Terminal deoxyribonucleotide transferase and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to terminal deoxyribonucleotide transferase and application thereof. The present invention provides a terminal transferase or a mutant thereof; wherein both the terminal transferase and the terminal transferase mutant have catalytic activity independent of template synthesis of nucleic acid molecules, the total cost of synthesizing custom nucleic acid is reduced, and nucleic acid can be synthesized faster, cheaper and higher in quality even if modified nucleotide is used; especially, compared with the terminal transferase, most of the terminal transferase mutants have The present invention relates to a primer chain having a higher catalytic activity (i.e., the activity of catalyzing the addition of nucleotides (preferably 3 '-O-allyl modified nucleotides, preferably RNA) to the primer chain) and / or a wider extension / incorporation ability (i.e., more species of nucleotides (preferably 3'-O-allyl modified nucleotides, preferably RNA) can be incorporated to the primer chain).

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a terminal deoxyribonucleotidyl transferase and an application thereof. Background Art

[0002] Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase found in humans and other vertebrates. It plays a crucial role in DNA synthesis, particularly during the development of B and T cells of the immune system. The primary function of TdT is to add deoxyribonucleotides to the 3' end of a DNA chain without a template.

[0003] Over the past 40 years, a novel chemical synthesis method for nucleic acids based on solid-phase phosphoramidite chemistry has been widely used and continuously improved. This technique involves a four-step chain extension cycle, with each cycle adding a single base to a growing oligonucleotide chain attached to a solid support matrix. Although this method has been the preferred method for nucleic acid synthesis for decades, it has several significant limitations: it requires the use of multiple solvents and reagents, and due to chemical reaction efficiency limitations, the length of synthesized oligonucleotides typically does not exceed 150–200 bases. Furthermore, these short fragments require further assembly to provide the desired DNA sequence. An alternative to chemical synthesis is the use of template-independent DNA polymerases that add reversible terminator-modified nucleotides to the growing single-stranded nucleic acid chain. This allows for the controlled addition of one nucleotide type per cycle. Some natural enzymes are able to act on natural nucleotides in the absence of a template and can therefore catalyze the uncontrolled synthesis of nucleic acids. However, natural enzymes exhibit low extension efficiency when acting on nucleotides modified with blocking groups. To date, only a few DNA polymerases have been identified that can efficiently extend on single-stranded DNA (without a template). The most widely studied polymerase with this template-independent activity is terminal deoxyribonucleotidyl transferase (TdT). TdT has been widely used to synthesize single-stranded DNA for various applications, including biotechnology, biomedical research, and synthetic biology. Unfortunately, natural TdT binds to blocking group-modified nucleotides less efficiently than natural nucleotides. Therefore, a lot of work has been devoted to developing new TdT variants to improve the incorporation efficiency and enzyme stability of TdT for blocking group-modified nucleotides, such as U.S. Patent Publication US2019 / 0211315 by Champion et al. and International Patent Publication WO2017 / 216472 by Ybert et al.

[0004] In view of the above, it is necessary to develop an improved terminal transferase that can stably and efficiently perform extension using a blocking group-modified nucleotide in a non-template-dependent manner, which will promote the progress of the field of enzyme-based template-free polynucleotide synthesis. SUMMARY

[0005] An object of the first aspect of the present application is to provide a terminal transferase or a mutant thereof.

[0006] An object of the second aspect of the present application is to provide a recombinant protein.

[0007] An object of the third aspect of the present application is to provide a biological material related to the terminal transferase or the mutant thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application.

[0008] An object of the fourth aspect of the present application is to provide a terminal transferase-nucleotide complex.

[0009] An object of the fifth aspect of the present application is to provide use of the terminal transferase or the mutant thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the biological material of the third aspect of the present application, and / or the terminal transferase-nucleotide complex of the fourth aspect of the present application.

[0010] An object of the sixth aspect of the present application is to provide a kit.

[0011] An object of the seventh aspect of the present application is to provide a method for synthesizing a nucleic acid molecule.

[0012] An object of the eighth aspect of the present application is to provide a method for producing the terminal transferase or the mutant thereof of the first aspect of the present application, and / or the recombinant protein of the second aspect of the present application.

[0013] To achieve the above object, the technical solution adopted by the present application is as follows:

[0014] The first aspect of the present application provides a terminal transferase or a mutant thereof, wherein the amino acid sequence of the terminal transferase is as shown in SEQ ID NO: 16; and the amino acid sequence of the terminal transferase mutant has a mutation at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sites selected from the group consisting of V120, C162, G192, H202, D237, A255, D257, K261, L299, and G321 (the structure of the terminal transferase mutation site position is shown in FIG. 1). Figure 1

[0015] ​It is to be noted that V120, C162, G192, H202, D237, A255, D257, K261, L299, G321 represent the 120th, 162nd, 192nd, 202nd, 237th, 255th, 257th, 261st, 299th, 321st amino acid of SEQ ID NO: 16, respectively.

[0016] In some embodiments, the amino acid sequence of the terminal transferase mutant has a mutation at any one of the sites selected from V 120, C162, G192, H202, D237, A255, D257, K261, L299, G321.

[0017] In some embodiments, the amino acid sequence of the terminal transferase mutant, except for the mutation site, is identical to the corresponding amino acid sequence of the terminal transferase.

[0018] It is to be noted that at least 80% identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

[0019] It is to be noted that in the present application, “the amino acid sequence of the terminal transferase mutant, except for the mutation site, is identical to the corresponding amino acid sequence of the terminal transferase” refers to the amino acid sequence of the terminal transferase corresponding to the amino acid sequence of the mutant, except for the mutation site.

[0020] In some embodiments, the amino acid sequence of the terminal transferase mutant, except for the mutation site, is identical to the corresponding amino acid sequence of the terminal transferase.

[0021] In some embodiments, the terminal transferase or the mutant thereof has DNA polymerase and / or RNA polymerase activity.

[0022] In some embodiments, the terminal transferase or the mutant thereof has catalytic activity of adding nucleotides to the end of a nucleic acid molecule without template dependence.

[0023] In some embodiments, the nucleotide is a 3’-OH end modified nucleotide.

[0024] In some embodiments, the 3’-OH end modified nucleotide is modified by adding a blocking group to the 3’-OH end of the nucleotide.

[0025] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phosphato group; preferably O-allyl.

[0026] In some embodiments, the 5' end of the nucleotide contains 2 phosphate groups or 3 phosphate groups.

[0027] In some embodiments, the terminal transferase has catalytic activity for template-independent synthesis of nucleic acid molecules.

[0028] In some embodiments, the terminal transferase mutant has catalytic activity for template-independent synthesis of nucleic acid molecules.

[0029] In some embodiments, the nucleic acid molecule comprises at least one of DNA, RNA; further comprises RNA.

[0030] In some embodiments, the DNA comprises natural deoxynucleotides and / or unnatural deoxynucleotides.

[0031] In some embodiments, the RNA comprises natural nucleotides and / or unnatural nucleotides.

[0032] In some embodiments, the mutation comprises insertion, deletion, and / or substitution; preferably, the mutation is substitution.

[0033] In some embodiments, the mutation at the V120 site is substitution of V at position 120 to M.

[0034] In some embodiments, the mutation at the C162 site is substitution of C at position 162 to K, G, A, V, I, L, M, R, Q, N, S, W, Y, F, H, P, E, D, or T.

[0035] In some embodiments, the mutation at the G192 site is substitution of G at position 192 to S.

[0036] In some embodiments, the mutation at the H202 site is substitution of H at position 202 to K, G, A, V, I, L, M, R, Q, N, S, W, Y, F, D, P, E, C, or T.

[0037] In some embodiments, the mutation at the D237 site is substitution of D at position 237 to K, G, A, V, I, L, M, R, Q, N, S, W, Y, F, H, P, E, C, or T.

[0038] In some embodiments, the mutation at position A255 is to replace the A at position 255 with K, G, C, V, I, L, M, R, Q, N, S, W, Y, F, H, P, E, D, or T.

[0039] In some embodiments, the mutation at position D257 is to replace D at position 257 with M.

[0040] In some embodiments, the mutation at the K261 site is to replace the K at position 261 with Y, C, or F.

[0041] In some embodiments, the mutation at the L299 site is to replace L at position 299 with M.

[0042] In some embodiments, the mutation at the G321 site is to replace the G at position 321 with V or M.

[0043] In some embodiments, the terminal transferase has the function of incorporating A and U blocking modifications.

[0044] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-1) is that V at position 120 of SEQ ID NO: 16 is replaced by M, which has the function of incorporating blocking modified A and U.

[0045] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-2) is that the C at position 162 of SEQ ID NO: 16 is replaced by K, which has the function of incorporating blocking modified A and U.

[0046] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-3) is that the C at position 162 of SEQ ID NO: 16 is replaced by G, which has the function of incorporating blocking modified A and U.

[0047] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-4) is that the C at position 162 of SEQ ID NO: 16 is replaced by A, and it has the function of incorporating the blocking modified A and U.

[0048] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-5) is that the C at position 162 of SEQ ID NO: 16 is replaced by T, which has the function of incorporating blocking modified A and U.

[0049] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-6) is that the C at position 162 of SEQ ID NO: 16 is replaced by V, which has the function of incorporating blocking modified A and U.

[0050] In some embodiments, the terminal transferase mutant (RP-7) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with I, which has incorporation function for blocked modified A and U.

[0051] In some embodiments, the terminal transferase mutant (RP-8) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with L, which has incorporation function for blocked modified A and U.

[0052] In some embodiments, the terminal transferase mutant (RP-9) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with M, which has incorporation function for blocked modified A and U.

[0053] In some embodiments, the terminal transferase mutant (RP-10) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with R, which has incorporation function for blocked modified A and U.

[0054] In some embodiments, the terminal transferase mutant (RP-11) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with Q, which has incorporation function for blocked modified A and U.

[0055] In some embodiments, the terminal transferase mutant (RP-12) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with N, which has incorporation function for blocked modified A and U.

[0056] In some embodiments, the terminal transferase mutant (RP-13) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with S, which has incorporation function for blocked modified A and U.

[0057] In some embodiments, the terminal transferase mutant (RP-14) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with W, which has incorporation function for blocked modified A and U.

[0058] In some embodiments, the terminal transferase mutant (RP-15) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with Y, which has incorporation function for blocked modified A and U.

