WO2025140014A1 - Use of terminal deoxynucleotidyl transferase mutant in nucleic acid synthesis - Google Patents

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

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

Driven by high-throughput sequencing and gene editing technologies, synthetic biology has risen rapidly, and DNA synthesis has become an emerging industry. At present, there are two main methods for synthesizing oligonucleotides from scratch: chemical synthesis (phosphoramidite synthesis) and biosynthesis (enzymatic synthesis). Phosphoramidite synthesis is the mainstream method for synthesizing short-chain DNA, which can reliably provide short DNA chains of <200 nucleotides. The phosphoramidite synthesis method mainly completes a single base addition cycle through four steps of deprotection, coupling, capping and oxidation. The steps are cumbersome, and there are problems such as a long single cycle time (6-8 minutes), high consumption of chemical reagents, and high cost. In addition, a large amount of toxic and flammable organic reagents are used in the reaction process, which causes great pollution. At present, the synthesis of nucleotide sequences >200bp by phosphoramidite synthesis is still a heavy burden. For this reason, researchers have tried to develop alternative technologies such as Gibbs assembly to produce longer DNA chains.

Enzymatic de novo DNA synthesis is a promising technology that has attracted attention since the 1950s. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that is widely used and has obvious advantages in enzymatic de novo DNA synthesis technology. TdT can indiscriminately extend four natural nucleotides (A, T, C, G) to the 3' end of the starting chain without a template. Studies have shown that TdT can extend several nucleotides within 1 second and can extend the synthesized DNA to several thousand bases. The superior synthesis length and speed far exceed the range of commercially available phosphoramidite synthesis technology.

Controlling nucleotide incorporation through the "reversible termination" mechanism has become the main strategy for de novo DNA synthesis of TdT: adding a reversibly removable protecting group "Protecting Group, PG" at the 3rd position of the pentose of the nucleotide to ensure that each reaction step is terminated after extending one nucleotide, and in the subsequent reaction, PG can be removed and restored to a hydroxyl group, thereby extending the next required nucleotide (Figure 1). This strategy only requires two steps of coupling and deprotection to complete a single base addition cycle. Compared with the phosphoramidite synthesis method, it greatly improves the possible limit of single-cycle efficiency, and the entire reaction is completed in the aqueous phase, with mild reaction conditions and more green characteristics.

Since the reversible termination incorporation scheme uses non-natural nucleotide monomers with PG modification, and the natural TdT has too low activity towards it, resulting in low synthesis efficiency, it is necessary to improve the polymerization activity of TdT towards the modified nucleotide monomers through molecular modification. For example, CN114921436A discloses a terminal deoxynucleotidyl transferase mutant with improved thermal stability, which mutates the sites of the amino acid sequence of the wild-type terminal deoxynucleotidyl transferase shown in SEQ ID NO.1 to obtain a mutant with six mutations of N135P, S138H, Q229D, K232D, L234R, and V366M.

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

The embodiments of the present application provide a terminal deoxynucleotidyl transferase mutant and a preparation method and application thereof, in order to solve the problems of low efficiency, reaction energy consumption, high pollution, etc. in synthesizing DNA by the traditional phosphoramidite synthesis method, as well as the low catalytic efficiency of wild terminal deoxynucleotidyl transferase.

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:

(1) the following mutations occur based on the sequence shown in SEQ ID NO:2: any one or at least two of the following sequences: 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 of the sequence described in (1), and having the same or similar functions 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 functions as the sequence described in (1).

In the present application, the terminal deoxynucleotidyl transferase GeTdT (SEQ ID NO. 2) derived from the gecko is molecularly modified to adjust the polarity and spatial size of the active pocket 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 dNTP substrates to the 3'-OH end of a single oligonucleotide chain in the absence of a template, providing a new and effective tool enzyme for the enzymatic de novo synthesis of nucleic acids.

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.

