Use of terminal deoxynucleotidyl transferase mutant in nucleic acid synthesis

Abstract

A method for synthesizing nucleic acids is provided, said method comprising: utilizing a terminal deoxynucleotidyl transferase mutant to catalyze the ligation of dNTPs to the 3'-OH end of an oligodeoxynucleotide single strand, the amino acid sequence of the terminal deoxynucleotidyl transferase mutant comprising any one of the following sequences: (1) a sequence having a mutation at the following site(s) in the sequence shown in SEQ ID NO: 2: a. position 459, or b. positions 459 and 460; (2) a sequence obtained by substitution, deletion or addition of one or at least two amino acid residues in the sequence as shown in (1), which has the same or similar function as the sequence shown in (1); or (3) a sequence having at least 90% sequence homology with the sequence shown in (1) or (2), which has the same or similar function as the sequence shown in (1).

Description

Application of terminal deoxynucleotidyl transferase mutants in nucleic acid synthesis Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the application of a terminal deoxynucleotidyl transferase mutant in nucleic acid synthesis. 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. Currently, there are two main methods for synthesizing oligonucleotides from scratch: chemical synthesis (phosphoramidite synthesis) and biosynthesis (enzymatic synthesis). Phosphoramidite synthesis is the current mainstream method for synthesizing short-chain DNA, and can reliably provide short DNA chains of less than 200 nucleotides. The phosphoramidite synthesis method mainly completes a single base addition cycle through four steps: deprotection, coupling, capping, and oxidation. The steps are cumbersome, and there are problems such as a long single cycle time (6-8 minutes), high chemical reagent consumption, and high cost. In addition, a large amount of toxic and flammable organic reagents are used during the reaction process, resulting in high 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 DNA polymerase widely used and possesses significant advantages 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 chain without a template. Studies have demonstrated 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.

[0004] Controlling nucleotide incorporation through a "reversible termination" mechanism has become the primary strategy for de novo DNA synthesis of TdT: a reversibly removable protecting group (PG) is added to the 3rd position of the pentose sugar of the nucleotide, ensuring that each reaction step is terminated after the extension of a single nucleotide. In subsequent reactions, the PG can be cleaved and restored to a hydroxyl group, allowing the next desired nucleotide to be extended (Figure 1). This strategy requires only two steps, coupling and deprotection, to complete a single base addition cycle. Compared to phosphoramidite synthesis, this significantly increases the potential limit of single-cycle efficiency. Furthermore, the entire reaction is completed in aqueous phase, resulting in mild reaction conditions and a greener environment.

[0005] Because the reversible termination incorporation scheme uses non-natural nucleoside monomers with PG modifications, natural TdT has low activity against them, resulting in low synthesis efficiency. Therefore, molecular modification is necessary to enhance TdT's polymerization activity toward the modified nucleoside monomers. For example, CN114921436A discloses a terminal deoxynucleotidyl transferase mutant with improved thermal stability. By mutating the amino acid sequence of the wild-type terminal deoxynucleotidyl transferase shown in SEQ ID NO. 1, six mutations were obtained, namely, N135P, S138H, Q229D, K232D, L234R, and V366M.

[0006] In summary, discovering and designing terminal deoxynucleotidyl transferases with high polymerization activity and exploring efficient nucleic acid synthesis schemes based on them are of great significance to the field of nucleic acid synthesis. Summary of the Invention

[0007] The embodiments of the present application provide a terminal deoxynucleotidyl 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 traditional phosphoramidite synthesis of DNA, as well as the low catalytic efficiency of wild-type terminal deoxynucleotidyl transferase.

[0008] In a first aspect, the present application provides a terminal deoxynucleotidyl transferase mutant, wherein the amino acid sequence of the terminal deoxynucleotidyl transferase mutant comprises any one of the following sequences:

[0009] (1) Based on the sequence of SEQ ID NO: 2, the following mutations occur: 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

[0010] (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

[0011] (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).

[0012] In the present application, the terminal deoxynucleotidyl transferase GeTdT (SEQ ID NO. 2) derived from the gecko is molecularly modified. Through molecular modification, the polarity and spatial size of the active pocket are adjusted to enable it to 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 dNTP substrates to the 3'-OH end of an oligonucleotide single chain in the absence of a template, providing a new and effective tool enzyme for enzymatic de novo nucleic acid synthesis.

[0013] In the present application, the introduction of specific mutations into the wild-type terminal deoxynucleotidyl transferase can improve its polymerization activity for modified nucleotide monomers. It can be understood that based on the terminal deoxynucleotidyl 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.