[0059] In some embodiments, the terminal transferase mutant (RP-16) has an amino acid sequence in which the C at position 162 of SEQ ID NO: 16 is replaced with F, which has incorporation function for blocked modified A and U.

[0060] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-17) is that the C at position 162 of SEQ ID NO: 16 is replaced by H, which has the function of incorporating blocking modified A and U.

[0061] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-18) is that the C at position 162 of SEQ ID NO: 16 is replaced by P, which has the function of incorporating blocking modified A and U.

[0062] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-19) is that C at position 162 of SEQ ID NO: 16 is replaced by E, which has the function of incorporating blocking modified A and U.

[0063] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-20) is that C at position 162 of SEQ ID NO: 16 is replaced by D, which has the function of incorporating blocking modified A and U.

[0064] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-21) is that the G at position 192 of SEQ ID NO: 16 is replaced by S, and it has the function of incorporating blocking modified A, U, and G.

[0065] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-22) is that H at position 202 of SEQ ID NO: 16 is replaced by K, which has the function of incorporating the blocking modified C.

[0066] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-23) is that H at position 202 of SEQ ID NO: 16 is replaced by G, which has the function of incorporating the blocking modified C.

[0067] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-24) is that H at position 202 of SEQ ID NO: 16 is replaced by A, and it has the function of incorporating the blocking modified C.

[0068] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-25) is that H at position 202 of SEQ ID NO: 16 is replaced by T, which has the function of incorporating the blocking modified C.

[0069] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-26) is that H at position 202 of SEQ ID NO: 16 is replaced by V, which has the function of incorporating the blocking modified C.

[0070] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-27) is that H at position 202 of SEQ ID NO: 16 is replaced by I, which has the function of incorporating the blocking modified C.

[0071] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-28) is that H at position 202 of SEQ ID NO: 16 is replaced by L, which has the function of incorporating the blocking modified C.

[0072] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-29) is that H at position 202 of SEQ ID NO: 16 is replaced by M, which has the function of incorporating the blocking modified C.

[0073] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-30) is that H at position 202 of SEQ ID NO: 16 is replaced by R, which has the function of incorporating the blocking modified C.

[0074] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-31) is that H at position 202 of SEQ ID NO: 16 is replaced by Q, which has the function of incorporating the blocking modified C.

[0075] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-32) is that H at position 202 of SEQ ID NO: 16 is replaced by N, which has the function of incorporating the blocking modified C.

[0076] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-33) is that H at position 202 of SEQ ID NO: 16 is replaced by S, which has the function of incorporating the blocking modified C.

[0077] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-34) is that H at position 202 of SEQ ID NO: 16 is replaced by W, which has the function of incorporating the blocking modified C.

[0078] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-35) is that H at position 202 of SEQ ID NO: 16 is replaced by Y, which has the function of incorporating the blocking modified C.

[0079] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-36) is that H at position 202 of SEQ ID NO: 16 is replaced by F, which has the function of incorporating the blocking modified C.

[0080] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-37) is that H at position 202 of SEQ ID NO: 16 is replaced by C, and it has the function of blocking the incorporation of the modified C.

[0081] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-38) is that H at position 202 of SEQ ID NO: 16 is replaced by P, which has the function of incorporating the blocking modified C.

[0082] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-39) is that H at position 202 of SEQ ID NO: 16 is replaced by E, which has the function of incorporating the blocking modified C.

[0083] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-40) is that H at position 202 of SEQ ID NO: 16 is replaced by D, which has the function of incorporating the blocking modified C.

[0084] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-41) is that D at position 237 of SEQ ID NO: 16 is replaced by K, which has the function of incorporating the blocking modified C.

[0085] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-42) is that D at position 237 of SEQ ID NO: 16 is replaced by G, which has the function of incorporating blocking modified C and U.

[0086] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-43) is that D at position 237 of SEQ ID NO: 16 is replaced by A, and it has the function of incorporating the blocking modified C.

[0087] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-44) is that D at position 237 of SEQ ID NO: 16 is replaced by T, which has the function of incorporating blocking modified C and U.

[0088] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-45) is that D at position 237 of SEQ ID NO: 16 is replaced by V, and it has the function of incorporating the blocking modified C.

[0089] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-46) is that D at position 237 of SEQ ID NO: 16 is replaced by I, and it has the function of incorporating the blocking modified C.

[0090] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-47) is that D at position 237 of SEQ ID NO: 16 is replaced by L, and it has the function of incorporating the blocking modified C.

[0091] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-48) is that D at position 237 of SEQ ID NO: 16 is replaced by M, and it has the function of incorporating the blocking modified C.

[0092] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-49) is that D at position 237 of SEQ ID NO: 16 is replaced by R, and it has the function of incorporating the blocking modified C.

[0093] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-50) is that D at position 237 of SEQ ID NO: 16 is replaced by Q, which has the function of incorporating blocking modified A, U, G, and C.

[0094] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-51) is that D at position 237 of SEQ ID NO: 16 is substituted with N, which has the function of incorporating the blocking modified C.

[0095] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-52) is that D at position 237 of SEQ ID NO: 16 is replaced by S, and it has the function of incorporating the blocking modified C.

[0096] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-53) is that D at position 237 of SEQ ID NO: 16 is replaced by W, which has the function of incorporating the blocking modified C.

[0097] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-54) is that D at position 237 of SEQ ID NO: 16 is replaced by Y, which has the function of incorporating the blocking modified C.

[0098] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-55) is that D at position 237 of SEQ ID NO: 16 is replaced by F, which has the function of incorporating the blocking modified C.

[0099] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-56) is that D at position 237 of SEQ ID NO: 16 is replaced by H, which has the function of incorporating the blocking modified C.

[0100] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-57) is that D at position 237 of SEQ ID NO: 16 is replaced by P, which has the function of incorporating the blocking modified C.

[0101] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-58) is that D at position 237 of SEQ ID NO: 16 is replaced by E, and it has the function of incorporating the blocking modified C.

[0102] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-59) is that D at position 237 of SEQ ID NO: 16 is replaced by C, and it has the function of incorporating the blocking modified C.

[0103] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-60) is that the A at position 255 of SEQ ID NO: 16 is replaced by K, which has the function of incorporating the blocking modified C.

[0104] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-61) is that the A at position 255 of SEQ ID NO: 16 is replaced by G, which has the function of incorporating the blocking modified C.

[0105] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-62) is that the A at position 255 of SEQ ID NO: 16 is replaced by C, and it has the function of blocking the incorporation of the modified C.

[0106] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-63) is that the A at position 255 of SEQ ID NO: 16 is replaced by V, which has the function of incorporating the blocking modified C.

[0107] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-64) is that the A at position 255 of SEQ ID NO: 16 is replaced by I, and it has the function of incorporating the blocking modified C.

[0108] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-65) is that the A at position 255 of SEQ ID NO: 16 is replaced by L, which has the function of incorporating the blocking modified C.

[0109] In some embodiments, the amino acid sequence of the terminal transferase mutant (RP-66) is that the A at position 255 of SEQ ID NO: 16 is replaced by M, which has the function of incorporating the blocking modified C.

[0110] In some embodiments, the terminal transferase mutant (RP-67) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with R, which has incorporation function for a blocked modified C.

[0111] In some embodiments, the terminal transferase mutant (RP-68) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with Q, which has incorporation function for a blocked modified C.

[0112] In some embodiments, the terminal transferase mutant (RP-69) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with N, which has incorporation function for a blocked modified C.

[0113] In some embodiments, the terminal transferase mutant (RP-70) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with S, which has incorporation function for a blocked modified C.

[0114] In some embodiments, the terminal transferase mutant (RP-71) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with W, which has incorporation function for a blocked modified C.

[0115] In some embodiments, the terminal transferase mutant (RP-72) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with Y, which has incorporation function for a blocked modified C.

[0116] In some embodiments, the terminal transferase mutant (RP-73) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with F, which has incorporation function for a blocked modified C.

[0117] In some embodiments, the terminal transferase mutant (RP-74) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with H, which has incorporation function for a blocked modified C.

[0118] In some embodiments, the terminal transferase mutant (RP-75) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with P, which has incorporation function for a blocked modified C.

[0119] In some embodiments, the terminal transferase mutant (RP-76) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with E, which has incorporation function for a blocked modified C.

[0120] In some embodiments, the terminal transferase mutant (RP-77) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with T, which has incorporation function for a blocked modified C.

[0121] In some embodiments, the terminal transferase mutant (RP-78) has an amino acid sequence in which the A at position 255 of SEQ ID NO: 16 is replaced with D, which has incorporation function for a blocked modified C.

[0122] In some embodiments, the terminal transferase mutant (RP-79) has an amino acid sequence in which the D at position 257 of SEQ ID NO: 16 is replaced with M, which has incorporation function for a blocked modified A and U.

[0123] In some embodiments, the terminal transferase mutant (RP-80) has an amino acid sequence in which the K at position 261 of SEQ ID NO: 16 is replaced with Y, which has incorporation function for a blocked modified C.

[0124] In some embodiments, the terminal transferase mutant (RP-81) has an amino acid sequence in which the K at position 261 of SEQ ID NO: 16 is replaced with C, which has incorporation function for a blocked modified C.

[0125] In some embodiments, the terminal transferase mutant (RP-82) has an amino acid sequence in which the K at position 261 of SEQ ID NO: 16 is replaced with F, which has incorporation function for a blocked modified C.

[0126] In some embodiments, the terminal transferase mutant (RP-83) has an amino acid sequence in which the L at position 299 of SEQ ID NO: 16 is replaced with M, which has incorporation function for a blocked modified C.

[0127] In some embodiments, the terminal transferase mutant (RP-84) has an amino acid sequence in which the G at position 321 of SEQ ID NO: 16 is replaced with M, which has incorporation function for a blocked modified A.

[0128] In some embodiments, the terminal transferase mutant (RP-85) has an amino acid sequence in which the G at position 321 of SEQ ID NO: 16 is replaced with V, which has incorporation function for a blocked modified A and U.

[0129] In some embodiments, the group of the blocked modification comprises at least one of O-amino, O-allyl, O-azido, O-phospho group; preferably O-allyl.

[0130] In some embodiments, the terminal transferase mutant has higher catalytic activity (i.e., higher activity in catalyzing the addition of nucleotides (preferably nucleotides with modified 3'-OH ends) to the primer chain) and / or broader extension / incorporation ability (i.e., more types of nucleotides (preferably nucleotides with modified 3'-OH ends) can be incorporated into the primer chain) compared to the terminal transferase.

