Terminal deoxynucleotidyl transferase and use thereof

Abstract

Provided are a terminal deoxynucleotidyl transferase (TdT) or a biologically active fragment thereof and the use thereof. The terminal deoxynucleotidyl transferase or the biologically active fragment thereof comprises a catalytic domain, the catalytic domain comprising: a. an amino acid sequence shown as SEQ ID NO: 1 or 2; b. an amino acid sequence having one or more amino acid substitutions, deletions and / or additions as compared to the amino acid sequence shown as SEQ ID NO: 1 or 2; or c. an amino acid sequence having at least 60% identity as compared to the amino acid sequence shown as SEQ ID NO: 1 or 2. The catalytic domain has the function of catalyzing a single nucleotide to be polymerized on the 3'-OH end of a polynucleotide chain.

Description

Terminal deoxynucleotidyl transferases and their applications Technical Field

[0001] This disclosure relates to the field of biotechnology, specifically to novel terminal deoxynucleotidyl transferases and their applications. Background Technology

[0002] Compared to chemical synthesis methods based on solid-phase phosphorous acid synthesis, biosynthesis mediated by the enzyme terminal deoxynucleotidyl transferase (TdT) provides an effective approach for de novo DNA synthesis. Unlike most nucleic acid polymerases, TdT can exhibit base polymerization activity without a template, randomly adding new bases to the 3' end of single-stranded DNA molecules. The rate and sequence of base addition can be controlled by reversibly blocking modified bases, thereby achieving controllable synthesis of DNA sequences.

[0003] Although several types of TdT have been proposed in related technologies, most are natural TdTs, resulting in a limited variety and simple enzymatic characteristics. Furthermore, the nucleotide monomer substrates used in TdT-dominated de novo DNA synthesis schemes are generally non-natural nucleotides modified with protecting groups (PGs). The polarity and steric hindrance of PGs do not adequately accommodate the catalytic activity pockets of most natural TdTs, leading to low TdT activity and inefficient DNA synthesis. While partial mutations of TdTs to improve their performance have been reported, these mutations are still based on the existing framework of a few natural TdT sequences, thus limiting the scope for modification.

[0004] Therefore, it is of great significance to develop novel TdT with novel enzymatic properties and high catalytic efficiency for applications such as the biosynthesis of DNA and TdT-mediated biosensors. Summary of the Invention

[0005] The first aspect of this disclosure provides a terminal deoxynucleotidyl transferase or a bioactive fragment thereof, comprising a catalytic domain, said catalytic domain: a. having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; b. having one or more amino acid sequences with amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and said catalytic domain having the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain, said mutation including substitution, deletion and / or addition; or c. having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and said catalytic domain having the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

[0006] In some embodiments, the catalytic domain has an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the terminal deoxynucleotidyltransferase or its bioactive fragment is derived from Bagarius yarrelli.

[0007] In some embodiments, the catalytic domain has an amino acid mutation at at least one of positions 310 and 311 of SEQ ID NO: 1, wherein the mutation is preferably a substitution.

[0008] In some embodiments, compared to SEQ ID NO: 1, the catalytic domain has the following mutation: D at position 310 is replaced with R; and / or R at position 311 is replaced with E.

[0009] In some embodiments, the catalytic domain has an amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the TdT or its bioactive fragment is derived from the cichlid (Amphilophus citrinellus).

[0010] In some embodiments, the catalytic domain has an amino acid mutation at at least one of positions 312 and 313 of SEQ ID NO: 2, wherein the mutation is preferably a substitution.

[0011] In some embodiments, compared to SEQ ID NO: 2, the catalytic domain has the following mutation: E at position 312 is replaced with R; and / or R at position 313 is replaced with E.

[0012] In some embodiments, the terminal deoxynucleotidyltransferase or its bioactive fragment further comprises an N-terminal or C-terminal noncatalytic domain, the N-terminal or C-terminal noncatalytic domain comprising an α-helix and / or a β-sheet. In some embodiments, the N-terminal noncatalytic domain has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0013] In some embodiments, the terminal deoxynucleotidyl transferase or its bioactive fragment has an amino acid sequence as shown in SEQ ID NO: 5 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5, and the terminal deoxynucleotidyl transferase or its bioactive fragment has the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

[0014] In some embodiments, the terminal deoxynucleotidyl transferase or its bioactive fragment has an amino acid sequence as shown in SEQ ID NO: 6 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6, and the terminal deoxynucleotidyl transferase or its bioactive fragment has the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

[0015] A second aspect of this disclosure provides a fusion protein comprising a terminal deoxynucleotidyl transferase or a biologically active fragment thereof as described in any embodiment of the first aspect of this disclosure, and an additional portion fused thereto. In some embodiments, the additional portion is a tag protein. In some embodiments, the tag protein is at least one selected from PolyHis, FLAG, GFP, Strep-Tag II, Poly Arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, Ubiquitin, and NusA. In some embodiments, the additional portion is located at the N-terminus and / or C-terminus of the terminal deoxynucleotidyl transferase or its biologically active fragment thereof.