In this application, the term "homology" can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). When a position in the compared sequences is occupied by the same base or amino acid, 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 evaluate the homology between related sequences. A polynucleotide sequence or amino acid sequence has a certain percentage (for example, 90%, 95%, 98% or 99%) of "sequence homology" with another sequence means that when the sequences are aligned, the percentage of bases or amino acids in the two compared sequences are the same.

In the present application, single point mutation of E459 and combined mutation of two points 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.

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

In some embodiments, the mutation may include any of 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 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.

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

In a third aspect, the present application provides a recombinant vector, wherein the recombinant vector contains the nucleic acid molecule described in the second aspect.

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

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.

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 a product for nucleic acid synthesis.

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.

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.

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

In a tenth aspect, the present application provides a method for synthesizing nucleic acid, the method comprising: using the terminal deoxynucleotidyl transferase mutant described in the first aspect to catalyze the connection of dNTP to the 3'-OH end of a single-stranded oligonucleotide.

In the embodiments of the present application, the dNTP includes natural dNTP and non-natural dNTP, preferably non-natural dNTP. In some embodiments, the dNTP includes 3'-O-blocking modified dNTP, and the blocking modification includes a protecting group such as "Protecting Group, PG". In some embodiments, the reversible protecting group can be selected from alkyl, aralkyl, alkenyl, alkynyl, allyl (e.g., 3'-O-allyl), aryl, heteroaryl, heterocyclic, benzyl, azide group, azido (e.g., 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 embodiments of the present application, the nucleotides may also carry a label to facilitate their detection. Preferably, the label is a fluorescent label. Each nucleotide type may carry a different fluorescent label. However, the detectable label is not necessarily 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.

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 each modified nucleotide shown in FIG8, such as 3'-O-azidomethyl-dATP, 3'-O-azidomethyl dCTP, 3'-O-azidomethyl-dGTP, 3'-O-azidomethyl-dTTP, 3'-ONH ₂ -dATP, 3'-ONH ₂ -dCTP, 3'-ONH ₂ -dGTP and/or 3'-ONH ₂ -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.

In some embodiments, the terminal deoxynucleotidyl transferase mutant performs the catalysis in the absence of a template to synthesize nucleic acids. In some embodiments, the nucleic acids include RNA and DNA. It is understood that the nucleic acid synthesis method 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 the present application and then converted to an RNA array in vitro, or the synthesized RNA array can be converted to a DNA array by reverse transcription, etc. The present application does not limit the specific type of nucleic acid array constructed.

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

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

ii) contacting dNTP and the terminal deoxynucleotidyl transferase mutant as described in the first aspect of the present application with the solid phase carrier under conditions suitable for the polymerization reaction, 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, wherein the dNTP is a non-natural dNTP with a reversible protective 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.

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 more 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 in a temperature range of 35-70°C, for example, the reaction system can be treated at 45-65°C, such as 50-60°C, for example, 51°C, 52°C, 53°C, 54°C, 55°C56°C, 57°C, 58°C or any temperature therebetween. The treatment duration can be determined depending on the reaction system, the amount of enzyme input, etc. For example, any duration from 30s to 48 hours can be selected for incubation, and the present application does not limit the specific incubation duration.

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

In the embodiment of the present application, based on the starting sequence connected to the solid phase carrier, 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, so that the target sequence (target nucleic acid) can be synthesized environmentally and efficiently through multiple cycles of the steps of "enzymatic coupling, cleaning, deprotection, cleaning". 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 synthesizing the target sequence more efficiently.

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, pH value of the buffer, ion concentration and suitable concentration of each component, etc.; and a suitable reaction time and reaction temperature. Conditions suitable for terminal transferase-catalyzed polymerization reactions are well known in the art, and the present 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 phase reaction system, with mild conditions, no need to introduce harmful reagents, simple steps, and fast synthesis speed, and can be effectively used for biological synthesis of nucleic acids.

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.

In the embodiments of the present application, the starting sequence may also be referred to as the starting chain, the initiating chain or the 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: surface modification treatment of 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 with 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.