[0014] 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.

[0015] In the present application, single mutation of E459 and combined mutation of E459 and R460 are involved, that is, the mutant mutates at position 459 of the sequence shown in SEQ ID NO: 2, or mutates at positions 459 and 460.

[0016] In some embodiments, the mutation comprises any one of E459R, E459K, E459Q and E459V, preferably E459R.

[0017] In some embodiments, the mutation may include any one of an E459R and R460Q combined mutation (which can be written as E459R / R460Q), an E459R and R460K combined mutation, an E459R and R460E combined mutation, an E459R and R460H combined mutation, an E459R and R460N combined mutation, an E459R and R460D combined mutation, an E459R and R460S combined mutation, an E459R and R460L combined mutation, an E459R and R460M combined mutation, an E459R and R460W combined mutation, an E459R and R460F combined mutation, an E459R and R460Y combined mutation, an E459R and R460C combined mutation, or an E459R and R460G combined mutation.

[0018] In a second aspect, the present application provides a nucleic acid molecule encoding the terminal deoxynucleotidyl transferase mutant described in the first aspect.

[0019] In a third aspect, the present application provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0020] In a fourth aspect, the present application provides a recombinant cell, wherein the recombinant cell contains the recombinant vector described in the third aspect.

[0021] In a fifth aspect, the present application provides a method for preparing the terminal deoxynucleotidyl transferase mutant described in the first aspect, the preparation method comprising: inserting a nucleic acid molecule encoding the terminal deoxynucleotidyl transferase mutant described in the first aspect 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 isolating and purifying to obtain the terminal deoxynucleotidyl transferase mutant.

[0022] In a sixth aspect, the present application provides the use of the terminal deoxynucleotidyl transferase mutant described in the first aspect in the preparation of products for nucleic acid synthesis.

[0023] In a seventh aspect, the present application provides a product (eg, a kit) for nucleic acid synthesis, wherein the product comprises the terminal deoxynucleotidyl transferase mutant described in the first aspect.

[0024] In an eighth aspect, the present application provides the use of the terminal deoxynucleotidyl transferase mutant described in the first aspect in nucleic acid synthesis.

[0025] In a ninth aspect, the present application provides use of a kit comprising the terminal deoxynucleotidyl transferase mutant described in the first aspect in nucleic acid synthesis or solid-phase nucleic acid synthesis based on an enzymatic reaction.

[0026] In a tenth aspect, the present application provides a method for synthesizing nucleic acids, comprising: using the terminal deoxynucleotidyl transferase mutant described in the first aspect to catalyze the ligation of dNTPs to the 3'-OH end of a single-stranded oligonucleotide.

[0027] In the present application embodiment, the dNTP includes natural dNTP and non-natural dNTP, preferably non-natural dNTP. In certain embodiments, the dNTP includes 3'-O-blocking modified dNTP, and the blocking modification includes such as protecting group "Protecting Group, PG". In certain embodiments, the reversible protecting group can be selected from alkyl, aralkyl, alkenyl, alkynyl, allyl (such as 3'-O-allyl), aryl, heteroaryl, heterocyclic group, benzyl, azide group, azido (such as 3'-O-azidomethyl), amino, ketone, isocyanate, phosphate, carbonate, sulfhydryl, acyl, oxime, cyano, alkoxy, aryloxy, heteroaryloxy or amide, etc., and these reversible blocking groups dissociate under aqueous conditions to produce molecules with free 3'-OH. In the present application embodiment, nucleotides can also carry labels to facilitate their detection. Preferably, the label is a fluorescent marker. Each nucleotide type can carry different fluorescent markers. However, the detectable label does not necessarily have to be a fluorescent label. Any label that allows detection of the incorporation of a nucleotide into a DNA sequence can be used, and the present disclosure is not limited to a specific type of label.

[0028] In some embodiments, the reversible protecting group may include at least one of an O-alkyl, an O-amide, an O-amino, an O-allyl, an O-oxime, an O-azido (e.g., an O-azidomethyl), or an O-phosphate group. In some embodiments, the reversible protecting group is an O-amino and / or an O-azidomethyl. In some embodiments, the dNTP may be the modified nucleotides shown in FIG8 , such as 3'-O-azidomethyl-dATP, 3'-O-azidomethyl dCTP, 3'-O-azidomethyl-dGTP, 3'-O-azidomethyl-dTTP, 3'-ONH2-dATP, 3'-ONH2-dCTP, 3'-ONH2-dGTP, and / or 3'-ONH2-dTTP. It is understood that the method proposed in the embodiment of the present application is based on a TdT mutant, which can effectively catalyze the polymerization of each modified nucleotide at the end of the starting sequence, thereby achieving efficient synthesis of the target sequence.