[0131] In some embodiments, the amino acid sequence of the terminal transferase mutant is:

[0132] a1) the amino acid sequence shown in SEQ ID NO: 27; or

[0133] a2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 27 and having the same function (e.g., having higher catalytic activity (i.e., the activity of catalyzing the addition of nucleotides (preferably nucleotides with modified 3'-OH termini) to a primer chain) and / or broader extension / incorporation capacity (i.e., the ability to incorporate a wider variety of nucleotides (preferably nucleotides with modified 3'-OH termini) into a primer chain) than the terminal transferase).

[0134] In some embodiments, the amino acid sequence of the terminal transferase mutant is:

[0135] b1) the amino acid sequence shown in SEQ ID NO: 20; or

[0136] b2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 20 and having the same function (e.g., having higher catalytic activity (i.e., the activity of catalyzing the addition of nucleotides (preferably nucleotides with modified 3'-OH termini) to a primer chain) and / or broader extension / incorporation capacity (i.e., the ability to incorporate a wider variety of nucleotides (preferably nucleotides with modified 3'-OH termini) into a primer chain) than the terminal transferase).

[0137] The second aspect of the present invention provides a recombinant protein comprising a modified portion and the terminal transferase or a mutant thereof according to the first aspect of the present invention.

[0138] In some embodiments, the modifying moiety is a protein tag.

[0139] In some embodiments, the protein tag is selected from at least one of Poly his (His), FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA; further selected from at least one of Poly his (His), FLAG, Strep-Tag II, Poly arg, C-myc, SUMO, GST, MBP, and NusA; further selected from Poly his (His) and SUMO.

[0140] In some embodiments, the modification moiety is located at the N-terminus and / or C-terminus of the terminal transferase or a mutant thereof.

[0141] In some embodiments, the modification moiety is located at the N-terminus of the terminal transferase or a mutant thereof.

[0142] In some embodiments, a connecting peptide (eg, a connecting peptide having an amino acid sequence of amino acids 1 to 13 of SEQ ID NO: 1) is further included between Poly his (His) and SUMO in the modified portion.

[0143] In some embodiments, a connecting peptide (such as a connecting peptide having an amino acid sequence of amino acids 111-112 of SEQ ID NO: 1) is further included between the SUMO in the modification portion and the terminal transferase or a mutant thereof.

[0144] In some embodiments, the amino acid sequence of the recombinant protein is:

[0145] c1) the amino acid sequence shown in SEQ ID NO: 15; or

[0146] c2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 15 and having the same function (e.g., having catalytic activity (i.e., the activity of catalyzing the addition of nucleotides (preferably nucleotides with modified 3'-OH termini, such as A and U) to a primer chain).

[0147] In some embodiments, the amino acid sequence of the recombinant protein is:

[0148] m1) the amino acid sequence shown in SEQ ID NO: 26; or

[0149] m2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 26 and has the same function (for example, compared to terminal transferase, it has higher catalytic activity (i.e., higher activity in catalyzing the addition of nucleotides (preferably nucleotides with modified 3'-OH termini) to the primer chain) and / or broader extension / incorporation capacity (i.e., it can incorporate more types of nucleotides (preferably nucleotides with modified 3'-OH termini) into the primer chain).

[0150] In some embodiments, the amino acid sequence of the recombinant protein is:

[0151] 11) the amino acid sequence shown in SEQ ID NO: 19; or

[0152] l2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:19 and having the same function (for example, compared to terminal transferase, having higher catalytic activity (i.e., higher activity in catalyzing the addition of nucleotides (preferably nucleotides with modified 3'-OH termini) to a primer chain) and / or broader extension / incorporation capacity (i.e., being able to incorporate a wider variety of nucleotides (preferably nucleotides with modified 3'-OH termini) into a primer chain).

[0153] The third aspect of the present invention provides a biomaterial related to the terminal transferase or a mutant thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention, wherein the biomaterial comprises any one of d1) to d12):

[0154] d1) a nucleic acid molecule encoding the terminal transferase or a mutant thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention;

[0155] d2) an expression cassette comprising the nucleic acid molecule described in d1);

[0156] d3) a vector comprising the nucleic acid molecule described in d1);

[0157] d4) a vector comprising the expression cassette described in d2);

[0158] d5) a transgenic cell line comprising the nucleic acid molecule described in d1);

[0159] d6) a transgenic cell line comprising the expression cassette described in d2);

[0160] d7) a transgenic cell line comprising the vector described in d3);

[0161] d8) a transgenic cell line comprising the vector of d4);

[0162] d9) a recombinant bacteria comprising the nucleic acid molecule of d1);

[0163] d10) a recombinant bacteria comprising the expression cassette of d2);

[0164] d11) a recombinant bacteria comprising the vector of d3);

[0165] d12) a recombinant bacteria comprising the vector of d4).

[0166] In some embodiments, the transgenic cell line does not comprise reproductive material.

[0167] In some embodiments, the vector can further comprise a promoter operably linked to the nucleic acid molecule.

[0168] In some embodiments, the vector in d3), d4) is independently selected from the group consisting of a non-pathogenic viral vector and a non-viral vector.

[0169] In some embodiments, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0170] In some embodiments, the non-viral vector comprises, but is not limited to, a plasmid vector.

[0171] In some embodiments, the recombinant bacteria in d9) to d12) comprises, but is not limited to, Escherichia coli.

[0172] In some embodiments, the nucleic acid molecule encoding the terminal transferase has a nucleotide sequence of:

[0173] e1) a nucleotide sequence as set forth in SEQ ID NO: 14; or

[0174] e2) a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence as set forth in SEQ ID NO: 14 and having the same function.

[0175] In some embodiments, the nucleic acid molecule encoding the terminal transferase mutant has a nucleotide sequence of:

[0176] f1) a nucleotide sequence as set forth in SEQ ID NO: 23; or

[0177] f2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 23 and has the same function.

[0178] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the terminal transferase mutant is:

[0179] n1) the nucleotide sequence shown in SEQ ID NO: 25; or

[0180] n2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 25 and has the same function.

[0181] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein is:

[0182] g1) the nucleotide sequence shown in SEQ ID NO: 13; or

[0183] g2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 13 and having the same function.

[0184] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein is:

[0185] h1) the nucleotide sequence shown in SEQ ID NO: 22; or

[0186] h2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 22 and having the same function.

[0187] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein is:

[0188] o1) the nucleotide sequence shown in SEQ ID NO: 24; or

[0189] o2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO: 24 and has the same function.

[0190] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0191] A fourth aspect of the present invention provides a terminal transferase-nucleotide complex comprising:

[0192] i1) at least one of the terminal transferase or a mutant thereof according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention; and

[0193] i2) Nucleotides.

[0194] In some embodiments, the nucleotide is connected to the terminal transferase or a mutant thereof according to the first aspect of the present invention via a linker; and / or, the nucleotide is connected to the recombinant protein according to the second aspect of the present invention via a linker.

[0195] In some embodiments, the nucleotides comprise at least one of natural deoxyribonucleotides, non-natural deoxyribonucleotides, natural ribonucleotides, and non-natural ribonucleotides.

[0196] In some embodiments, the nucleotides are dNTPs and / or NTPs.

[0197] A fifth aspect of the present invention provides the use described in any one of (g1) to (g4):

[0198] (g1) Use of the terminal transferase or a mutant thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, and / or the biomaterial according to the third aspect of the present invention in the preparation of a DNA polymerase (preferably a terminal transferase) and / or an RNA polymerase;

[0199] (g2) Use of the terminal transferase or a mutant thereof according to the first aspect of the present invention, and / or the recombinant protein according to the second aspect of the present invention as a DNA polymerase (preferably a terminal transferase) and / or an RNA polymerase;

[0200] (g3) Use of the terminal transferase or a mutant thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, and / or the terminal transferase-nucleotide complex according to the fourth aspect of the present invention in at least one of j1) to j5);

[0201] (g4) Use of the terminal transferase or a mutant thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the biomaterial according to the third aspect of the present invention, and / or the terminal transferase-nucleotide complex according to the fourth aspect of the present invention in j6);

[0202] j1) catalyzing single-base or multi-base extension of a nucleic acid molecule, preferably catalyzing single-base or multi-base extension of a nucleic acid molecule in the absence of a template strand;

[0203] j2) synthesizing nucleic acid molecules, preferably in the absence of a template strand;

[0204] j3) catalyze DNA or RNA replication;

[0205] j4) catalyzing DNA or RNA amplification;

[0206] j5) performing nucleic acid sequencing;

[0207] j6) preparing a product, wherein the product is used for at least one of j1) to j5).

[0208] In some embodiments, catalyzing single-base or multi-base extension of a nucleic acid molecule is performed in the absence of a template strand.

[0209] In some embodiments, synthesis of nucleic acid molecules is performed in the absence of a template strand.

[0210] In some embodiments, the product is a kit.

[0211] In some embodiments, the base comprises at least one of a natural base and an unnatural base.

[0212] In some embodiments, the nucleic acid molecule comprises at least one of DNA and RNA; and further comprises RNA.

[0213] In some embodiments, the DNA comprises natural deoxynucleotides and / or non-natural deoxynucleotides.

[0214] In some embodiments, the RNA comprises natural nucleotides and / or non-natural nucleotides.

[0215] In some embodiments, the nucleotides used to synthesize the nucleic acid molecules are nucleotides with modified 3'-OH termini.

[0216] In some embodiments, the nucleotide with a modified 3'-OH end is obtained by adding a blocking group to the 3'-OH end of the nucleotide.

[0217] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phospho group; preferably O-allyl.

[0218] In some embodiments, the 5' end of the nucleotide used for synthesizing the nucleic acid molecule contains 2 phosphate groups or 3 phosphate groups.

[0219] In some embodiments, the sequencing is SBS sequencing.

[0220] In a sixth aspect of the present application, there is provided a kit of any one of (h1) to (h2):

[0221] (h1) A kit comprising at least one of the terminal transferase or mutant thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application;

[0222] (h2) A kit comprising the terminal transferase-nucleotide complex of the fourth aspect of the present application.

[0223] In some embodiments, the kit of (h1) further comprises at least one of nucleotide, cobalt ion, NaCl, buffer.

[0224] In some embodiments, the nucleotide comprises at least one of natural deoxyribonucleotide, unnatural deoxyribonucleotide, natural ribonucleotide, unnatural ribonucleotide.