[0016] The third aspect of this disclosure provides a polynucleotide that encodes a terminal deoxynucleotidyl transferase or a biologically active fragment thereof as described in any embodiment of the first aspect of this disclosure, or a fusion protein or a complementary sequence thereof as described in any embodiment of the second aspect.

[0017] In some embodiments, the polynucleotide comprises a sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8.

[0018] A fourth aspect of this disclosure provides a vector comprising a polynucleotide as described in any embodiment of a third aspect of this disclosure.

[0019] A fifth aspect of this disclosure provides a cell comprising a polynucleotide as described in any embodiment of the third aspect of this disclosure or a vector as described in any embodiment of the fourth aspect of this disclosure, or expressing a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure or a fusion protein as described in any embodiment of the second aspect.

[0020] A sixth aspect of this disclosure provides a kit comprising: a terminal deoxynucleotidyl transferase or a biologically active fragment thereof as described in any embodiment of the first aspect of this disclosure, or a fusion protein as described in any embodiment of the second aspect.

[0021] In some embodiments, the kit further includes an enzyme buffer and / or nucleotides. In some embodiments, the enzyme buffer is optionally selected from at least one of the following: phosphate buffer, borate buffer, citrate buffer, Tris buffer, arsenate buffer, and Hepes buffer.

[0022] In some embodiments, the nucleotide comprises at least one of a natural nucleotide and a non-natural nucleotide. In some embodiments, the non-natural nucleotide is a nucleotide modified at the 3'-OH end. In some embodiments, the 3'-OH modified nucleotide is obtained by modifying the nucleotide by adding a blocking group to the 3'-OH end, the blocking group comprising at least one of alkyl, aralkyl, alkenyl, alkynyl, allyl, aryl, heteroaryl, heterocyclic, benzyl, azide, azido, amino, ketone, isocyanate, phosphate, carbonate, thio, acyl, oxime, cyano, alkoxy, aryloxy, heteroaryloxy, and amide.

[0023] The seventh aspect of this disclosure proposes the use of terminal deoxynucleotidyl transferases or their bioactive fragments as described in any embodiment of the first aspect of this disclosure, fusion proteins as described in any embodiment of the second aspect of this disclosure, or kits as described in any embodiment of the sixth aspect of this disclosure in one or more of the following: i) catalyzing the polymerization between nucleotides to extend mononucleotides or polynucleotides, preferably under template-free conditions; ii) synthesizing polynucleotides, preferably under template-free conditions; iii) catalyzing the replication of polynucleotides; iv) labeling and / or detecting target nucleic acids; v) nucleic acid synthesis in bioinformatics storage; and vi) preparing products for any of i)-iv), wherein said nucleotides include at least one of natural nucleotides and non-natural nucleotides, optionally dNTPs and / or NTPs, preferably dNTPs.

[0024] An eighth aspect of this disclosure provides a method for synthesizing polynucleotides, comprising: contacting a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure or a fusion protein as described in any embodiment of the second aspect of this disclosure with nucleotides and primers, wherein the terminal deoxynucleotidyl transferase or its bioactive fragment or the fusion protein catalyzes the polymerization between nucleotides to elongate mononucleotides or polynucleotides, thereby synthesizing polynucleotides, wherein the nucleotides include at least one of natural nucleotides and non-natural nucleotides.

[0025] In some embodiments, the non-natural nucleotide is a nucleotide modified at the 3'-OH end. In some embodiments, the 3'-OH modified nucleotide is obtained by modifying the nucleotide by adding a blocking group to the 3'-OH end, the blocking group including at least one selected from O-alkyl, O-amide, O-amino, O-allyl, O-oxime, O-azido, and O-phosphate groups.

[0026] In some embodiments, the synthesis is performed in the absence of the template chain.

[0027] In some embodiments, the non-natural nucleotide further comprises a detectable marker, which may optionally be a fluorescent marker, biotin, digoxigenin, and / or a radiolabel.

[0028] A ninth aspect of this disclosure provides a method for labeling nucleic acids, comprising: contacting a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure or a fusion protein as described in any embodiment of the second aspect of this disclosure with a nucleotide and the nucleic acid, wherein the nucleotide is a non-natural nucleotide containing a detectable marker, and the terminal deoxynucleotidyl transferase or its bioactive fragment or the fusion protein catalyzes the polymerization of one or more of the non-natural nucleotides at the 3' end of the nucleic acid to label the nucleic acid.

[0029] A tenth aspect of this disclosure provides a method for detecting the presence of a target nucleic acid in a test sample, comprising: a) providing a test sample containing nucleic acid; b) contacting the test sample with a probe specifically targeting the target nucleic acid to perform in situ hybridization to obtain an in situ hybridization product, wherein the probe contains a detectable marker, the probe being prepared by a method for synthesizing polynucleotides according to an embodiment of this disclosure; c) removing probes that do not specifically bind to the target nucleic acid; and d) detecting the marker on the probe in the in situ hybridization product to determine whether the target nucleic acid is present in the test sample.

[0030] In some embodiments, the method further includes: performing qualitative, quantitative, and / or localization analysis on the target nucleic acid based on the presence of the target nucleic acid in the sample to be tested, using the signal intensity of the marker.