[0029] In some embodiments, the terminal deoxynucleotidyl transferase mutant performs the catalysis to synthesize nucleic acids in the absence of a template. In some embodiments, the nucleic acids include RNA and DNA. It is understood that the nucleic acid synthesis methods proposed in the embodiments of the present application can be used for the in vitro construction of RNA arrays or DNA arrays. For example, a DNA array can be synthesized based on the TdT mutant proposed in this application and then converted into an RNA array in vitro, or the synthesized RNA array can be converted into a DNA array through reverse transcription, etc. This application does not limit the specific type of nucleic acid array constructed.

[0030] In an eleventh aspect, the present application provides a method for solid-phase synthesis of nucleic acids based on an enzymatic reaction, comprising:

[0031] i) providing a solid phase support, wherein the solid phase support is bound to a starting sequence;

[0032] ii) contacting dNTPs and the terminal deoxynucleotidyl transferase mutant as described in the embodiment of the first aspect of the present application with the solid phase support under conditions suitable for the polymerization reaction, wherein the terminal deoxynucleotidyl transferase mutant catalyzes the polymerization of the dNTPs at the 3'-OH end of the starting sequence to obtain an extended polynucleotide sequence, wherein the dNTPs are non-natural dNTPs whose 3'-OH end is modified with a reversible protecting group;

[0033] iii) removing the reversible protecting group of the dNTP at the 3'-OH end of the extended polynucleotide sequence; and

[0034] iv) repeating steps ii) and iii) one or more times until the target nucleic acid is obtained.

[0035] In some embodiments, the method further comprises: iii-a) after step iii), treating the reaction system with a protease; and iv') repeating steps ii), iii) and iii-a) once or multiple times until the target nucleic acid is obtained. In some embodiments, cysteine ​​proteases, metalloproteases, serine proteases, aspartate amino acids and / or serine proteases can be used, preferably serine proteases, such as proteinase K. In some embodiments, the treatment can be carried out at a temperature range of 35-70°C, for example, the treatment can be carried out at 45-65°C, such as 50-60°C, for example, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C or any temperature therebetween. The treatment time can be determined depending on the reaction system, the amount of enzyme input, etc. For example, any time from 30s to 48 hours can be selected for incubation, and the present application does not limit the specific incubation time.

[0036] In some embodiments, the method further comprises: after each step in steps ii) to iv) and before the next step begins, using a cleaning reagent to clean the reaction system. In some embodiments, the cleaning can be repeated once or multiple times. In some embodiments, the cleaning reagent is optionally a salt buffer and / or an enzyme degradation solution. In some embodiments, the cleaning reagent may include Tris-HCl buffer, EDTA buffer, etc., which may also include ingredients such as Tween, DTT, NaCl. In some embodiments, the cleaning reagent may also include proteinase K and / or RNase, etc., to reduce the reaction background and improve the synthesis efficiency.

[0037] In the embodiment of the present application, based on the starting sequence connected to the solid phase support, the terminal deoxynucleotidyl transferase mutant can catalyze the polymerization of a single nucleotide at the 3'-OH end of the starting sequence (starting chain), thereby achieving the extension of a single nucleotide. In the case where the polymerized nucleotide is a non-natural nucleotide, such as a nucleotide modified with a protecting group, its protecting group will prevent the continued extension of the nucleotide, thereby achieving the controlled addition of the nucleotide; then, by removing the protecting group, the polymerization and extension of the nucleotide can be continued on the polymerized terminal nucleotide, thereby through multiple cycles of the steps of "enzymatic coupling, cleaning, deprotection, cleaning", the target sequence (target nucleic acid) can be synthesized in an environmentally friendly and efficient manner. In addition, by introducing a protease treatment step into the solid phase synthesis method, the conversion rate of the multi-step synthesis can be further improved, thereby more efficiently synthesizing the target sequence.

[0038] In the embodiments of the present application, the conditions suitable for the polymerization reaction may include: a reaction system suitable for the reaction, such as a suitable buffer, the pH value of the buffer, the ion concentration and the concentration of each component suitable for the reaction; and a suitable reaction time and reaction temperature. Conditions suitable for terminal transferase-catalyzed polymerization reactions are well known in the art, and this application does not limit the reaction conditions. It is understandable that the solid-phase synthesis method proposed in the embodiments of the present application is based on an aqueous reaction system, has mild conditions, does not require the introduction of harmful reagents, has simple steps, and has a fast synthesis rate, and can be effectively used for biological synthesis of nucleic acids.