[0225] In some embodiments, the nucleotide is dNTP and / or NTP.

[0226] In some embodiments, the nucleotide is a 3'-OH end modified nucleotide. In some embodiments, the 3'-OH end modified nucleotide is modified by adding a blocking group to the 3'-OH end of the nucleotide.

[0227] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phospho group; preferably O-allyl.

[0228] In some embodiments, the 5' end of the nucleotide contains 2 phosphate groups or 3 phosphate groups.

[0229] In some embodiments, the kit of (h2) further comprises at least one of cobalt ion, buffer, NaCl.

[0230] In some embodiments, the kit of (h2) further comprises a substance for removing the linker.

[0231] In some embodiments, the buffer in (h1) and (h2) comprises at least one of phosphate buffer, borate buffer, citrate buffer, Tris buffer, and Hepes buffer; further comprises at least one of phosphate buffer and Tris buffer; and further comprises Tris buffer.

[0232] In some embodiments, the kit is used for at least one of j1) to j5).

[0233] The seventh aspect of the present invention provides a method for synthesizing nucleic acid molecules, comprising the steps of using the terminal transferase or a mutant thereof of the first aspect of the present invention, the recombinant protein of the second aspect of the present invention, the terminal transferase-nucleotide complex of the fourth aspect of the present invention, and / or the kit of the sixth aspect of the present invention.

[0234] In some embodiments, the synthesis of nucleic acid molecules is performed in the absence of a template strand.

[0235] In some embodiments, the method is k1) or k2):

[0236] k1) The method comprises the following steps: mixing the terminal transferase or a mutant thereof according to the first aspect of the present invention, and / or the recombinant protein according to the second aspect of the present invention, with a primer chain and nucleotides, and reacting the resulting system;

[0237] k2) The method comprises the following steps: mixing the terminal transferase-nucleotide complex of the fourth aspect of the present invention with a primer chain, and reacting the resulting system.

[0238] In some embodiments, the nucleotide described in k1) is the nucleotide in the sixth aspect of the present invention.

[0239] In some embodiments, the system described in k1) and k2) further comprises cobalt ions, a buffer, and NaCl.

[0240] In some embodiments, the buffer is the buffer in the sixth aspect of the present invention.

[0241] In some embodiments, the reaction temperature is 25-70°C; preferably, the reaction temperature is 25-40°C.

[0242] The eighth aspect of the present invention provides a method for preparing the terminal transferase or its mutant according to the first aspect of the present invention, and / or the recombinant protein according to the second aspect of the present invention, which is obtained by culturing the transgenic cell line and / or recombinant bacteria according to the third aspect of the present invention.

[0243] The beneficial effects of the present invention are:

[0244] The present invention provides terminal transferases or mutants thereof; wherein, both the terminal transferases and the terminal transferase mutants have catalytic activity for synthesizing nucleic acid molecules independently of a template, thereby reducing the overall cost of synthesizing custom nucleic acids. Even when modified nucleotides are used, nucleic acid synthesis can be achieved faster, cheaper, and with higher quality. In particular, most terminal transferase mutants have higher catalytic activity (i.e., activity in catalyzing the addition of nucleotides (preferably 3'-O-allyl-modified nucleotides, preferably RNA) to a primer chain) and / or broader extension / incorporation capacity (i.e., the ability to incorporate a wider variety of nucleotides (preferably 3'-O-allyl-modified nucleotides, preferably RNA) into a primer chain) than terminal transferases. BRIEF DESCRIPTION OF THE DRAWINGS

[0245] Figure 1 This is a diagram showing the location structure of the terminal transferase mutation site.

[0246] Figure 2 This is the plasmid map of pET28a-SUMO-terminal transferase.

[0247] Figure 3 The figure shows the SDS-PAGE results of the purified terminal transferase (RP-02) and mutant (RP-50) recombinant proteins.

[0248] Figure 4 The Qsep400 peak graph shows a portion of a crude enzyme single base extension functional test of a terminal transferase recombinant protein (RP02) comprising terminal transferase (RP01) or its mutants (RP-1 to RP-85) and terminal transferase mutant recombinant proteins (RP-1-SUMO to RP-85-SUMO).

[0249] Figure 5 This is a graph showing the UREA-PAGE results of the pure enzyme single-base extension activity of the terminal transferase mutant (RP-50) recombinant protein (RP-50-SUMO). DETAILED DESCRIPTION

[0250] In this application, unless otherwise indicated, the term "amino acid" is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0251] In this application, unless otherwise specified, nucleotide sequences are displayed from 5' end to 3' end.

[0252] In this application, unless otherwise specified, amino acid sequences are displayed in the N-terminus → C-terminus format.

[0253] In this application, unless otherwise specified, non-natural dNTPs or modified dNTPs have the same meaning, including but not limited to dNTPs with labels (eg, fluorescent labels) and / or dNTPs with an O-reversible terminator at the 3' end.

[0254] In this application, unless otherwise specified, non-natural NTP or modified NTP have the same meaning, including but not limited to NTP with a label (eg, fluorescent label) and / or NTP with an O-reversible terminator at the 3' end.

[0255] In this application, unless otherwise indicated, "identity" has the conventional meaning in the art and refers to the "homology" between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared after best alignment, and the differences between the two sequences are randomly distributed over their entire length.

[0256] By studying the use of terminal transferase for de novo nucleic acid synthesis without template control sequences, the inventors discovered that certain amino acid residues within the catalytic domain of terminal transferase can be specifically modified to enhance the ability of the modified terminal transferase to synthesize nucleic acids. More specifically, the inventors have developed improved terminal transferases with specific amino acid substitutions. These substitutions reduce the overall cost of synthesizing custom nucleic acids, enabling faster, cheaper, and higher-quality nucleic acid synthesis, even when using modified nucleotides.

[0257] The present invention is further described in detail below through specific examples.

[0258] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0259] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0260] Example 1 Plasmid Construction and Expression Purification of Terminal Deoxynucleotidyl Transferase (TdT) and TdT Mutants

[0261] In this example, the terminal transferase mutant was constructed using the Mut Express II Fast Mutagenesis Kit V2 (Norvozymes, Cat. No. C214) for mutant expression vector construction, and the His tag was used for Ni column affinity purification. The terminal transferase and terminal transferase mutant in this example were each added with a SUMO tag containing a linker peptide at the N-terminus ( SSGLVPRGSHMAS MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQ GKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG GS, SEQ ID NO: 1, wherein the single underlined portion (amino acids 1-13 of SEQ ID NO: 1) is the amino acid sequence of the connecting peptide, the double underlined portion (amino acids 111-112 of SEQ ID NO: 1) is the amino acid sequence of the connecting peptide, and the wavy portion (amino acids 14-110 of SEQ ID NO: 1) is the amino acid sequence of the SUMO tag. The application of SUMO (Small Ubiquitin-like Modifier) ​​tag is mainly based on its unique properties, which can significantly improve the expression level, solubility and purification efficiency of the target protein. It is a conventional means of protein expression and purification and has no special enzymatic function) and His tag (HHHHHH, SEQ ID NO:2) to form a terminal transferase recombinant protein and a terminal transferase mutant recombinant protein to promote the soluble expression of the terminal transferase / terminal transferase mutant and facilitate the purification of the terminal transferase / terminal transferase mutant; those skilled in the art are well aware that the addition or absence of a tag has no effect on the performance of the terminal transferase / terminal transferase mutant (the functions of the terminal transferase recombinant protein and the terminal transferase mutant recombinant protein (such as thermal stability, catalytic activity, extension / incorporation ability) are brought by the terminal transferase or the terminal transferase mutant itself and are not related to the tag), and the tag can be added to the C-terminus and / or N-terminus of the terminal transferase / terminal transferase mutant, and the tag can also be another His tag (Poly his, such as: HH, HHH, HHHH (SEQ ID NO: 3), HHHHH (SEQ ID NO: 4), HHHHHHH (SEQ ID NO: 5), HHHHHHHH (SEQ ID NO: 6), HHHHHHHHH (SEQ ID NO: 7), HHHHHHHHH (SEQ ID NO: 8), HHHHHHHHH (SEQ ID NO: 9), HHHHHHHHH (SEQ ID NO: 10), HHHHHHHHH (SEQ ID NO: 11), HHHHHHHHH (SEQ ID NO: 12), HHHHHHHHH (SEQ ID NO: 13), HHHHHHHHH (SEQ ID NO: 14), HHHHHHHHH (SEQ ID NO: 15), HHHHHHHHH (SEQ ID NO: 16), HHHHHHHHH (SEQ ID NO: 17), HHHHHHHHHH (SEQ ID NO: 18), HHHHHHHHH (SEQ ID NO: 19), HHHHHHHHH NO:8)), FLAG (DYKDDDDK, SEQ ID NO:9), Strep-Tag II (WSHPQFEK, SEQ ID NO:10), Poly arg (for example: RRRRR, SEQ ID NO:11), C-myc (EQKLISEEDL, SEQ ID NO:12), GST, MBP, NusA.

[0262] 1. Preparation of Terminal Transferase Recombinant Protein

[0263] 1. Construction of expression vector for terminal transferase recombinant protein (terminal transferase is referred to as RP01 and terminal transferase recombinant protein is referred to as RP02)

[0264] The recombinant expression vector (pET28a-sumo-terminal transferase) is a gene encoding a terminal transferase fused with a His tag and a SUMO tag (the nucleotide sequence of the gene encoding the terminal transferase fused with a His tag and a SUMO tag is a nucleotide sequence of a gene encoding a terminal transferase fused with a His tag (six histidines (His)) and a SUMO tag connected to the 5' end of the nucleotide sequence of the terminal transferase encoding gene, wherein the nucleotide sequence of the gene encoding the terminal transferase fused with a His tag and a SUMO tag is shown in SEQ ID NO: 13; the nucleotide sequence of the gene encoding the terminal transferase fused with a His tag and a SUMO tag is shown in SEQ ID NO: 14; the amino acid sequence of the terminal transferase fused with a His tag and a SUMO tag (terminal transferase recombinant protein RP 02) is a His tag (six histidines (His)) and a SUMO tag connected to the N-terminus of the terminal transferase, and the amino acid sequence of the terminal transferase fused with a His tag and a SUMO tag (terminal transferase recombinant protein) is shown in SEQ ID NO: 15; wherein the amino acid sequence of the terminal transferase is shown in SEQ ID NO: 16, according to Seamless Cloning. The kit instructions and operation steps were used to recombinant the protein into the pET28a vector (with the insertion sites of BamHI and XhoI). The resulting recombinant expression vector (pET28a-SUMO-terminal transferase) was a vector for expressing the terminal transferase recombinant protein RP02 (see the schematic diagram). Figure 2 shown).