In some embodiments, the first active substance can be selected from hydroxyl, amino, carboxyl, silane, antigen, antibody and/or streptavidin, etc.; the second active substance can be selected from carboxyl, hydroxyl, amino, antigen, antibody and/or biotin, etc. Therefore, in some embodiments, the first active substance and the second active substance can be combined by "antigen-antibody", "biotin-streptavidin", "amino-carboxyl condensation", etc., so as to fix the starting sequence on the solid phase carrier. The present application does not limit the connection method between the solid phase carrier and the starting sequence.

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

In the embodiment of the present application, a deprotecting agent is used in step iii) to remove the reversible protecting group. In some embodiments, the deprotecting agent can be sodium nitrite, tris (2-carboxyethyl) phosphine (TCEP) and/or a palladium complex. In some 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 nucleotide at the end of the synthetic chain has 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 nucleotide 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.

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 where the polymerization has not occurred. In some embodiments, TdT is used to couple ddNTP (i.e., 2', 3'-dideoxynucleoside triphosphate) at the 3'-OH 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. At the same time, since its 3' end is hydrogen (-H), TdT catalytic coupling cannot be further performed in the next round of extension reaction, thereby achieving the blocking effect of the capping reaction. Subsequently, the oligonucleotide chain that has not reached the target fragment length can be separated and removed through a purification step, so that the enzymatic synthesis product of the oligonucleotide chain has a higher purity and yield.

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

In some embodiments, the separation method of the target nucleic acid can be determined according to 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 changes of the reaction system, such as detaching the target nucleic acid from the surface of the solid phase carrier by ultrasound, vibration, etc., or the connection bond between the two can be broken by temperature changes such as high temperature treatment, etc., to release the target nucleic acid. In some embodiments, the chemical method can include using a reducing agent or a pH value 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 reagent, and the cutting reagent is optionally a site-specific cutting enzyme or protease, such as 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 a protein, then a protease can be used to treat 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.

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

In some embodiments, the target nucleic acid can be purified by electrophoresis, centrifugation, hydrolysis, silica gel column, chloroform precipitation, centrifugal column, biomagnetic beads, etc. This application does not limit the purification method.

In the examples of the present application, the method for solid phase synthesis of nucleic acids based on enzymatic reaction can be performed by a biosynthesizer. It is understandable that the method proposed in the examples of the present application can be used as the basic synthesis principle of a biosynthesizer to synthesize nucleic acids in vitro, and the TdT mutant proposed in the examples of the present application also provides an efficient synthesis basis for nucleic acid synthesis in a biosynthesizer.

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

The present application molecularly modifies the wild-type terminal deoxynucleotidyl transferase, improves its polymerization activity for modified nucleotide monomers, greatly improves the catalytic efficiency for modified nucleic acid monomers, achieves efficient single-base addition efficiency, and can add a variety of 3'-O-blocked modified dNTPs substrates to the 3'-OH end of the oligonucleotide single chain without a template, providing a new and effective tool enzyme for enzymatic de novo synthesis of nucleic acids. In addition, the present application also proposes a method for efficient nucleic acid synthesis using the new enzyme, providing a basis for enzymatic in vitro nucleic acid synthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. 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 paying creative work.

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

FIG. 2 is an SDS-PAGE image of the purified protein of the GeTdT mutant according to an embodiment of the present application.

FIG. 3 is a Urea-PAGE diagram of the terminal deoxynucleotidyl transferase product according to an example of the present application.

FIG. 4 is a capillary electrophoresis diagram of transformation products of GeTdT wild type and its E459R and E459R/R460Q mutants according to an embodiment of the present application.

FIG. 5 is a Urea-PAGE diagram of the terminal deoxynucleotidyl transferase product according to an example of the present application.

FIG6 shows a process of solid phase nucleic acid synthesis based on enzymatic reaction according to an embodiment of the present application.

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

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.