[0039] In the embodiments of the present application, the solid phase carrier may include glass, silicon, polylysine coating material, nitrocellulose, polystyrene, cyclic olefin copolymer, cyclic olefin polymer, polypropylene, polyethylene or polycarbonate or a composite material formed by a combination thereof. In some embodiments, the solid phase carrier is in the form of a chip, a microplate or microbeads. It is understood that the solid phase carrier proposed in the embodiments of the present application is not limited to the specific material and form of the carrier as long as it can fix the starting sequence and realize the polymerization reaction based on it.

[0040] In the embodiments of the present application, the starting sequence may also be referred to as a starting chain, a triggering chain or an initiator, which may be an oligonucleotide sequence, and the length may be 3-100nt, for example, 3-50nt, 5-20nt or any value therebetween. In some embodiments, the starting sequence may be connected to the solid phase carrier via a partner affinity molecule. Thus, in some embodiments, before the starting sequence is bound to the solid phase carrier, the method further comprises: performing a surface modification treatment on the solid phase carrier so that the surface of the solid phase carrier is modified with a first active substance. In some embodiments, the starting sequence may be modified with a second active substance, which is a partner affinity molecule to the first active substance on the surface of the solid phase carrier, thereby achieving the connection between the solid phase carrier and the starting sequence.

[0041] In some embodiments, the first active substance can be selected from hydroxyl groups, amino groups, carboxyl groups, silane groups, antigens, antibodies, and / or streptavidin groups, etc.; the second active substance can be selected from carboxyl groups, hydroxyl groups, amino groups, antigens, antibodies, and / or biotin groups, etc. Therefore, in some embodiments, the first active substance and the second active substance can be bound to each other through "antigen-antibody," "biotin-streptavidin," "amino-carboxyl condensation," etc., thereby fixing the starting sequence on the solid support. This application does not limit the connection method between the solid support and the starting sequence.

[0042] In the embodiments of the present application, the surface modification treatment of the solid phase support can be performed by at least one of plasma, acid, and base, so that the surface of the solid phase support is modified with a first active substance. In some embodiments, the acid is optionally sulfuric acid, and the base is optionally hydrogen peroxide. It is understood that as long as the surface modification treatment modifies the surface of the solid phase support with an active substance that can be used for connection and / or protection, the specific treatment method of the surface modification treatment is not limited in this application.

[0043] In the embodiment of the present application, a deprotecting agent is used in step iii) to remove the reversible protecting group. In certain embodiments, the deprotecting agent can be sodium nitrite, tris (2-carboxyethyl) phosphine (TCEP) and / or a palladium complex. In certain embodiments, the deprotecting agent is acidic sodium nitrite, wherein the oxidizing property of sodium nitrite under acidic conditions can react with the aminooxy (-ONH2) protecting group at the 3' end to generate a hydroxyl group, so that the nucleotides at the end of the synthetic chain have extensibility, thereby achieving the purpose of deprotection. The deprotecting agent proposed in the embodiment of the present application can remove the protecting group (blocking group) of the nucleotides at the end of the synthetic chain, so that it is restored to a hydroxyl group, thereby providing a basis for the next round of extension.

[0044] In an embodiment of the present application, the method may further include: blocking the 3'-OH end of the starting sequence or the extended polynucleotide sequence in which the polymerization has not occurred. In some embodiments, TdT is used to couple ddNTP (i.e., 2', 3'-dideoxynucleoside triphosphate) at the 3' end of the oligonucleotide chain where base extension has not occurred to achieve a blocking reaction, thereby improving the overall synthesis efficiency. It is understandable that, compared to dNTP, due to the smaller spatial position of ddNTP, TdT has a higher catalytic coupling efficiency at its 3'-OH end, so it can react more efficiently with the hydroxyl group that did not react in the extension step, and because its 3' end is hydrogen (-H), TdT catalytic coupling cannot occur further in the next round of extension reaction, thereby achieving the blocking effect of the capping reaction, and the oligonucleotide chain that has not reached the target fragment length can be separated and removed by a purification step subsequently, so that the enzymatic synthesis product of the oligonucleotide chain has higher purity and yield.

[0045] In an embodiment of the present application, the method further comprises: v) separating the target nucleic acid from the solid phase carrier.