[0265] 2. Construction of recombinant bacteria

[0266] The recombinant expression vector (pET28a-SUMO-terminal transferase) constructed above was introduced into Escherichia coli BL21 competent cells (BL21 (DE 3), Beijing Solaibao Technology Co., Ltd.), plated onto LB solid medium containing 50 μg / mL kanamycin (Kana), and incubated in a 37°C constant temperature incubator for 12 hours to screen for positive monoclonal colonies. Three to five positive monoclonal colonies were selected and identified by colony PCR using primers T7 (sequence: TAATACGACTCACTATAGGG (5'-3'), SEQ ID NO: 17) and T7-Ter (sequence: GCTAGTTATTGCTCA GCGG (5'-3'), SEQ ID NO: 18). Colonies that produced a fragment that was substantially consistent with the expected theoretical size were considered positive clones and were designated BL21 / pET28a-SUMO-terminal transferase (BL21 / pET28a-SUMO-Wild).

[0267] 3. Expression of Terminal Transferase Recombinant Protein RP02 and Preparation of Crude Extract

[0268] A single BL21 / pET28a-sumo-terminal transferase (BL21 / pET28a-sumo-Wild) colony was selected and cultured overnight in 150 μL LB liquid medium (containing 50 μg / mL kanamycin) at 37°C, 200 rpm / min. The next day, the colony was diluted 1:100 and transferred to 150 μL LB liquid medium (containing 50 μg / mL kanamycin). The culture was shaken at 37°C, 220 rpm / min to an OD600 of 0.6-0.8. IPTG was added to a final concentration of 0.5 mM and the culture was induced overnight at 16°C. The induced BL21 / pET28a-sumo-Wild culture was collected.

[0269] Add 1.5 μL of BL21 / pET28a-sumo-Wild bacterial solution after the above induction 10X Protein Extraction Reagent (Brand: Merck Millipore Catalog number: 70921-3), mix well, and incubate at room temperature for 20 minutes to lyse the bacteria to obtain a crude recombinant protein extract. This crude recombinant protein extract can be used for subsequent protein activity assays.

[0270] Expression and purification of recombinant terminal transferase protein RP02

[0271] A single BL21 / pET28a-sumo-Wild colony was picked and cultured in 10 mL of LB liquid medium (containing 50 μg / mL of kanamycin) at 37°C and 220 rpm overnight. The next day, the colony was diluted 1:100 and transferred to 1 L of LB liquid medium (containing 50 μg / mL of kanamycin). The colony was shaken and cultured at 37°C and 220 rpm until the OD600 was 0.8-1.0. IPTG was added at a final concentration of 0.5 mM and the culture was induced at 16°C overnight.

[0272] The induced BL21 / pET28a-sumo-Wild cells were collected by centrifugation at 6000 rpm for 10 min. Buffer C (50 mM Tris-HCl, 100 mM NaCl, pH 8) was added to the centrifuged cells and resuspended. The cells were disrupted using a high-pressure, low-temperature disruptor at 800 bar and 4°C. After disruption, the cells were centrifuged at 14000 rpm for 1 h, and the supernatant was filtered through a 0.22 μm filter.

[0273] Next, a 5mL nickel gravity column was used for protein purification. Before the supernatant was loaded onto the column, 10 column volumes of buffer A (50mM Tris-HCl, 100mM NaCl, pH 8) were used to balance the Ni column. The supernatant was slowly passed through the Ni column at a flow rate of 0.8mL / min. The supernatant flow-through was collected at the same time. Buffer A was then used to wash the Ni column 4 times, with 4 column volumes each time. Buffer B containing a high concentration of imidazole (50mM Tris-HCl, 100mM NaCl, 0.25M Imidazole, pH 8) was then used to wash the Ni column 4 times, with 1 column volume each time to elute the target protein. Protein expression and purification results were detected using 12% SDS-PAGE. The results are shown in Figure 4. Figure 3 Shown: M is a protein marker (PageRuler Prestained Protein Ladder, 26616, Thermo Fisher Scientific), the box is the eluted target protein, and its molecular weight is consistent with the expected size of 56.7 kDa.

[0274] The collected target protein was desalted and concentrated by centrifugal ultrafiltration at low temperature (4°C). The ultrafiltered protein sample was added with glycerol to a final concentration of 20% and then aliquoted and stored at -80°C.

[0275] 2. Preparation of Terminal Transferase Mutant Recombinant Protein

[0276] 1. Preparation of recombinant proteins of terminal transferase mutants (RP-21, RP-50)

[0277] (1) Construction of terminal transferase mutant recombinant protein expression vector

[0278] The vector expressing the terminal transferase recombinant protein (i.e., the above-mentioned recombinant expression vector (pET28a-SUMO-terminal transferase)) was used as a template and the Mut Express II Fast Mutagenesis Kit was used. The V2 kit manual operating steps were used to perform site-directed mutagenesis to obtain terminal transferase mutant (RP-21, RP-50) recombinant protein expression vectors (i.e., pET28a into which the terminal transferase mutant (RP-21, RP-50) encoding gene fused with a His tag (6 histidines His) and a SUMO tag was inserted, with the insertion sites NdeI and XhoI: wherein the terminal transferase mutant (RP-21, RP-50) encoding gene fused with a His tag (6 histidines His) and a SUMO tag was connected to the nucleotide sequence of the terminal transferase mutant (RP-21, RP-50) encoding gene at the 5' end of the nucleotide sequence of the terminal transferase mutant (RP-21, RP-50) encoding gene, wherein the nucleotide sequence of the terminal transferase mutant (RP-21) encoding gene fused with a His tag (6 histidines His) and a SUMO tag was as shown in SEQ ID NO: 24; the nucleotide sequence of the terminal transferase mutant (RP-21) encoding gene was as shown in SEQ ID NO: ID NO: 25; the nucleotide sequence of the gene encoding the terminal transferase mutant (RP-50) fused with a His tag (6 histidines His) and a SUMO tag is shown in SEQ ID NO: 22; the nucleotide sequence of the gene encoding the terminal transferase mutant (RP-50) is shown in SEQ ID NO: 23; the amino acid sequence of the terminal transferase mutant (RP-21) fused with a His tag (6 histidines His) and a SUMO tag (terminal transferase mutant RP-21 recombinant protein) is the terminal transferase mutant RP-21 with a His tag (6 histidines His) and a SUMO tag connected to its N-terminus, and the amino acid sequence of the terminal transferase mutant RP-21 recombinant protein is shown in SEQ ID NO: 26: wherein the amino acid sequence of the terminal transferase mutant RP-21 is shown in SEQ ID NO: 27, that is, SEQ ID NO:16, G at position 192 is replaced by S); the amino acid sequence of the terminal transferase mutant (RP-50) fused with a His tag (6 histidines His) and a SUMO tag (terminal transferase mutant RP-50 recombinant protein) is the terminal transferase mutant RP-50 with a His tag (6 histidines His) and a SUMO tag connected to the N-terminus, and the amino acid sequence of the terminal transferase mutant RP-50 recombinant protein is shown in SEQ ID NO:19: wherein the amino acid sequence of the terminal transferase mutant RP-50 is shown in SEQ ID NO:20, i.e., D at position 237 of SEQ ID NO:16 is replaced by Q).

[0279] (2) Construction of recombinant bacteria

[0280] The recombinant bacteria expressing the mutant terminal transferase (RP-21, RP-50) recombinant protein were obtained by introducing the mutant terminal transferase (RP-21, RP-50) recombinant protein expression vector prepared in the above (1) into the BL21 competent cells (BL21 (DE3), Beijing Solabio Technology Co., Ltd.) in the same manner as the method of "2, Construction of recombinant bacteria" in "I, Preparation of terminal transferase recombinant protein" above.

[0281] (3) Expression of mutant terminal transferase (RP-21, RP-50) recombinant protein and preparation of crude extract

[0282] The recombinant bacteria (BL21 / pET28a-Mut-RP-21, BL21 / pET28a-Mut-RP-50) prepared in the above (2) were expressed and lysed in the same manner as the method of "3, Expression of terminal transferase recombinant protein RP02 and preparation of crude extract" in "I, Preparation of terminal transferase recombinant protein" above, and the obtained recombinant protein crude extract can be used for subsequent protein activity determination.

[0283] (4) Expression and purification of mutant terminal transferase (RP-50) recombinant protein

[0284] A single colony of BL21 / pET28a-Mut-RP-50 was picked and inoculated in 10 mL of LB liquid medium (containing kanamycin 50 μg / mL) and incubated at 37°C, 220 rpm overnight. The next day, the culture was diluted at a dilution of 1:100 and inoculated in 1 L of LB liquid medium (containing kanamycin 50 μg / mL) and incubated at 37°C, 220 rpm until the OD600 was 0.8-1.0. IPTG was added at a final concentration of 0.5 mM, and the culture was induced at 16°C overnight.

[0285] The BL21 / pET28a-Mut-RP-50 bacteria after induction were collected by centrifugation at a speed of 6000 rpm for 10 min. The bacteria were resuspended in buffer C (50 mM Tris-HCl, 100 mM NaCl, pH 8) after centrifugation. The bacteria were crushed using a high-pressure low-temperature crusher at a pressure of 800 bar and a temperature of 4°C. The crushed bacteria were centrifuged at a speed of 14000 rpm for 1 h, and the supernatant was filtered using a 0.22 μm filter membrane.

[0286] Next, protein purification was performed using a 5 mL nickel gravity column. The Ni column was equilibrated with 10 column volumes of buffer A (50 mM Tris-HCl, 100 mM NaCl, pH 8) before loading the supernatant. The supernatant was slowly passed through the Ni column at a flow rate of 0.8 mL / min while collecting the flow-through. The Ni column was then washed with buffer A four times at 4 column volumes each. The target protein was eluted from the Ni column using buffer B (50 mM Tris-HCl, 100 mM NaCl, 0.25 M imidazole, pH 8) four times at 1 column volume each. Protein expression and purification results were detected using 12% SDS-PAGE, as shown in FIG. 1. M is the protein marker (PageRuler Prestained Protein Ladder, 26616, Thermo Fisher Scientific), and the target protein eluted is boxed. The molecular weight of the target protein is consistent with the expected size of 56.7 kDa. Figure 3

[0287] The collected target protein was desalted and concentrated by centrifugal ultrafiltration at low temperature (4°C). The ultrafiltrated protein sample was then added with glycerol at a final concentration of 20% and aliquoted, and stored at -80°C.