FIG. 9 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

To further illustrate the technical means and effects adopted by the present application, the present application is further described below in conjunction with the embodiments and drawings. It is understood that the specific implementation methods described herein are only used to explain the present application, rather than to limit the present application.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

Example 1: Construction of terminal deoxynucleotidyl transferase GeTdT mutant

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. Using the recombinant expression plasmid pGS-21a/GeTdT _{(G144—A512)} 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) 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 technology using the corresponding mutation primer pairs shown in Table 1, and Sanger sequencing was used to confirm whether the encoding gene of the GeTdT mutant was correct.

Table 1 Corresponding primers for constructing GeTdT mutants

The PCR reaction system was: 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.

The PCR amplification program was set as follows: first, pre-denaturation at 94°C for 5 min; then 21 cycles (denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 7 min 50 s); finally, extension at 72°C for 7 min, and insulation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.

Dpn I was added to the PCR product verified by gel electrophoresis, and the template was degraded by water bath at 37°C for 2 hours, and 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, cultured at 37°C for 11 hours, and 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 clones with correct sequencing were subjected to plasmid extraction and transformed into the expression host Escherichia coli BL21 (DE3) competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.), and 19 recombinant Escherichia coli strains capable of expressing 18 mutants and GeTdT wild-type proteins were obtained.

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

The 19 recombinant E. coli obtained in Example 1 were inoculated into LB medium respectively, and after culturing at 37°C for 8 hours, they were transferred to 300mL TB fermentation medium at an inoculation amount of 5% of the volume of the fermentation medium. First, they were placed at 37°C and 200rpm for constant temperature culture for 3 hours. When the bacterial OD ₆₀₀ was 0.5-0.7, IPTG was added to a final concentration of 1μM and the culture was induced at 25°C and 200rpm for 24 hours. After the fermentation was completed, the fermentation broth was crushed by high-pressure homogenization (crushing conditions were 4°C and 80MPa) and then centrifuged (12000rpm, 30min, 4°C), and the supernatant was the crude enzyme solution of the terminal deoxynucleotidyl transferase mutant produced by the recombinant bacteria.

The enzyme solution sample prepared in the previous step was subjected to protein purification, as follows: the terminal deoxynucleotidyl transferase protein was purified by nickel ion affinity chromatography. The crude enzyme solution of the terminal deoxynucleotidyl transferase mutant was loaded onto a nickel ion affinity chromatography column (nickel column) respectively. The recombinant protein was purified by gradient elution method: purification buffer A and purification buffer B were used to prepare 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), and then the protein bound to the nickel column was gradually eluted with the eluents in the order of imidazole concentration from low to high. During the purification process, all eluents were precooled to 4°C to ensure that the recombinant protein maintained biological activity. The eluent 4 containing the purified target protein was ultrafiltered and concentrated using an ultrafiltration tube that could retain 30 kDa protein, and the purified eluent was replaced 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. According to Figure 2, it can be seen that the target protein with higher purity was obtained.

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

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

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

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

Using single-stranded Oligo(dT) ₁₈ (SEQ ID NO.40) with a polymerization degree of 18 as an oligonucleotide substrate, and using four nucleotides with azidomethyl modification at the 3' end (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, 3'-O-Azidomethyl-dGTP) as nucleic acid monomer substrates (structures shown in Figure 3), the terminal transfer activity of the purified wild-type GeTdT and its mutants was determined. Oligo(dT) _18, an oligonucleotide substrate, was extended by one base under the action of terminal deoxynucleotidyl transferase to obtain the product Oligo(dT) _18- dNTP-3'-O-Azidomethyl. The reaction system for the activity test of the wild-type GeTdT enzyme and its mutants is shown in Table 2. After the reaction system is mixed, react at 37°C for 10 minutes. After the reaction is completed, the reaction is terminated by heating at 95°C for 10 minutes. Urea polyacrylamide gel electrophoresis (20% denaturing gel) was used to semi-quantitatively analyze the reaction products of GeTdT wild type and its mutants, and the results are shown in Figure 3. The formula of the 5× reaction buffer used was: 0.5M Na-Cacodylate, 5mM CoCl ₂ , pH 7.2.