[0046] In some embodiments, the method for separating the target nucleic acid can be determined based on the connection mode between the solid phase carrier and the starting sequence. In some embodiments, the target nucleic acid can be separated from the solid phase carrier by physical, chemical or biological methods. In some embodiments, the physical method can include ultrasonic treatment or temperature change of the reaction system, for example, by ultrasound, vibration, etc., to detach the target nucleic acid from the surface of the solid phase carrier, or by temperature change, such as high temperature treatment, to break the connection bond between the two, to release the target nucleic acid. In some embodiments, the chemical method can include using a reducing agent or pH regulator to break the connection bond between the target nucleic acid and the solid phase carrier to release the target nucleic acid. In some embodiments, the biological method includes the use of a cutting agent, which can optionally be a site-specific cutting enzyme or protease, for example, by cutting a specific site in its starting sequence, or based on the connection between the target nucleic acid and the solid phase carrier through a substance such as protein, a protease can be used to degrade the protein component in the active substance used for connection to release the target nucleic acid. In some embodiments, the site-specific cutting enzyme can include User enzyme, restriction endonuclease and / or modified base cutting enzyme, such as RNase.

[0047] In the embodiment of the present application, the method further comprises: vi) purifying the target nucleic acid.

[0048] In some embodiments, the target nucleic acid can be purified by electrophoresis, centrifugation, hydrolysis, silica gel column method, chloroform precipitation method, centrifugal column method, biological magnetic bead method, etc. This application does not limit the purification method.

[0049] In the examples of this application, the enzymatic solid-phase nucleic acid synthesis method described herein can be performed using a biosynthesizer. It is understood that the method described herein can serve as the basic synthesis principle for in vitro nucleic acid synthesis using a biosynthesizer, and the TdT mutants described herein also provide a highly efficient synthesis basis for nucleic acid synthesis using a biosynthesizer.

[0050] Compared with the prior art, this application has the following beneficial effects:

[0051] This application molecularly modifies wild-type terminal deoxynucleotidyl transferase (TDase), enhancing its polymerization activity toward modified nucleotide monomers and significantly improving its catalytic efficiency toward modified nucleic acid monomers, achieving efficient single-base addition efficiency. This enzyme is capable of adding a variety of 3'-O-blocked modified dNTPs substrates to the 3'-OH end of single-stranded oligonucleotides without a template, providing a novel and effective tool enzyme for enzymatic de novo nucleic acid synthesis. Furthermore, this application proposes a method for efficient nucleic acid synthesis using this novel enzyme, providing a foundation for enzymatic in vitro nucleic acid synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0053] FIG1 is a flow chart of a terminal deoxynucleotidyl transferase-directed enzymatic synthesis scheme according to an embodiment of the present application.

[0054] FIG2 is an SDS-PAGE image of the purified protein of the GeTdT mutant according to an embodiment of the present application.

[0055] FIG3 is a Urea-PAGE image of the terminal deoxynucleotidyl transferase product according to an example of the present application.

[0056] FIG4 is a capillary electrophoresis diagram of the transformation products of GeTdT wild type and its E459R and E459R / R460Q mutants according to an embodiment of the present application.

[0057] FIG5 is a Urea-PAGE image of the terminal deoxynucleotidyl transferase product according to an example of the present application.

[0058] FIG6 shows a process for solid-phase synthesis of nucleic acids based on an enzymatic reaction according to an embodiment of the present application.

[0059] FIG7 shows the product detection results of solid-phase nucleic acid synthesis based on an enzymatic reaction according to an embodiment of the present application;

[0060] FIG8 shows a modified nucleotide with a protecting group that can be used for solid phase synthesis according to an embodiment of the present application.

[0061] FIG9 shows the polynucleotide sequences synthesized under the catalysis of GeTdT mutants with or without protease treatment according to an embodiment of the present application. DETAILED DESCRIPTION

[0062] 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.

[0063] 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.

[0064] Example 1: Construction of a terminal deoxynucleotidyl transferase GeTdT mutant

[0065] Based on the terminal deoxynucleotidyl transferase GeTdT from Gecko, the nucleotide sequence is shown in SEQ ID NO.1 and the 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 complete peptide chain sequence expressed is shown in SEQ ID NO. 3), mutations were introduced at characteristic sites by rapid PCR using the corresponding mutation primer pairs shown in Table 1, and the coding gene of the GeTdT mutant was confirmed to be correct by Sanger sequencing.