[0288] 2. Preparation of recombinant proteins of terminal transferase mutants (RP-1 to RP-20, RP-22 to RP-49, RP-51 to RP-85)

[0289] ​The method is the same as the above-described method of "II. Preparation of a terminal transferase mutant recombinant protein", except that the amino acid sequence and the nucleotide sequence of the terminal transferase mutant (RP-50) are replaced with the amino acid sequence and the nucleotide sequence of the terminal transferase mutant (RP-1 to RP-20, RP-22 to RP-49, RP-51 to RP-85), respectively, wherein the amino acid sequence of the terminal transferase mutant (RP-1) is that V at position 120 of SEQ ID NO: 16 is replaced with M; the amino acid sequence of the terminal transferase mutant (RP-2) is that C at position 162 of SEQ ID NO: 16 is replaced with K; the amino acid sequence of the terminal transferase mutant (RP-3) is that C at position 162 of SEQ ID NO: 16 is replaced with G; the amino acid sequence of the terminal transferase mutant (RP-4) is that C at position 162 of SEQ ID NO: 16 is replaced with A; the amino acid sequence of the terminal transferase mutant (RP-5) is that C at position 162 of SEQ ID NO: 16 is replaced with T; the amino acid sequence of the terminal transferase mutant (RP-6) is that C at position 162 of SEQ ID NO: 16 is replaced with V; the amino acid sequence of the terminal transferase mutant (RP-7) is that C at position 162 of SEQ ID NO: 16 is replaced with I; the amino acid sequence of the terminal transferase mutant (RP-8) is that C at position 162 of SEQ ID NO: 16 is replaced with L; the amino acid sequence of the terminal transferase mutant (RP-9) is that C at position 162 of SEQ ID NO: 16 is replaced with M; the amino acid sequence of the terminal transferase mutant (RP-10) is that C at position 162 of SEQ ID NO: 16 is replaced with R; the amino acid sequence of the terminal transferase mutant (RP-11) is that C at position 162 of SEQ ID NO: 16 is replaced with Q; the amino acid sequence of the terminal transferase mutant (RP-12) is that C at position 162 of SEQ ID NO: 16 is replaced with N; the amino acid sequence of the terminal transferase mutant (RP-13) is that C at position 162 of SEQ ID NO: 16 is replaced with S; the amino acid sequence of the terminal transferase mutant (RP-14) is that C at position 162 of SEQ ID NO: 16 is replaced with W; the amino acid sequence of the terminal transferase mutant (RP-15) is that C at position 162 of SEQ ID NO: 16 is replaced with Y; the amino acid sequence of the terminal transferase mutant (RP-16) is that C at position 162 of SEQ ID NO: 16 is replaced with F; the amino acid sequence of the terminal transferase mutant (RP-17) is that C at position 162 of SEQ ID NO: 16 is replaced with H; the amino acid sequence of the terminal transferase mutant (RP-18) is that C at position 162 of SEQ ID NO: 16 is replaced with P;The amino acid sequence of the terminal transferase mutant (RP-19) is that the C at position 162 of SEQ ID NO: 16 is replaced by E; the amino acid sequence of the terminal transferase mutant (RP-20) is that the C at position 162 of SEQ ID NO: 16 is replaced by D; the amino acid sequence of the terminal transferase mutant (RP-22) is that the H at position 202 of SEQ ID NO: 16 is replaced by K; the amino acid sequence of the terminal transferase mutant (RP-23) is that the H at position 202 of SEQ ID NO: 16 is replaced by G; the amino acid sequence of the terminal transferase mutant (RP-24) is that the H at position 202 of SEQ ID NO: 16 is replaced by A; the amino acid sequence of the terminal transferase mutant (RP-25) is that the H at position 202 of SEQ ID NO: 16 is replaced by T; the amino acid sequence of the terminal transferase mutant (RP-26) is that the H at position 202 of SEQ ID NO: The amino acid sequence of the terminal transferase mutant (RP-27) is that the H at position 202 of SEQ ID NO: 16 is substituted with V; the amino acid sequence of the terminal transferase mutant (RP-28) is that the H at position 202 of SEQ ID NO: 16 is substituted with L; the amino acid sequence of the terminal transferase mutant (RP-29) is that the H at position 202 of SEQ ID NO: 16 is substituted with M; the amino acid sequence of the terminal transferase mutant (RP-30) is that the H at position 202 of SEQ ID NO: 16 is substituted with R; the amino acid sequence of the terminal transferase mutant (RP-31) is that the H at position 202 of SEQ ID NO: 16 is substituted with Q; the amino acid sequence of the terminal transferase mutant (RP-32) is that the H at position 202 of SEQ ID NO: 16 is substituted with N; the amino acid sequence of the terminal transferase mutant (RP-33) is that the H at position 202 of SEQ ID NO: 16 is substituted with The amino acid sequence of the terminal transferase mutant (RP-34) is that the H at position 202 of SEQ ID NO: 16 is substituted with S; the amino acid sequence of the terminal transferase mutant (RP-35) is that the H at position 202 of SEQ ID NO: 16 is substituted with Y; the amino acid sequence of the terminal transferase mutant (RP-36) is that the H at position 202 of SEQ ID NO: 16 is substituted with F; the amino acid sequence of the terminal transferase mutant (RP-37) is that the H at position 202 of SEQ ID NO: 16 is substituted with C; the amino acid sequence of the terminal transferase mutant (RP-38) is that the H at position 202 of SEQ ID NO: 16 is substituted with P; the amino acid sequence of the terminal transferase mutant (RP-39) is that the H at position 202 of SEQ ID NO: 16 is substituted with E;The amino acid sequence of the terminal transferase mutant (RP-40) is a substitution of H at position 202 of SEQ ID NO: 16 with D; the amino acid sequence of the terminal transferase mutant (RP-41) is a substitution of D at position 237 of SEQ ID NO: 16 with K; the amino acid sequence of the terminal transferase mutant (RP-42) is a substitution of D at position 237 of SEQ ID NO: 16 with G; the amino acid sequence of the terminal transferase mutant (RP-43) is a substitution of D at position 237 of SEQ ID NO: 16 with A; the amino acid sequence of the terminal transferase mutant (RP-44) is a substitution of D at position 237 of SEQ ID NO: 16 with T; the amino acid sequence of the terminal transferase mutant (RP-45) is a substitution of D at position 237 of SEQ ID NO: 16 with V; the amino acid sequence of the terminal transferase mutant (RP-46) is a substitution of D at position 237 of SEQ ID NO: 16 with I; the amino acid sequence of the terminal transferase mutant (RP-47) is a substitution of D at position 237 of SEQ ID NO: 16 with L; the amino acid sequence of the terminal transferase mutant (RP-48) is a substitution of D at position 237 of SEQ ID NO: 16 with M; the amino acid sequence of the terminal transferase mutant (RP-49) is a substitution of D at position 237 of SEQ ID NO: 16 with R; the amino acid sequence of the terminal transferase mutant (RP-51) is a substitution of D at position 237 of SEQ ID NO: 16 with N; the amino acid sequence of the terminal transferase mutant (RP-52) is a substitution of D at position 237 of SEQ ID NO: 16 with S; the amino acid sequence of the terminal transferase mutant (RP-53) is a substitution of D at position 237 of SEQ ID NO: 16 with W; the amino acid sequence of the terminal transferase mutant (RP-54) is a substitution of D at position 237 of SEQ ID NO: 16 with Y; the amino acid sequence of the terminal transferase mutant (RP-55) is a substitution of D at position 237 of SEQ ID NO: 16 with F; the amino acid sequence of the terminal transferase mutant (RP-56) is a substitution of D at position 237 of SEQ ID NO: 16 with H; the amino acid sequence of the terminal transferase mutant (RP-57) is a substitution of D at position 237 of SEQ ID NO: 16 with P; the amino acid sequence of the terminal transferase mutant (RP-58) is a substitution of D at position 237 of SEQ ID NO: 16 with E; the amino acid sequence of the terminal transferase mutant (RP-59) is a substitution of D at position 237 of SEQ ID NO: 16 with C; the amino acid sequence of the terminal transferase mutant (RP-60) is a substitution of A at position 255 of SEQ ID NO: 16 with K;The amino acid sequence of the terminal transferase mutant (RP-61) is that the A at position 255 of SEQ ID NO: 16 is replaced by G; the amino acid sequence of the terminal transferase mutant (RP-62) is that the A at position 255 of SEQ ID NO: 16 is replaced by C; the amino acid sequence of the terminal transferase mutant (RP-63) is that the A at position 255 of SEQ ID NO: 16 is replaced by V; the amino acid sequence of the terminal transferase mutant (RP-64) is that the A at position 255 of SEQ ID NO: 16 is replaced by I; the amino acid sequence of the terminal transferase mutant (RP-65) is that the A at position 255 of SEQ ID NO: 16 is replaced by L; the amino acid sequence of the terminal transferase mutant (RP-66) is that the A at position 255 of SEQ ID NO: 16 is replaced by M; the amino acid sequence of the terminal transferase mutant (RP-67) is that the A at position 255 of SEQ ID NO: The amino acid sequence of the terminal transferase mutant (RP-68) is that the A at position 255 of SEQ ID NO: 16 is substituted with R; the amino acid sequence of the terminal transferase mutant (RP-69) is that the A at position 255 of SEQ ID NO: 16 is substituted with N; the amino acid sequence of the terminal transferase mutant (RP-70) is that the A at position 255 of SEQ ID NO: 16 is substituted with S; the amino acid sequence of the terminal transferase mutant (RP-71) is that the A at position 255 of SEQ ID NO: 16 is substituted with W; the amino acid sequence of the terminal transferase mutant (RP-72) is that the A at position 255 of SEQ ID NO: 16 is substituted with Y; the amino acid sequence of the terminal transferase mutant (RP-73) is that the A at position 255 of SEQ ID NO: 16 is substituted with F; the amino acid sequence of the terminal transferase mutant (RP-74) is that the A at position 255 of SEQ ID NO: The amino acid sequence of the terminal transferase mutant (RP-75) is that the A at position 255 of SEQ ID NO: 16 is substituted with H; the amino acid sequence of the terminal transferase mutant (RP-76) is that the A at position 255 of SEQ ID NO: 16 is substituted with E; the amino acid sequence of the terminal transferase mutant (RP-77) is that the A at position 255 of SEQ ID NO: 16 is substituted with T; the amino acid sequence of the terminal transferase mutant (RP-78) is that the A at position 255 of SEQ ID NO: 16 is substituted with D; the amino acid sequence of the terminal transferase mutant (RP-79) is that the D at position 257 of SEQ ID NO: 16 is substituted with M; the amino acid sequence of the terminal transferase mutant (RP-80) is that the K at position 261 of SEQ ID NO: 16 is substituted with Y;The amino acid sequence of the terminal transferase mutant (RP-81) is that K at position 261 of SEQ ID NO: 16 is replaced by C; the amino acid sequence of the terminal transferase mutant (RP-82) is that K at position 261 of SEQ ID NO: 16 is replaced by F; the amino acid sequence of the terminal transferase mutant (RP-83) is that L at position 299 of SEQ ID NO: 16 is replaced by M; the amino acid sequence of the terminal transferase mutant (RP-84) is that G at position 321 of SEQ ID NO: 16 is replaced by M; and the amino acid sequence of the terminal transferase mutant (RP-85) is that G at position 321 of SEQ ID NO: 16 is replaced by V.