[0031] In some embodiments, the nucleic acids in the sample to be tested are single-stranded and are directly or indirectly immobilized.

[0032] In some embodiments, the probe is a single-stranded nucleic acid probe and contains non-natural nucleotides. In some embodiments, the probe is synthesized via a terminal deoxynucleotidyl transferase mediated by embodiments of this disclosure.

[0033] In some embodiments, the detectable marker may optionally be a fluorescent marker, biotin, digoxigenin, and / or a radiolabeled marker.

[0034] The technical solution disclosed herein achieves the following technical effects:

[0035] This disclosure presents novel terminal deoxynucleotidyl transferases (referred to as BaTdT and AcTdT, with the amino acid sequences of their catalytic domains shown in SEQ ID NO: 1-2 and the full-length amino acid sequences shown in SEQ ID NO: 5-6), which exhibit excellent catalytic activity for nucleotide polymerization and can be used in various fields such as the biosynthesis of polynucleotides, polynucleotide characterization, and the fabrication of biosensors. Furthermore, the novel terminal deoxynucleotidyl transferase sequences provided in this disclosure show significant differences from traditional terminal deoxynucleotidyl transferases, thus providing a terminal deoxynucleotidyl transferase framework containing novel enzymatic catalytic structures and properties, possessing extremely high application prospects and modification value. In addition, this disclosure also proposes mutant enzymes based on BaTdT and AcTdT. Compared to BaTdT and AcTdT, each mutant exhibits higher catalytic activity, thus also possessing extremely high application value. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 shows the three-dimensional structural models of BaTdT and AcTdT according to Embodiment 2 of this disclosure and their comparison with MuTdT;

[0038] Figure 2 shows the purified BaTdT according to Example 3 of this disclosure. (P118-A480) and AcTdT (T74-A438) SDS-PAGE detection image;

[0039] Figure 3 illustrates BaTdT according to Embodiment 4 of this disclosure. (P118-A480) and AcTdT (T74-A438) A detection graph of the polymerization products obtained by polymerizing natural nucleotides;

[0040] Figure 4 illustrates BaTdT according to Embodiment 5 of this disclosure. (P118-A480) and AcTdT (T74-A438) A detection graph of the polymerization products obtained by polymerizing modified nucleotides;

[0041] Figure 5 illustrates the chemical structures of some modified nucleotides according to embodiments of the present disclosure;

[0042] Figure 6 shows the purified BaTdT according to Example 6 of this disclosure. (P118-A480) -D310R / R311E mutants and AcTdT (T74-A438)SDS-PAGE analysis of the -E312R / R313E mutant;

[0043] Figure 7 illustrates BaTdT according to Embodiment 7 of this disclosure. (P118-A480) -D310R / R311E mutants and AcTdT (T74- A438) Detection graph of polymerization products of -E312R / R313E mutant.

[0044] Figure 8 illustrates BaTdT according to Embodiment 8 of this disclosure. (P118-A480) -D310R / R311E mutant (A) and AcTdT (T74-A438) A detection diagram of DNA products synthesized by the -E312R / R313E mutant (B) through continuous polymerization modification of nucleotides.

[0045] Figure 9 illustrates BaTdT according to Embodiment 9 of this disclosure. (P118-A480) -D310R / R311E mutant (A) and AcTdT (T74-A438) The detection diagram of the synthesis of fluorescently labeled DNA products by the -E312R / R313E mutant (B) through continuous polymerization of fluorescently labeled modified nucleotides. Detailed Implementation

[0046] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present disclosure in any way.

[0047] This disclosure is based on the inventor's following understanding:

[0048] Compared to the low efficiency, high energy consumption, and heavy pollution associated with traditional phosphorus amide synthesis of DNA, TdT-dominated enzymatic de novo DNA synthesis offers significant advantages and is considered a promising third-generation DNA biosynthesis technology. This TdT-catalyzed de novo DNA synthesis strategy requires only two steps—coupling and deprotection—to complete a single-base addition cycle, significantly improving single-cycle efficiency and the potential limit of DNA product length compared to the phosphorus amide method. Furthermore, it eliminates the need for large amounts of toxic and harmful reagents, operates under mild reaction conditions, and allows for the extension of relatively long single strands in vitro. The de novo synthesis of single-stranded DNA using TdT to integrate dNTPs into the ends of oligonucleotide chains has been validated. However, this technology currently faces several challenges, such as the limited number of reported TdT species and their singular enzymatic properties, which severely restricts the selection of TdTs for enzymatic DNA synthesis. Moreover, current TdT modifications are mostly based on their natural framework, offering extremely limited modification potential.

[0049] Based on this, the inventors, through numerous experiments and tests, discovered two novel terminal deoxynucleotidyl transferases, BaTdT and AcTdT, from deep-sea metagenomics. The novel TdT of this disclosure exhibits excellent template-independent DNA polymerization activity and low sequence similarity (less than 60%) to existing TdT sequences. Furthermore, by performing two-site mutations on BaTdT and AcTdT respectively, the resulting mutant TdT also showed good catalytic activity against a variety of modified substrates. The novel TdT proposed in this disclosure has good potential for applications such as de novo DNA synthesis via biological methods, and its novel sequence backbone provides a new framework and significant room for performance improvement in TdT modification.