[0066] Table 1 Corresponding primers for constructing GeTdT mutants

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Example 2: Expression and purification of terminal deoxynucleotidyl transferase GeTdT and its mutants

[0071] 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 fermentation, 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 deoxynucleotidyl transferase mutant produced by the recombinant bacteria.

[0072] The enzyme solution sample prepared in the previous step was subjected to protein purification as follows: Terminal deoxynucleotidyl transferase protein was purified using nickel affinity chromatography. The crude enzyme solutions of the terminal deoxynucleotidyl transferase mutants were loaded onto nickel affinity chromatography columns. 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 increasing 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 eluate 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 / R460V. 0N; 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. As shown in Figure 2, a target protein with high purity was obtained.

[0073] Purification buffer A (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole.

[0074] Purification buffer B (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole.

[0075] 2× enzyme storage buffer (pH 7.2): 40 mM Tris-HCl, 400 mM NaCl, 5% glycerol.

[0076] Example 3: Semi-quantitative analysis of the activity of terminal deoxynucleotidyl transferase GeTdT and its mutants

[0077] 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 deoxynucleotidyl 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.

[0078] Table 2 Activity test reaction system of wild-type GeTdT enzyme and its mutants

[0079] 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) 19Lanes 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, E4 59R / R460D; 12, E459R / R460S; 13, E459R / R460L; 14, E459R / R460M; 15, E459R / R 460W; 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.

[0080] Example 4: Determination of the conversion rate of terminal deoxynucleotidyl transferase GeTdT and its mutants

[0081] 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 deoxynucleotidyl 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.

[0082] Table 3 Conversion rate determination reaction system of wild-type GeTdT enzyme and its mutants

[0083] The conversion rate of terminal deoxynucleotidyl transferase reaction products 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 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.

[0084] Table 4 Activity test results of wild-type GeTdT enzyme and its mutants

[0085] 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%.

[0086] Example 5: Activity testing of terminal deoxynucleotidyl transferase GeTdT and its mutants on different 3'-O-blocked modified dTTPs

[0087] 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 deoxynucleotidyl 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.

[0088] Table 5. Terminal transfer activity test reaction system of wild-type GeTdT enzyme and its mutants

[0089] As shown in Figure 5, the GeTdT mutants of this example have better activities than the wild type for the four nucleotides modified with different protecting groups at the 3' end. That is, the dominant mutants represented by E459R and E459R / R460Q all show higher polymerization efficiency for various types of modified nucleic acid monomers. Among them, for the modified nucleotide type 3'-O-Amide-dTTP, E459R / R460Q shows a significant improvement in the polymerization efficiency of this modified nucleotide type compared to the wild type and E459R.

[0090] Example 6: Terminal deoxynucleotidyl transferase mutant catalyzed oligonucleotide solid phase synthesis

[0091] In this example, magnetic beads were used as solid phase carriers and oligonucleotide solid phase synthesis was performed under the catalysis of terminal deoxynucleotidyl transferase mutants. The specific process is shown in FIG6 , and the specific steps are as follows.

[0092] 6.1 Magnetic Bead Pretreatment

[0093] Dynabeads TMAfter mixing, transfer 6.6 μL of the solution to a centrifuge tube. Wash the magnetic beads with 6.6 μL of 2× Binding and Washing (B&W) Buffer, then place the centrifuge tube on a magnetic rack. Once the solution has clarified, discard the supernatant and resuspend the magnetic beads in 6.6 μL of 1× B&W Buffer. Wash the beads again. After washing one to two times, discard the supernatant and resuspend the magnetic beads in 6.6 μL of 2× B&W Buffer.

[0094] The preparation method of B&W Buffer (2×) is: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA 2M NaCl, 0.01–0.1% Tween-20.

[0095] 6.2 Fixing the starting sequence

[0096] Add 0.4 μL of 100 μM starting sequence (Oligo(dT) 18 , SEQ ID NO.40), and then 6.2 μL of water was added, mixed, and then placed on a shaking instrument for 15 minutes for incubation.

[0097] After incubation, place the centrifuge tube on a magnetic stand and discard the supernatant in the system with the help of the magnetic stand. Then, wash the magnetic beads three times with 6.6μL 1× B&W Buffer, wash once with Reaction Buffer, and discard the supernatant for subsequent use.

[0098] The formula of 5×Reaction buffer is: 0.5M Na-Cacodylate, 5mM CoCl2, pH 7.2.