[0290] Example 2 Single-step RNA synthesis function detection of terminal transferase and its mutants

[0291] In this example, first, the single-step RNA synthesis function of the terminal transferase recombinant protein (RP02) containing the terminal transferase (RP01) or its mutants (RP-1 to RP-85) obtained in Example 1 or the terminal transferase mutant recombinant protein was detected using the Qsep400 nucleic acid protein analysis system (Qsep400, QIAgencies). The single-base incorporation function of allyl-modified ribonucleotides 3'-O-Allyl-NTPs (NTP refers to any one of ribonucleotides A, U, C, and G) was detected. Subsequently, the terminal transferase mutant recombinant protein (RP-50-SUMO) containing the superior mutant RP-50 was selected for UREA-PAGE detection of the single-base incorporation function of the pure enzyme, and the function was compared with the control (terminal transferase recombinant protein RP02 containing the terminal transferase). The specific experimental method is as follows:

[0292] This example verifies the function of the terminal transferase recombinant protein (RP02) or the terminal transferase mutant recombinant protein containing terminal transferase (RP01) or its mutants (RP-1 to RP-85) obtained in Example 1 to incorporate single 3'-O-Allyl-NTPs (NTP refers to any one of the ribonucleotides A, U, C, and G). The reaction system includes 1 μM primer (primer sequence: CGCTTGCACAGGTGCGTTGG (5'-3',), SEQ ID NO: 21), 250 μM 3'-O-Allyl-NTPs (3'-O-Allyl-ATP or 3'-O-Allyl-UTP or 3'-O-Allyl-CTP or 3'-O-Allyl-GTP, which can be abbreviated as A or U or C or G), 0.25 mM CoCl2, 100 mM NaCl and 20 mM Tris buffer (pH 7.9) (the concentration of each component is the final concentration, and the total volume of the reaction system is 10 μL), 3 μL crude enzyme solution (crude extract of the recombinant protein (terminal transferase or terminal transferase mutant) obtained in Example 1), the reaction is carried out at 30°C for 30 minutes, the reaction is quenched by incubation at 95°C for 2 minutes, 5 μL ddH2O is added to each tube of reaction solution, and the reaction is analyzed using Qsep400.

[0293] In this example, the terminal transferase recombinant protein (RP02) comprising terminal transferase (RP01) or its mutants (RP-1 to RP-85) and the terminal transferase mutant recombinant protein were assayed using Qsep400 to determine the function of blocking the incorporation of modified A, U, C, or G. The results are shown in Tables 1 and Figure 4 As shown: Figure 4In the figure, the main peak is the primer n without incorporation of a single base, and the small peak immediately to the right of the main peak (if any, the extended base is marked in the figure) is the primer n+1 with incorporation of a single base; the terminal transferase recombinant protein (RP02) containing terminal transferase (RP01) has the incorporation function (extension function) for A and U; the terminal transferase mutant recombinant protein containing terminal transferase mutants (RP-1 to RP-20, RP-79, RP-85) has the incorporation function (extension function) for A and U; the terminal transferase mutant recombinant protein containing terminal transferase mutants (RP-42, RP-44) has the incorporation function (extension function) for C and U; the terminal transferase mutant recombinant protein containing terminal transferase mutants (RP-1 to RP-20, RP-79, RP-85) has the incorporation function (extension function) for C and U. The terminal transferase mutant recombinant proteins of the terminal transferase mutants (RP-22 to RP-41, RP-43, RP-45 to RP-49, RP-51 to RP-78, RP-80 to RP-83) have the incorporation function (extension function) of C; the terminal transferase mutant recombinant protein including the terminal transferase mutant (RP-84) has the incorporation function (extension function) of A; the terminal transferase mutant recombinant protein including the terminal transferase mutant (RP-21) has the incorporation function (extension function) of A, U, and G; the terminal transferase mutant recombinant protein including the terminal transferase mutant (RP-50) has the incorporation function (extension function) of A, U, C, and G.

[0294] Table 1 Functional tests of terminal transferase recombinant protein (RP02) containing terminal transferase (RP01) or its mutants (RP-1 to RP-85) and terminal transferase mutant recombinant proteins

[0295]

[0296]

[0297] The present embodiment first verifies the function of incorporating single 3'-O-Allyl-NTPs by the purified terminal transferase mutant (RP-50) recombinant protein obtained in Example 1. The reaction system comprises 1 μM primer (the primer sequence is: CGCTTGCACAGGTGCGTTGG (5'-3'), SEQ ID NO: 21), 250 μM 3'-O-Allyl-NTPs (3'-O-Allyl-ATP or 3'-O-Allyl-UTP or 3'-O-Allyl-CTP or 3'-O-Allyl-GTP, which can be simply written as A or U or C or G), 0.25 mM CoCl2, 100 mM NaCl and 20 mM Tris buffer (pH 7.9) (the concentration of each component is the final concentration, and the total volume of the reaction system is 10 μL), 0.1 mg / mL pure enzyme solution (the purified terminal transferase mutant (RP-50) recombinant protein obtained in Example 1 and the terminal transferase recombinant protein RP02), and the reaction is carried out at 30°C for 30 minutes. The reaction is analyzed by 20% polyacrylamide gel electrophoresis, and the results are shown in Figure 5 Figure 1. Compared with the control comprising the terminal transferase recombinant protein RP02, the terminal transferase mutant (RP-50) recombinant protein comprising the terminal transferase mutant (RP-50) has the ability to extend all four allyl-modified ribonucleotides and has higher extension activity than the control, which is manifested by the fact that the four n+1 bands of the terminal transferase mutant (RP-50) recombinant protein comprising the terminal transferase mutant (RP-50) are more obvious than those of the control. In addition, the terminal transferase mutant (RP-50) recombinant protein comprising the terminal transferase mutant (RP-50) has preference for the extension activity of the four allyl-modified ribonucleotides, and the extension effect of 3'-O-Allyl-CTP is the best (n represents the primer, and n+1 represents the primer after the addition of 3'-O-Allyl-NTPs, i.e., the product of the above reaction system).

[0298] Industrial applications

[0299] The experiments of the present application demonstrate that the present application has improved the terminal transferase by improving the ability of the terminal transferase to modify nucleotides in a single step (such as allyl-modified ribonucleotides) and other improvement goals: compared with the unmutated terminal transferase, the mutant has a wider extension ability (i.e., can incorporate more types of nucleotides (preferably 3'-OH end modified nucleotides) into the primer chain) and higher extension activity (i.e., higher activity of catalyzing the addition of nucleotides (preferably 3'-OH end modified nucleotides) to the primer chain).

[0300] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A terminal transferase or a mutant thereof, wherein the amino acid sequence of the terminal transferase is shown in SEQ ID NO: 16; Compared to the amino acid sequence of the terminal transferase, the amino acid sequence of the terminal transferase mutant has a mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten positions selected from the group consisting of V120, C162, G192, H202, D237, A255, D257, K261, L299, and G321; Preferably, the amino acid sequence of the terminal transferase mutant other than the mutation site is at least 80% identical to the corresponding amino acid sequence of the terminal transferase; Preferably, the amino acid sequence of the terminal transferase mutant other than the mutation site is identical to the corresponding amino acid sequence of the terminal transferase.