[0050] Unless otherwise stated, all technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures described below in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization are well-known and commonly used in the art. Nucleic acid and peptide synthesis is performed using standard techniques. These techniques and procedures are performed according to conventional methods described in the art and various general references (see, generally, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which are incorporated herein by reference), which are incorporated herein by reference throughout. The nomenclature used herein and the laboratory procedures described below in analytical chemistry and organic synthesis are well-known and commonly used in the art. Chemical synthesis or chemical analysis may also be performed using standard techniques or variations thereof.

[0051] In this disclosure, "naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that has not been intentionally modified by human manipulation. A "mutant" means a sequence that has a change of at least one amino acid relative to a natural or wild-type amino acid sequence. In some embodiments, the change (mutation) includes at least one of substitution, deletion, and insertion.

[0052] In this disclosure, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds with the same basic chemical structure as naturally occurring amino acids, i.e., carbon atoms bonded to hydrogen atoms, carboxyl groups, amino groups, and R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (such as ortholeucine) or modified peptide backbones, but retain a substantially identical chemical structure to naturally occurring amino acids. Amino acid mimics are compounds with structures different from the common chemical structure of amino acids, but functioning similarly to naturally occurring amino acids. The amino acid sequences proposed in this disclosure (e.g., the amino acid sequences shown in SEQ ID NO: 1 or 2) may include the aforementioned amino acid analogs and mimics or related modifications, as long as they do not affect the basic properties of the corresponding amino acid or the activity of the entire enzyme or its active fragment.

[0053] In this disclosure, the term "nucleotide" can refer to naturally occurring nucleotides as well as xeno-nucleic acids (XNAs), such as modified nucleotides or xeno-nucleic acid aptamers, as disclosed in WO2022 / 083686A1. In some embodiments, the modified nucleotide can be a nucleotide modified with a reversible blocking group, wherein the reversible blocking group modification can occur at the 3' sugar ring hydroxyl group and / or base, and the reversible blocking group can be selected from one or more of the following: alkyl, aralkyl, alkenyl, alkynyl, allyl (e.g., 3'-O-allyl), aryl, heteroaryl, heterocyclic, benzyl, azide group, azide group (e.g., 3'-O-azidomethyl), amino, ketone, isocyanate group, phosphate ester group, carbonate group, thio, acyl, oxime, cyano, alkoxy, aryloxy, heteroaryloxy, or amide group, etc., which dissociate under aqueous conditions to produce a molecule with a free 3'-OH.

[0054] In embodiments of this disclosure, nucleotides may also be labeled to facilitate their detection. In some embodiments, the label is a fluorescent label. In some embodiments, the fluorescent label may be selected from one or more of AF532, ATTO532, Cy3B, Cy3, Cy5, ATTO647N, ATTO647, and AF647. In some embodiments, each nucleotide type may be labeled with a different fluorescent label. In other embodiments, each nucleotide type or a subset of nucleotide types may be labeled with the same fluorescent label. It is understood that the fluorescent label modified in the nucleotide may also be any fluorescent dye used in the art for nucleic acid labeling / detection, and this disclosure does not limit the specific type of fluorescent label. However, the detectable label is not necessarily a fluorescent label. Any label that allows the detection of nucleotide incorporation in the DNA sequence can be used, such as digoxigenin, biotin, or radiolabeling, and this disclosure does not limit the specific type of label.

[0055] In this disclosure, the term "percentage of identity" for nucleic acid or polypeptide sequences is defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to a known polypeptide after arranging the sequence to obtain the maximum percentage of identity and introducing gaps (if necessary) to achieve the maximum percentage of homology. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using algorithms employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA. 87, 2264-2268), programs tailored for sequence similarity searches.

[0056] In this disclosure, the terms "terminal transferase" and "terminal deoxynucleotidyl transferase (TdT)" are used interchangeably. TdT is a polymerase belonging to the polymerase family X and is therefore also referred to as a "polymerase." Unlike most template-dependent polymerases, terminal transferases are special polymerases whose native activity is to catalyze template-independent polymerization reactions. For example, they can randomly add dNTPs to the free 3' end of single-stranded DNA to achieve the elongation of single or polynucleotides. The "TdT function" or "TdT activity" referred to in this disclosure refers to its property of catalyzing the polymerization between nucleotides to elongate the synthetic chain.

[0057] In this disclosure, the term "bioactive fragment" refers to any fragment, derivative, homology, or analog of terminal deoxynucleotidyl transferase (TdT) and its mutants, possessing biomolecule-specific in vivo or in vitro activity, including, for example, terminal transferase activity. In some embodiments, the bioactive fragment, derivative, homology, or analog of the terminal deoxynucleotidyl transferase possesses any degree of biological activity of the terminal deoxynucleotidyl transferase in any in vivo or in vitro assay of interest. In some embodiments, the bioactive fragment may include any number of consecutive amino acid residues of BaTdT or AcTdT.