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

In Figure 3, the products were obtained by transforming four nucleotide monomer substrates with azidomethyl modification at the 3' end (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, and 3'-O-Azidomethyl-dGTP) by GeTdT and its mutants; NC represents Oligo(dT) ₁₈ ; PC represents 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/R460N. 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 activities of the GeTdT mutants shown in the figure for the four nucleotides with azidomethyl modification at the 3' end are better than those of the wild type, among which 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.

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

Using 5'-ROX-Oligo(dT) ₉₈ (SEQ ID NO.41) with a single-stranded 5' end modified with ROX fluorescent group with a polymerization degree of 98 as an oligonucleotide substrate, and 3'-O-Azidomethyl-dTTP with an azidomethyl modification at the 3' end 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. The oligonucleotide substrate 5'-ROX-Oligo(dT) ₉₈ was extended by one base under the action of terminal deoxynucleotidyl transferase to obtain the product 5'-ROX-Oligo(dT) ₉₉ -3'-O-Azidomethyl. The reaction system for determining the conversion rate of the GeTdT wild type and its mutants is shown in Table 3. After the reaction system is mixed, react at 37°C for 10 minutes, and then heat at 95°C for 10 minutes to terminate the reaction.

Table 3. Reaction system for determining the conversion rate of wild-type GeTdT enzyme and its mutants

The conversion rate of the terminal deoxynucleotidyl transferase reaction product was determined by capillary electrophoresis: the reaction product was diluted 200 times with ddH ₂ O, 1 μL was taken as a sample, and mixed with 9 μL of HiDi formamide mixed with internal standard, and the fragment length was analyzed by Applied Biosystems 3730xl Genetic Analyzer. The conversion rate of the reaction product can characterize the reaction activity, and the calculation formula is: conversion rate = fragment 99 peak area/(fragment 98 peak area + fragment 99 peak area). The results of the GeTdT wild type and mutant activity test are shown in Table 4 and Figure 4, 98 represents the peak of the substrate 5'-ROX-Oligo(dT) ₉₈ , and 99 represents the peak of the product 5'-ROX-Oligo(dT) ₉₉ -3'-O-Azidomethyl.

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

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

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

Using single-stranded Oligo(dT) ₁₈ (SEQ ID NO.40) with a degree of polymerization of 18 as an oligonucleotide substrate, and using four nucleotides (3'-O-Amide-dTTP, 3'-O-Allyl-dTTP, 3'-O-Methy-dTTP, 3'-O-Oxime-dTTP) with different protective groups (PG) modified at the 3' end as nucleic acid monomer substrates (structures shown in Figure 5), the terminal transfer activity of the purified wild-type GeTdT and its mutants was determined. Oligo(dT) _18, an oligonucleotide substrate, was extended by one base under the action of terminal deoxynucleotidyl transferase 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, it is reacted at 37°C for 10 minutes. After the reaction is completed, it is heated at 95°C for 10 minutes to terminate the reaction. The reaction products of GeTdT wild type and its mutants were semi-quantitatively analyzed by urea polyacrylamide gel electrophoresis (20% denaturing gel). The results are shown in FIG5 . The products were obtained by GeTdT and its mutants transforming 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 is formulated as: 0.5M Na-Cacodylate, 5mM CoCl ₂ , pH 7.2.

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

As shown in the results in Figure 5, the activity of the GeTdT mutant of this example for the four nucleotides modified with different protective groups at the 3' end is better than that of the wild type, that is, the dominant mutants represented by E459R and E459R/R460Q show higher polymerization efficiency for each type of modified nucleic acid monomer, among which 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 with the wild type and E459R.

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

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.