2. The terminal transferase or a mutant thereof according to claim 1, characterized in that: The mutation comprises insertion, deletion, and / or substitution; Preferably, the mutation at the V120 site is to replace the V at position 120 with M; Preferably, the mutation at position C162 is to replace the C at position 162 with K, G, A, V, I, L, M, R, Q, N, S, W, Y, F, H, P, E, D, or T; Preferably, the mutation at the G192 site is to replace the G at position 192 with S; Preferably, the mutation at position H202 is to replace H at position 202 with K, G, A, V, I, L, M, R, Q, N, S, W, Y, F, D, P, E, C, or T; Preferably, the mutation at position D237 is to replace D at position 237 with K, G, A, V, I, L, M, R, Q, N, S, W, Y, F, H, P, E, C, or T; Preferably, the mutation at position A255 is to replace the A at position 255 with K, G, C, V, I, L, M, R, Q, N, S, W, Y, F, H, P, E, D, or T; Preferably, the mutation at position D257 is to replace D at position 257 with M; Preferably, the mutation at the K261 site is to replace the K at position 261 with Y, C, or F; Preferably, the mutation at the L299 site is to replace L at position 299 with M; Preferably, the mutation at the G321 site is to replace the G at position 321 with V or M; Preferably, the amino acid sequence of the terminal transferase mutant is a substitution of V at position 120 of SEQ ID NO: 16 with M; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of C at position 162 of SEQ ID NO: 16 with K; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of C at position 162 of SEQ ID NO: 16 with G; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of C at position 162 of SEQ ID NO: 16 with A; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of C at position 162 of SEQ ID NO: 16 with T; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by V; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by I; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by L; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by M; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by R; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by Q; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by N; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by S; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by W; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by Y; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by F; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by H; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by P; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by E; Preferably, the amino acid sequence of the terminal transferase mutant is that C at position 162 of SEQ ID NO: 16 is replaced by D; Preferably, the amino acid sequence of the terminal transferase mutant is that G at position 192 of SEQ ID NO: 16 is replaced by S; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by K; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by G; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by A; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by T; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by V; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by I; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by L; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by M; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by R; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by Q; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by N; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by S; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by W; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by Y; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by F; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by C; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by P; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by E; Preferably, the amino acid sequence of the terminal transferase mutant is that H at position 202 of SEQ ID NO: 16 is replaced by D; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by K; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by G; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by A; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by T; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by V; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by I; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by L; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by M; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by R; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by Q; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by N; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by S; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by W; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by Y; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by F; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by H; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by P; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by E; Preferably, the amino acid sequence of the terminal transferase mutant is that D at position 237 of SEQ ID NO: 16 is replaced by C; Preferably, the amino acid sequence of the terminal transferase mutant is that A at position 255 of SEQ ID NO: 16 is replaced by K; Preferably, the amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is replaced by G; Preferably, the amino acid sequence of the terminal transferase mutant is that A at position 255 of SEQ ID NO: 16 is replaced by C; Preferably, the amino acid sequence of the terminal transferase mutant is that A at position 255 of SEQ ID NO: 16 is replaced by V; Preferably, the amino acid sequence of the terminal transferase mutant is that A at position 255 of SEQ ID NO: 16 is replaced by I; Preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with L; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with M; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with R; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with Q; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with N; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with S; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with W; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of A at position 255 of SEQ ID NO: 16 with Y; preferably, the amino acid sequence of the terminal transferase mutant is a substitution of The amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is substituted with F; preferably, the amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is substituted with H; preferably, the amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is substituted with P; preferably, the amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is substituted with E; preferably, the amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is substituted with T; preferably, the amino acid sequence of the terminal transferase mutant is that the A at position 255 of SEQ ID NO: 16 is substituted with D; preferably, the amino acid sequence of the terminal transferase mutant is that the D at position 257 of SEQ ID NO: 16 is substituted with M; preferably, the amino acid sequence of the terminal transferase mutant is that the K at position 261 of SEQ ID NO: 16 is substituted with Y; preferably, the amino acid sequence of the terminal transferase mutant is that the The K at position 261 of SEQ ID NO: 16 is substituted with C; preferably, the amino acid sequence of the terminal transferase mutant is that the K at position 261 of SEQ ID NO: 16 is substituted with F; preferably, the amino acid sequence of the terminal transferase mutant is that the L at position 299 of SEQ ID NO: 16 is substituted with M; preferably, the amino acid sequence of the terminal transferase mutant is that the G at position 321 of SEQ ID NO: 16 is substituted with M; preferably, the amino acid sequence of the terminal transferase mutant is that the G at position 321 of SEQ ID NO: 16 is substituted with V;Preferably, the amino acid sequence of the terminal transferase mutant is: a1) the amino acid sequence shown in SEQ ID NO: 27; or a2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 27 and having the same function; Preferably, the amino acid sequence of the terminal transferase mutant is: b1) the amino acid sequence shown in SEQ ID NO: 20; or b2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 20 and having the same function.

3. A recombinant protein comprising a modified portion and the terminal transferase or a mutant thereof according to any one of claims 1 to 2; preferably, the modified portion is a protein tag; Preferably, the protein tag is selected from at least one of Poly his, FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA; Preferably, the modified portion is located at the N-terminus and / or C-terminus of the terminal transferase or its mutant; Preferably, the amino acid sequence of the recombinant protein is: c1) the amino acid sequence shown in SEQ ID NO: 15; or c2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 15 and having the same function; Preferably, the amino acid sequence of the recombinant protein is: m1) the amino acid sequence shown in SEQ ID NO: 26; or m2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 26 and having the same function; Preferably, the amino acid sequence of the recombinant protein is: 11) the amino acid sequence shown in SEQ ID NO: 19; or l2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 19 and having the same function.

4. A biomaterial related to the terminal transferase or a mutant thereof according to any one of claims 1 to 2, or the recombinant protein according to claim 3, wherein the biomaterial comprises any one of d1) to d12): d1) a nucleic acid molecule encoding the terminal transferase or a mutant thereof according to any one of claims 1 to 2, or the recombinant protein according to claim 3; d2) an expression cassette comprising the nucleic acid molecule described in d1); d3) a vector comprising the nucleic acid molecule described in d1); d4) a vector comprising the expression cassette described in d2); d5) a transgenic cell line comprising the nucleic acid molecule described in d1); d6) a transgenic cell line comprising the expression cassette described in d2); d7) a transgenic cell line comprising the vector described in d3); d8) a transgenic cell line comprising the vector described in d4); d9) a recombinant bacterium comprising the nucleic acid molecule described in d1); d10) a recombinant bacterium comprising the expression cassette described in d2); d11) a recombinant bacterium comprising the vector described in d3); d12) a recombinant bacterium comprising the vector described in d4); Preferably, the vectors in d3) and d4) are independently selected from non-pathogenic viral vectors and non-viral vectors; Preferably, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector; Preferably, the non-viral vector comprises a plasmid vector; Preferably, the recombinant bacteria in d9) to d12) are Escherichia coli; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the terminal transferase is: e1) the nucleotide sequence shown in SEQ ID NO: 14; or e2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 14 and having the same function; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the terminal transferase mutant is: f1) the nucleotide sequence shown in SEQ ID NO: 23; or f2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 23 and having the same function; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the terminal transferase mutant is: n1) the nucleotide sequence shown in SEQ ID NO: 25; or n2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 25 and having the same function; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein is: g1) the nucleotide sequence shown in SEQ ID NO: 13; or g2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 13 and having the same function; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein is: h1) the nucleotide sequence shown in SEQ ID NO: 22; or h2) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO: 22 and having the same function; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the recombinant protein is: o1) the nucleotide sequence shown in SEQ ID NO: 24; or o2) has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO: 24 and has the same function.

5. A terminal transferase-nucleotide complex comprising: f1) at least one of the terminal transferase according to any one of claims 1 to 2 or a mutant thereof, and the recombinant protein according to claim 3; and f2) nucleotides; Preferably, the nucleotide is connected to the terminal transferase or a mutant thereof via a linker; and / or The nucleotide and the recombinant protein are connected via a linker; Preferably, the nucleotide comprises at least one of natural deoxyribonucleotides, non-natural deoxyribonucleotides, natural ribonucleotides, and non-natural ribonucleotides.

6. The use described in any one of (g1) to (g4): (g1) Use of the terminal transferase or a mutant thereof according to any one of claims 1 to 2, the recombinant protein according to claim 3, and / or the biomaterial according to claim 4 in the preparation of DNA polymerase and / or RNA polymerase; (g2) Use of the terminal transferase or a mutant thereof according to any one of claims 1 to 2, and / or the recombinant protein according to claim 3 as a DNA polymerase and / or RNA polymerase; (g3) Use of the terminal transferase or a mutant thereof according to any one of claims 1 to 2, the recombinant protein according to claim 3, and / or the terminal transferase-nucleotide complex according to claim 5 in at least one of j1) to j5); (g4) Use of the terminal transferase or mutant thereof according to any one of claims 1 to 2, the recombinant protein according to claim 3, the biomaterial according to claim 4, and / or the terminal transferase-nucleotide complex according to claim 5 in j6); j1) catalyzing single-base or multi-base extension of a nucleic acid molecule, preferably catalyzing single-base or multi-base extension of a nucleic acid molecule in the absence of a template chain; j2) synthesizing nucleic acid molecules, preferably in the absence of a template strand; j3) catalyze DNA or RNA replication; j4) catalyzing DNA or RNA amplification; j5) performing nucleic acid sequencing; j6) preparing a product, wherein the product is used for at least one of j1) to j5); Preferably, the product is a kit; Preferably, the base comprises at least one of a natural base and a non-natural base; Preferably, the nucleic acid molecule comprises at least one of DNA and RNA.

7. The kit according to any one of (h1) to (h2): (h1) A kit comprising at least one of the terminal transferase or a mutant thereof according to any one of claims 1 to 2 and the recombinant protein according to claim 3; (h2) A kit comprising the terminal transferase-nucleotide complex according to claim 5.

8. The kit according to claim 7, wherein: The kit described in (h1) further comprises at least one of nucleotides, cobalt ions, NaCl, and a buffer; Preferably, the nucleotide comprises at least one of natural deoxyribonucleotides, non-natural deoxyribonucleotides, natural ribonucleotides, and non-natural ribonucleotides; Preferably, the nucleotide is a nucleotide with a modified 3'-OH end; Preferably, the nucleotide with a modified 3'-OH end is obtained by adding a blocking group to the 3'-OH end of the nucleotide for modification; Preferably, the blocking group comprises at least one of an O-amino group, an O-allyl group, an O-azido group, and an O-phosphate group; Preferably, the kit described in (h2) further comprises at least one of cobalt ions, buffer, and NaCl; Preferably, the kit in (h2) further comprises a substance for removing the linker; Preferably, the buffer in (h1) and (h2) comprises at least one of phosphate buffer, borate buffer, citrate buffer, Tris buffer and Hepes buffer.

9. A method for synthesizing a nucleic acid molecule, comprising the steps of using the terminal transferase or a mutant thereof according to any one of claims 1 to 2, the recombinant protein according to claim 3, the terminal transferase-nucleotide complex according to claim 5, and / or the kit according to any one of claims 7 to 8; Preferably, the synthesis of nucleic acid molecules is carried out in the absence of a template strand; Preferably, the method is k1) or k2): k1) The method comprises the following steps: mixing the terminal transferase or a mutant thereof according to any one of claims 1 to 2, and / or the recombinant protein according to claim 3, with a primer chain and nucleotides, and reacting the resulting system; k2) The method comprises the steps of: mixing the terminal transferase-nucleotide complex according to claim 5 with a primer chain, and reacting the resulting system; Preferably, the nucleotide described in k1) is the nucleotide described in claim 8; Preferably, the system in k1) and k2) further comprises cobalt ions, a buffer solution, and NaCl; Preferably, the buffer solution is the buffer solution described in claim 8.

10. A method for preparing the terminal transferase or a mutant thereof according to any one of claims 1 to 2, and / or the recombinant protein according to claim 3, which is obtained by culturing the transgenic cell line and / or recombinant bacteria according to claim 4.

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