[0058] The first aspect of this disclosure provides a terminal deoxynucleotidyl transferase or a bioactive fragment thereof, comprising a catalytic domain having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or having one or more amino acid sequences with amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the catalytic domain having the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

[0059] In some embodiments, the catalytic domain of the terminal deoxynucleotidyl transferase or its bioactive fragment contains an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, for example, having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, 99.1%, 99.2%, etc. Sequences with 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, and 99.99% but less than 100% identity, and the catalytic domains of amino acid sequences with these identities still possess the catalytic function of terminal transferases.

[0060] In this embodiment of the disclosure, compared with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, the catalytic domain may have one or more amino acid substitutions, deletions, and / or additions, and the catalytic domain with the mutated amino acid sequence still has the catalytic function of a terminal transferase. For example, the catalytic domain has at least 1-70 amino acid mutations compared with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the catalytic domain has 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, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments of this disclosure, the catalytic domain has at least 31-60 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0061] In some embodiments, the catalytic domain has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the catalytic domain, or the terminal deoxynucleotidyl transferase containing it, or a bioactive fragment thereof, is derived from Bagarius yarrelli.

[0062] BaTdT (P118-A480) (SEQ ID NO: 1):

[0063] In some embodiments, the catalytic domain has an amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the catalytic domain or the terminal deoxynucleotidyl transferase containing it or a bioactive fragment thereof is derived from the cichlid (Amphilophus citrinellus).

[0064] AcTdT (T74-A438) (SEQ ID NO: 2):

[0065] In this embodiment of the disclosure, single-point or multi-point directed mutations can be performed on the catalytic domain to optimize its catalytic performance. In some embodiments, amino acid mutations can be performed at positions 310 and / or 311 of the catalytic domain shown in SEQ ID NO: 1, such as amino acid substitution. In some embodiments, D at position 310 of the catalytic domain shown in SEQ ID NO: 1 can be replaced with R; and / or R at position 311 can be replaced with E. In some embodiments, two-site mutations can be performed on the catalytic domain to optimize its catalytic performance, such as replacing D at position 310 of the catalytic domain shown in SEQ ID NO: 1 with R and replacing R at position 311 with E.

[0066] In other embodiments, amino acid mutations, such as amino acid substitutions, can be made at positions 312 and / or 313 of the catalytic domain shown in SEQ ID NO: 2. In some embodiments, the E at position 312 of the catalytic domain shown in SEQ ID NO: 2 can be replaced with R; and / or the R at position 313 can be replaced with E. In some embodiments, a two-site mutation can be made to the catalytic domain to optimize its catalytic performance, for example, replacing the E at position 312 of the catalytic domain shown in SEQ ID NO: 2 with R and replacing the R at position 313 with E.

[0067] It should be noted that the terminal deoxynucleotidyl transferase proposed in this embodiment catalyzes nucleotide polymerization based on its contained catalytic domain, thereby exerting its terminal transferase function. In other words, TdT containing only a single catalytic domain or its mutant form can function as a terminal transferase. Therefore, the catalytic domain proposed in this embodiment can also be directly referred to as a terminal deoxynucleotidyl transferase, which can directly function as a terminal transferase.

[0068] In this embodiment of the disclosure, in addition to the catalytic domain, the terminal deoxynucleotidyl transferase or its bioactive fragment may also contain other structures, such as N-terminal or C-terminal appendages, such as α-helices and / or β-sheets, which can be attached to the catalytic domain as non-catalytic domains. In some embodiments, the TdT or its bioactive fragment contains an N-terminal non-catalytic domain, which has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4. It is understood that the catalytic domain proposed in this embodiment of the disclosure can be attached with other amino acid sequences for the protection of the catalytic domain, protein screening and purification, and as a signal sequence for proteins (such as signal peptides), as long as it does not affect the catalytic function of the catalytic domain in the terminal deoxynucleotidyl transferase. This disclosure does not limit the other sequences attached to the catalytic domain.

[0069] In some embodiments, the catalytic domain shown in SEQ ID NO: 1 may have an amino acid sequence shown in SEQ ID NO: 3 attached to its N-terminus, in which case the TdT or its bioactive fragment may have an amino acid sequence shown in SEQ ID NO: 5, which is the full-length sequence of wild-type BaTdT.

[0070] IKFRDSTVYLVERRMGKTRRNFLAGLARSKGFCVENTLSSNVTHIVAEDNPADELWLWLQKQGIADLDKINVLDISWFTQSMSAGRPIPVEVQHRIEVSISVYPSVQPKIEAGPPKS (SEQ ID NO: 3, N-terminal non-catalytic domain of BaTdT)