6.1 Magnetic bead pretreatment

After mixing the magnetic beads (Dynabeads ^TM M-270 Streptavidin), take 6.6μL into a centrifuge tube, wash the magnetic beads with 6.6μL 2×Binding and washing (B&W) Buffer, and then place the centrifuge tube on the magnetic rack. After the liquid is clear, discard the supernatant, then add 6.6μL 1×B&W Buffer to resuspend the magnetic beads and wash the magnetic beads again. After washing 1 to 2 times, discard the supernatant and resuspend the magnetic beads with 6.6μL 2×B&W Buffer.

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

6.2 Fixing the starting sequence

0.4 μL of 100 μM starting sequence (Oligo(dT) ₁₈ , SEQ ID NO. 40) was added to the washed magnetic bead system, and then 6.2 μL of water was added. After mixing, the mixture was placed on a shaker for 15 minutes for incubation.

After incubation, place the centrifuge tube on a magnetic stand and use the magnetic stand to discard the supernatant in the system. Then use 6.6μL 1×B&W Buffer to wash the magnetic beads three times, then use Reaction Buffer to wash once, discard the supernatant and set aside for subsequent use.

The formula of 5×Reaction buffer is: 0.5M Na-Cacodylate, 5mM CoCl ₂ , pH 7.2.

6.3 Single Nucleotide Coupling

A polymerization reaction system was prepared on ice (the system is shown in Table 6 below), wherein the terminal transferase used was the mutant GeA49 of GeTdT with E459R/R460Q combined mutations, and the 3'O-modified dCTP used was dCTP-ONH _2. The reaction system was fully mixed and then placed in a PCR instrument for reaction at 37° C. for 1 minute.

Table 6

6.4 Cleaning

Remove the coupling reaction solution, use a magnetic stand and use 1×B&W Buffer to wash the magnetic bead system three times, and then completely remove the washing buffer.

6.5 Deprotection

Sodium nitrite buffer (700 mM, pH 5) was added to the washed magnetic beads and incubated for 1 min. 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.

6.6-a Proteinase K post-treatment

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.

6.6-b Cleaning

Remove the proteinase K reaction solution, use a magnetic stand and use 1×B&W Buffer to wash the magnetic bead system three times, and then completely remove the washing buffer.

6.7 Repeat the coupling, washing, deprotection and washing steps in steps 6.3-6.6 for a total of four times to synthesize the target sequence. After all are completed, heat at 95°C for 10 minutes to terminate the reaction.

6.8 Isolation of target sequences

After terminating the reaction, discard the supernatant with the help of a magnetic stand, wash the magnetic beads three times with 6.6μL 1×B&W Buffer, add 10μL 0.1% SDS solution to the magnetic beads, and heat at 98 degrees Celsius for 5 minutes to separate the synthesized target sequence, and then take the supernatant into a new EP tube.

6.9 Detection of synthetic sequences

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

In addition, this embodiment also sets up 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 of GeTdT mutant catalysis under the presence or absence of 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 in vitro nucleic acid synthesis.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (19)