[0099] 6.3 Single Nucleotide Coupling

[0100] A polymerization reaction system was prepared on ice (the system is shown in Table 6 below), wherein the terminal transferase used was the GeTdT mutant GeA49 with E459R / R460Q combined mutations, and the 3'O-modified dCTP used was dCTP-ONH2. The reaction system was thoroughly mixed and then placed in a PCR instrument at 37°C for 1 minute.

[0101] Table 6

[0102] 6.4 Cleaning

[0103] The coupling reaction solution was removed, and the magnetic bead system was washed three times with 1× B&W Buffer using a magnetic stand, and the washing buffer was completely removed.

[0104] 6.5 Deprotection

[0105] Sodium nitrite buffer (700 mM, pH 5) was added to the washed magnetic beads and incubated for 1 minute. The deprotection step was repeated twice to remove the protecting group of the 3'O-modified mononucleotide polymerized at the end of the starting sequence.

[0106] 6.6-a Proteinase K post-treatment

[0107] Add 1× B&W Buffer to the deprotected magnetic bead system, add 4 μL of proteinase K to the system, and treat at 53 degrees Celsius for 2 minutes.

[0108] 6.6-b Cleaning

[0109] Remove the proteinase K reaction solution, wash the magnetic bead system three times with 1× B&W Buffer using a magnetic stand, and then completely remove the wash buffer.

[0110] 6.7 Repeat the coupling, washing, deprotection, and cleaning steps in steps 6.3-6.6 four times to synthesize the target sequence. Terminate the reaction by heating at 95°C for 10 minutes.

[0111] 6.8 Isolation of target sequences

[0112] After terminating the reaction, the supernatant was discarded with the help of a magnetic stand. The magnetic beads were washed three times with 6.6 μL of 1× B&W Buffer. Then, 10 μL of 0.1% SDS solution was added to the magnetic beads and heated at 98 degrees Celsius for 5 minutes to separate the synthesized target sequence. The supernatant was then transferred to a new EP tube.

[0113] 6.9 Detection of Synthetic Sequences

[0114] The synthesized target sequence was detected by 20% Urea-PAGE, and the results are shown in Figure 7, where NC is the starting sequence, i.e., 18 nt of Oligo dT; lanes 1 to 4 are synthetic sequences with 1 to 8 single nucleotides added to the starting sequence, respectively. This demonstrates that the mutant TdT proposed in the examples of this application can effectively catalyze the de novo synthesis of the target sequence from single nucleotides modified with protecting groups under template-free conditions based on a solid-phase support.

[0115] In addition, this embodiment also provides a control group in which the protease treatment step is omitted, that is, compared with the experimental group process shown above, step 6.6 is not performed in the control group. Figure 9 shows the synthesis results under the catalysis of the GeTdT mutant with or without protease treatment according to this embodiment, wherein NC is the starting sequence, i.e., 18nt Oligo dT; the portion indicated in lanes A to D is the band of the synthetic sequence with 5 single nucleotides added to the starting sequence, wherein lanes B and D are the protease treatment group, i.e., the experimental group; lanes A and C are the control group not treated with protease. As can be seen from Figure 9, compared with the control group, lanes B and D have fewer non-target bands, and the bands of the target sequence are thicker and brighter, indicating that the method proposed in this embodiment improves the conversion rate of enzymatic synthesis by introducing the protease treatment step, and realizes the controllable and efficient synthesis of in vitro nucleic acid sequences. In summary, the present application obtains an advantageous TdT mutant by molecularly modifying natural TdT, greatly improving the catalytic efficiency of various types of modified nucleotide monomers and achieving efficient single base addition efficiency. In addition, the present application also proposes a method for efficiently synthesizing nucleic acids using the novel enzyme, thereby providing a basis for enzymatic synthesis of nucleic acids in vitro.

[0116] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0117] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for synthesizing nucleic acids, comprising: using a terminal deoxynucleotidyl transferase mutant to catalyze the ligation of dNTP to the 3'-OH end of a single-stranded oligonucleotide, wherein the amino acid sequence of the terminal deoxynucleotidyl transferase mutant comprises any one of the following sequences: (1) mutations occur at the following sites in the sequence shown in SEQ ID NO:2: a. position 459; or b. positions 459 and 460; (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 method according to claim 1, wherein the mutation comprises any one of E459R, E459K, E459Q and E459V, preferably E459R.

3. The method according to claim 1, wherein the mutation comprises 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, and E459R and R460G.

4. The method according to any one of claims 1 to 3, wherein the dNTP comprises natural dNTP and unnatural dNTP, preferably unnatural dNTP.