[0071] IKFRDSTVYLVERRMGKTRRNFLAGLARSKGFCVENTLSSNVTHIVAEDNPADELWLWLQKQGIADLDKINVLDISWFTQSMSAGRPIPVEVQHRIEVSISVYPSVQPKIEAGPPKSPCLT VSQYACQRRTTLNNHNKILTDALEVLAENYELIESVGPCLGFKKAASVLKSLPVPVRSIRDVEGLPCLGPETMAVIEDIFEFGSSSKVEDVLKDERYQTLKIFTSVFGVGPKTAEKWYRQG LRNLTQIVSDSTIHLNNMQKAGFQYYDDISKPVSKAEAEALSRIIVEIAGCVNPEVTVTLTGGFRRGKEFGHDVDFLLHVPGPGKEDGLLPAVIDRLRSQGVLLYLDFQESTFDISKLPSC RFEAMDHFQKCFLILKLKKEQVVGQQVEQRCGKDWKAVRVDLVAPPAECYPFALLGWSGSTQFDRDLRRFARLERKMMLDNHALYDKTTNTFLQAKTEEDIFTHLGLDYIEPWQRNA(SEQ ID NO: 5, BaTdT wild type - full length)

[0072] In some embodiments, the catalytic domain shown in SEQ ID NO: 2 may have an amino acid sequence shown in SEQ ID NO: 4 attached to its N-terminus, in which case the TdT or its bioactive fragment may have an amino acid sequence shown in SEQ ID NO: 6, which is the full-length sequence of wild-type AcTdT.

[0073] SDAVTHVVSEDSPASSVWXWLKGRHLKNLPVMHVLDISWFTDSMREGKPVAVETRHLIQVCDTLPALPEGTTP (SEQ ID NO: 4, N-terminal non-catalytic domain of AcTdT)

[0074] SDAVTHVVSEDSPASSVWXWLKGRHLKNLPVMHVLDISWFTDSMREGKPVAVETRHLIQVCDTLPALPEGTTPTPVSTVSQYACQRRTTTQNNNQIFTDAFEVLAESHEF NEMEGPCLAFRRAASVLKSLPWPVQCLRATDDLPCLGEHSNFVIEEILQYGRSFEVEKILSDERYQTLKLFTSVFGVGPKTAEKWYRRGLRSFSDVLAEPDIHLNRMQQSG FLHYGDISRAVSKAEARALGNIIDEAVHAITPDAILALTGGFRRGKEFGHDVDFIVTTPQLGKEDCLLTGTIDRLKQQGILLYCDYQASTFDELKLPSHRFEAMDHFAKC FLILRLEDSQVEEGLQSAEEDSRGWRAVRVDLVSPPVDRYAFALLGWTGSRQFERDLRRFARMERRMLLDNHSLYDKTKKEFLAATTEKDIFAHLGLEYIEPWQRNA(SEQ ID NO: 6, AcTdT wild type - full length)

[0075] In some embodiments, single-point or multi-point directed mutations may be performed on the full-length sequence of wild-type BaTdT or the full-length sequence of wild-type AcTdT to optimize its catalytic performance.

[0076] In some embodiments, the full-length sequence of wild-type BaTdT can be mutated at one or more points to the catalytic domain described in the above embodiments, for example, by mutating amino acids at positions 310 and / or 311 of the catalytic domain shown in SEQ ID NO: 1 (corresponding to positions 427 and 428 of SEQ ID NO: 5, respectively), such as by amino acid substitution. In some embodiments, corresponding to replacing D with R at position 310 and / or replacing R with E at position 311 of the catalytic domain shown in SEQ ID NO: 1, SEQ ID NO: 5 can have the mutation forms D427R and / or R428E.

[0077] In some embodiments, the full-length sequence of wild-type AcTdT can be mutated at one or more points to the catalytic domain described in the above embodiments, for example, by mutating amino acids at positions 312 and / or 313 of the catalytic domain shown in SEQ ID NO: 2 (corresponding to positions 385 and 386 of SEQ ID NO: 6, respectively), such as by amino acid substitution. In some embodiments, corresponding to replacing E with R at position 312 and / or R with E at position 313 of the catalytic domain shown in SEQ ID NO: 2, SEQ ID NO: 5 can have the mutation forms E385R and / or R386E.

[0078] In this embodiment of the disclosure, other mutations may also be made based on the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, so that the terminal deoxynucleotidyl transferase or its bioactive fragment has the same characteristics as SEQ ID NO: 5 or SEQ ID NO: 6. The amino acid sequence shown in NO:6 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99% but less than 100% identity with the sequence, and the terminal deoxynucleotidyl transferase or its bioactive fragment having these identical sequences still has the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain. It is understood that these mutations can occur in the catalytic domain and / or the N-terminal or C-terminal non-catalytic domains attached thereto, including synonymous substitution of amino acids, truncation or substitution of non-catalytic domains, and the introduction of any fragment of interest. These structural mutations or substitutions also fall within the scope of protection of this disclosure.

[0079] It is understood that the terminal deoxynucleotidyl transferases proposed in the embodiments of this disclosure may also have other modifications as needed, such as surface modifications like macromolecular modifications, small molecule modifications, cross-linking modifications, and / or immobilization modifications; internal modifications like modifications targeting non-catalytic groups, catalytic groups, the main chain, cofactors, and peptide chain extension; and / or chemical modifications combined with site-directed mutagenesis. The terminal deoxynucleotidyl transferases proposed in the embodiments of this disclosure may also have their primary structure modified as needed, such as by adding a terminal tag. These modifications can be achieved by conventional methods in the art as needed, and they also fall within the protection scope of this disclosure.