A method for synthesizing nucleic acid, comprising: Terminal deoxynucleotidyl transferase mutants are used to catalyze the ligation of dNTPs to the 3'-OH end of a single-stranded oligonucleotide. The amino acid sequence of the terminal deoxynucleotidyl transferase mutant comprises any one of the following sequences: (1) A mutation occurs at the following positions of 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 of the sequence described in (1), and having the same or similar functions 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 functions as the sequence described in (1). The method according to claim 1, wherein the mutation comprises any one of E459R, E459K, E459Q and E459V, preferably E459R. The method according to claim 1, wherein the mutation comprises any one of E459R and R460Q combined mutation, E459R and R460K combined mutation, E459R and R460E combined mutation, E459R and R460H combined mutation, E459R and R460N combined mutation, E459R and R460D combined mutation, E459R and R460S combined mutation, E459R and R460L combined mutation, E459R and R460M combined mutation, E459R and R460W combined mutation, E459R and R460F combined mutation, E459R and R460Y combined mutation, E459R and R460C combined mutation and E459R and R460G combined mutation. The method according to any one of claims 1 to 3, wherein the dNTPs include natural dNTPs and unnatural dNTPs, preferably unnatural dNTPs. The method according to claim 4, wherein the non-natural dNTP is a dNTP whose 3'-OH end is modified with a reversible protecting group, Optionally, the reversible protecting group includes O-alkyl, O-amide, O-amino, O-allyl, O-oxime, O-azido, and optionally at least one of O-azidomethyl and O-phosphoric acid groups. Preferably, the reversible protecting group is O-amino and/or O-azidomethyl. The method according to any one of claims 1 to 5, wherein the terminal deoxynucleotidyl transferase mutant performs the catalysis to synthesize nucleic acid in the absence of a template, Optionally, the nucleic acid includes RNA and DNA. A method for solid phase synthesis of nucleic acid based on enzymatic reaction, comprising: i) providing a solid phase support, wherein the solid phase support is bound to a starting sequence; ii) contacting dNTP and the terminal deoxynucleotidyl transferase mutant as defined in any one of claims 1 to 3 with the solid phase support under conditions suitable for the polymerization reaction, 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, wherein the dNTP is a non-natural dNTP with a reversible protective 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. 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. The method according to claim 8, further comprising: After each step in steps ii) to iv') and before the next step, the reaction system is cleaned with a cleaning agent. Optionally, the cleaning is repeated one or more times, The cleaning reagent may optionally be a salt buffer and/or an enzyme degradation solution. The method according to any one of claims 7 to 9, further comprising: v) separating the target nucleic acid from the solid phase carrier. The method according to any one of claims 7 to 10, further comprising: The 3'-OH end of the starting sequence or the extended polynucleotide sequence where the polymerization has not occurred is blocked, Optionally, the blocking is performed using ddNTPs. The method according to any one of claims 7 to 11, wherein the solid support comprises glass, silicon, poly-lysine coating material, nitrocellulose, polystyrene, cyclic olefin copolymer, cyclic olefin polymer, polypropylene, polyethylene or polycarbonate; or The solid phase carrier is in the form of a chip, a microplate or a microbead. 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: The solid phase carrier is subjected to surface modification treatment so that the surface of the solid phase carrier is modified with a first active substance, Optionally, the surface modification treatment of the solid phase carrier is performed by at least one of plasma, acid and base, The acid is optionally sulfuric acid, the base is optionally hydrogen peroxide, Optionally, the first active substance includes hydroxyl, amino, carboxyl, silane, antigen, antibody and/or streptavidin. The method according to claim 13, wherein the starting sequence comprises a second active substance, and the second active substance is linked to the first active substance to bind the starting sequence to the solid phase carrier; or The starting sequence is connected to the first active substance via the second active substance to bind the starting sequence to the solid phase carrier, Optionally, the second active substance includes carboxyl, hydroxyl, amino, antigen, antibody and/or biotin. The method according to claim 14, wherein the first active substance is an amino group and the second active substance is a carboxyl group; and/or the first active substance is streptavidin and the second active substance is biotin. The method according to any one of claims 7 to 15, wherein in step iii) the removal of the reversible protecting group is carried out using a deprotecting agent, The deprotection reagent may optionally be sodium nitrite, tris(2-carboxyethyl)phosphine (TCEP) and/or a palladium complex. The method according to claim 10, wherein in step v), the target nucleic acid is separated from the solid phase carrier using a physical, chemical or biological method, wherein The physical method includes ultrasonic treatment or temperature change of the reaction system; The chemical method includes using a reducing agent or a pH adjusting agent; The biological method comprises using a cleavage agent, which is optionally a site-specific cleavage enzyme or a protease, Optionally, the site-specific cutting enzyme includes User enzyme and restriction endonuclease. The method according to any one of claims 7 to 17, further comprising: vi) purifying the target nucleic acid. The method according to any one of claims 7 to 18, wherein the method is performed by a biosynthesizer.