting the nucleic acid molecule encoding the terminal deoxynucleotidyl transferase mutant according to claim 1 or 2 into an expression vector to obtain a recombinant vector, introducing the recombinant vector into a host cell, or directly integrating the nucleic acid molecule into the genome of the host cell to obtain a genetically engineered bacterium, culturing and separating and purifying to obtain the terminal deoxynucleotidyl transferase mutant.

7. Use of the terminal deoxynucleotidyl transferase mutant according to claim 1 or 2 in the preparation of a product for nucleic acid synthesis.

8. A product for nucleic acid synthesis, comprising the terminal deoxynucleotidyl transferase mutant according to claim 1 or 2.

9. Use of the terminal deoxynucleotidyl transferase mutant according to claim 1 or 2 in nucleic acid synthesis.

10. A method for synthesizing nucleic acid, comprising: Using the terminal deoxynucleotidyl transferase mutant according to claim 1 or 2 to catalyze the ligation of dNTPs to the 3'-OH end of an oligonucleotide single strand.

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