[0080] The terminal deoxynucleotidyl transferase proposed in this embodiment has excellent catalytic activity for nucleotide polymerization and can be used in many aspects such as the biosynthesis of polynucleotides, polynucleotide characterization, and the preparation of biosensors. In addition, through further optimization, the obtained terminal deoxynucleotidyl transferase mutant exhibits even higher catalytic activity, thus also having extremely high application value.

[0081] A second aspect of this disclosure provides a fusion protein comprising a terminal deoxynucleotidyl transferase or a bioactive fragment thereof as described in any embodiment of the first aspect of this disclosure, and an additional portion fused thereto. Optionally, in some embodiments, the additional portion is located at the N-terminus and / or C-terminus of the terminal deoxynucleotidyl transferase or its bioactive fragment.

[0082] In some embodiments, the additional portion may optionally be a tag protein, such as at least one of Poly His (e.g., 2×His, 3×His, 4×His, 5×His, 6×His, 7×His, 8×His, 9×His, etc.), FLAG, GFP, Strep-Tag II, Poly Arg (e.g., 5×Arg), C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, Ubiquitin, and NusA. It is understood that the specific sequences of these tag proteins and the methods of fusion with the target protein are well known in the art; and as those skilled in the art know, these tag proteins added to the N-terminus or C-terminus of the target protein facilitate purification, and the addition of the tag protein does not affect the performance of the terminal deoxynucleotidyl transferase.

[0083] In the embodiments of this disclosure, other tag sequences may be introduced as additional parts into the terminal deoxynucleotidyl transferase, or other small molecules, macromolecule conjugates, etc., may be introduced as additional parts to form a fusion protein. This disclosure does not limit the specific form and conjugation method of the additional parts.

[0084] A third aspect of this disclosure provides a polynucleotide encoding a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure, or a fusion protein or its complementary sequence as described in any embodiment of the second aspect of this disclosure. In some embodiments, the polynucleotide may comprise sequences as shown in SEQ ID NO: 7 or SEQ ID NO: 8, which respectively encode catalytic domains of BaTdT and AcTdT as shown in SEQ ID NO: 1 or SEQ ID NO: 2. It should be noted that, considering codon degeneracy, the polynucleotides in the embodiments of this disclosure are intended to cover the entire nucleotide sequence corresponding to the amino acid sequence of each terminal deoxynucleotidyl transferase.

[0085] The fourth aspect of this disclosure also proposes a carrier comprising a polynucleotide as proposed in any embodiment of the third aspect of this disclosure.

[0086] In some embodiments, the vector may be a non-pathogenic viral vector or a non-viral vector. In some embodiments, the non-pathogenic viral vector may be an adenovirus vector or a retroviral vector. In some embodiments, the non-viral vector includes plasmids, such as eukaryotic expression plasmids or prokaryotic expression plasmids. The vectors used in the embodiments of this disclosure are well known in the art, and this disclosure does not limit the specific type of vector.

[0087] The fifth aspect of this disclosure also provides a cell comprising a polynucleotide as described in any embodiment of the third aspect of this disclosure, a vector as described in any embodiment of the fourth aspect of this disclosure, or expressing a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure, or a fusion protein as described in any embodiment of the second aspect of this disclosure.

[0088] In some embodiments, the cell may be a commonly used bacterial strain in bioengineering, such as Escherichia coli, yeast, lactic acid bacteria, etc. This disclosure does not limit the specific type of recombinant bacteria.

[0089] The sixth aspect of this disclosure also provides a kit comprising: a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure, or a fusion protein as described in any embodiment of the second aspect of this disclosure. In some embodiments, the kit further comprises an enzyme buffer and / or dNTPs. In some embodiments, the enzyme buffer of the kit comprises magnesium ions, manganese ions, and / or NaCl. In some embodiments, the buffer may be a phosphate buffer, borate buffer, citrate buffer, Tris buffer, arsenate buffer, or Hepes buffer. It is understood that the buffer provides a reaction environment for the nucleotide polymerization catalyzed by the terminal deoxynucleotidyl transferase, and the buffer formulation is well known in the art; this disclosure does not limit its specific type and amount. It is understood that the kits proposed in the embodiments of this disclosure may also contain other components for nucleic acid synthesis, sequencing, or biosensor detection, as well as instructions for use, etc.; this disclosure does not limit other components in the kit.

[0090] The seventh aspect of this disclosure also proposes the use of terminal deoxynucleotidyl transferases or their bioactive fragments as described in any embodiment of the first aspect of this disclosure, or fusion proteins as described in any embodiment of the second aspect of this disclosure, or kits as described in any embodiment of the sixth aspect of this disclosure, in one or more of the following: i) catalyzing the polymerization of nucleotides to extend mononucleotides or polynucleotides, preferably under template-free conditions; ii) synthesizing polynucleotides, preferably under template-free conditions, such as de novo synthesis; iii) catalyzing the replication of polynucleotides; iv) nucleic acid sequencing; v) labeling and / or detection of target nucleic acids; vii) mediating analyte detection in biosensors; vii) nucleic acid synthesis in bioinformatics storage; and viii) preparing products for any of i)-v).