5. The method according to claim 4, wherein the unnatural dNTP is a dNTP with a reversible protecting group modified at the 3'-OH end, optionally, the reversible protecting group comprises O-alkyl, O-amido, O-amino, O-allyl, O-oxime, O-azide, optionally at least one of O-azidomethyl and O-phosphate group, preferably, the reversible protecting group is O-amino and / or O-azidomethyl.

6. The method according to any one of claims 1 to 5, wherein the terminal deoxynucleotidyl transferase mutant performs the catalysis to synthesize nucleic acids without a template, optionally, the nucleic acids comprise RNA and DNA.

7. A method for solid-phase synthesis of nucleic acids based on enzymatic reaction, comprising: i) providing a solid-phase support, wherein the solid-phase support is bound to a starting sequence; ii) Under conditions suitable for polymerization reaction, contacting dNTP and the terminal deoxynucleotidyl transferase mutant as defined in any one of claims 1 to 3 with the solid support, wherein the terminal deoxynucleotidyl transferase mutant catalyzes the polymerization of the dNTP at the 3'-OH end of the starting sequence to obtain an extended polynucleotide sequence, and wherein the dNTP is an unnatural dNTP with a reversible protecting group modified at the 3'-OH end; iii) Removing the reversible protecting group of the dNTP at the 3'-OH end of the extended polynucleotide sequence; and iv) Repeating steps ii) and iii) one or more times until the target nucleic acid is obtained.

8. The method according to claim 7, further comprising: iii-a) After step iii), treating the reaction system with a protease; and iv’) Repeating steps ii), iii) and iii-a) one or more times until the target nucleic acid is obtained, Optionally, the protease is a serine protease, preferably proteinase K.

9. The method according to claim 8, further comprising: After each of steps ii) to iv’), before the next step starts, washing the reaction system with a washing reagent, Optionally, the washing is repeated one or more times, The washing reagent is optionally a salt buffer and / or an enzyme degradation solution.

10. The method according to any one of claims 7 to 9, further comprising: v) Separating the target nucleic acid from the solid support.

11. The method according to any one of claims 7 to 10, further comprising: Blocking the 3'-OH end of the starting sequence or the extended polynucleotide sequence where polymerization has not occurred, Optionally, using ddNTP for the blocking.

12. The method according to any one of claims 7 to 11, wherein the solid support comprises glass, silicon, polylysine-coated material, nitrocellulose, polystyrene, cyclic olefin copolymer, cyclic olefin polymer, polypropylene, polyethylene or polycarbonate; or The solid support is in the form of a chip, a microplate or a microbead.

13. The method according to any one of claims 7 to 12, wherein before binding the starting sequence to the solid support, the method further comprises: Performing a surface modification treatment on the solid support so that the surface of the solid support is modified with a first active substance, Optionally, performing the surface modification treatment on the solid support by at least one of plasma, acid and base, The acid is optionally sulfuric acid, and the base is optionally hydrogen peroxide, Optionally, the first active substance comprises hydroxyl, amino, carboxyl, silyl, antigen, antibody and / or streptavidin-like.

14. The method according to claim 13, wherein the starting sequence contains a second active substance, and the second active substance is connected to the first active substance to bind the starting sequence to the solid support; or The starting sequence is connected to the first active substance via the second active substance to bind the starting sequence to the solid support, Optionally, the second active substance includes carboxyl, hydroxyl, amino, antigen, antibody, and / or biotin-like substances.

15. The method according to claim 14, wherein the first active substance is amino and the second active substance is carboxyl; and / or the first active substance is streptavidin and the second active substance is biotin.

16. The method according to any one of claims 7 to 15, wherein in step (iii), a deprotection reagent is used to remove the reversible protecting group. The deprotection reagent is optionally sodium nitrite, tris(2-carboxyethyl)phosphine (TCEP), and / or palladium complex.

17. The method according to claim 10, wherein in step (v), the target nucleic acid is separated from the solid support using physical, chemical, or biological methods, wherein the physical method includes sonication or temperature change of the reaction system; the chemical method includes using a reducing reagent or a pH regulator; the biological method includes using a cleavage reagent, and the cleavage reagent is optionally a site-specific cleavage enzyme or a protease. Optionally, the site-specific cleavage enzyme includes User enzyme and restriction endonuclease.

18. The method according to any one of claims 7 to 17 further includes: vi) purifying the target nucleic acid.

19. The method according to any one of claims 7 to 18, wherein the method is performed by a biosynthesizer.

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