[0091] The eighth aspect of this disclosure also provides a method for synthesizing polynucleotides, for example, synthesizing polynucleotides in the absence of the template strand, comprising: contacting a terminal deoxynucleotidyl transferase or its bioactive fragment as described in any embodiment of the first aspect of this disclosure, or a fusion protein as described in any embodiment of the second aspect of this disclosure, with nucleotides and primers under conditions suitable for polymerization, wherein the terminal deoxynucleotidyl transferase or its bioactive fragment or fusion protein catalyzes polymerization between nucleotides to elongate mononucleotides or polynucleotides, thereby synthesizing polynucleotides. In some embodiments, the conditions suitable for polymerization may include: a suitable reaction system, such as a suitable buffer solution, pH value, ion concentration, and suitable concentrations of each component; and suitable reaction time and reaction temperature. Conditions suitable for TdT-catalyzed polymerization are well known in the art, and this disclosure does not limit such reaction conditions.

[0092] In some embodiments, the nucleotide includes at least one of natural nucleotides and non-natural nucleotides, which may be ddNTP, dNTP and / or NTP, preferably dNTP.

[0093] In some embodiments, the non-natural nucleotide is a nucleotide modified with a 3'-OH end, such as a nucleotide modified with a reversible blocking group at the 3'-OH end. The reversible blocking group may include at least one of alkyl, aralkyl, alkenyl, alkynyl, allyl, aryl, heteroaryl, heterocyclic, benzyl, azide, azido, amino, ketone, isocyanate, phosphate, carbonate, thio, acyl, oxime, cyano, alkoxy, aryloxy, heteroaryloxy, or amide groups. It is understood that the TdT proposed in the embodiments of this disclosure can be used for highly efficient catalysis of the polymerization of nucleotides, especially modified nucleotides, thereby being effectively used for the biosynthesis of nucleic acids.

[0094] The ninth aspect of this disclosure also provides a method for labeling nucleic acids, comprising: contacting a terminal deoxynucleotidyl transferase or its bioactive fragment or fusion protein as described in any embodiment of this disclosure with a nucleotide and the nucleic acid under conditions suitable for polymerization, wherein the nucleotide is a non-natural nucleotide containing a detectable marker, and the terminal deoxynucleotidyl transferase or its bioactive fragment or fusion protein catalyzes the polymerization of one or more of the non-natural nucleotides at the 3' end of the nucleic acid to label the nucleic acid.

[0095] In some embodiments, the detectable marker may be a fluorescent marker, biotin, digoxigenin, and / or a radiolabel.

[0096] The tenth aspect of this disclosure also proposes a method for detecting the presence of a target nucleic acid in a test sample, comprising: a) providing a test sample containing nucleic acid; b) contacting the test sample with a probe specifically targeting the target nucleic acid to perform in situ hybridization to obtain an in situ hybridization product, wherein the probe contains a detectable marker, the probe being prepared by a method for synthesizing polynucleotides according to embodiments of this disclosure; c) removing probes that do not specifically bind to the target nucleic acid; and d) detecting the marker on the probe in the in situ hybridization product to determine whether the target nucleic acid is present in the test sample.

[0097] In some embodiments, the method further includes: performing qualitative, quantitative, and / or localization analysis on the target nucleic acid based on the presence of the target nucleic acid in the sample to be tested, using the signal intensity of the marker.

[0098] In some embodiments, the nucleic acids in the sample to be tested are single-stranded and are directly or indirectly immobilized.

[0099] In some embodiments, the probe is a single-stranded nucleic acid probe and contains non-natural nucleotides. In some embodiments, the probe is synthesized via a terminal deoxynucleotidyl transferase mediated by embodiments of this disclosure.

[0100] In some embodiments, the detectable marker may optionally be a fluorescent marker, biotin, digoxigenin, and / or a radiolabeled marker.

[0101] In this embodiment of the disclosure, when testing a sample, in situ hybridization (if present) can be performed on the target nucleic acid using a probe that specifically targets the target nucleic acid to obtain an in situ hybridization product. Then, probes that do not specifically bind to the target nucleic acid are removed. When the sample contains the target nucleic acid, the in situ hybridization product contains a hybrid of the target nucleic acid and the probe; when the sample does not contain the target nucleic acid, due to the binding specificity of the probe, the in situ hybridization product does not contain a hybrid of the target nucleic acid and the probe. Therefore, by detecting the presence of a detectable marker in the probe within the in situ hybridization product, it can be determined whether the sample contains the target nucleic acid; furthermore, based on the intensity of the detected marker signal, the content and location of the target nucleic acid can be determined, thereby completing the qualitative, quantitative, and / or localization analysis of the target nucleic acid in the sample.

[0102] It should be noted that the above explanations and descriptions of the terminal deoxynucleotidyl transferase or its bioactive fragments are also applicable to the fusion proteins, polynucleotides, vectors, cells, kits and their applications in the embodiments of this disclosure, and will not be repeated here.

[0103] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0104] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0105] Example 1: Obtaining a novel terminal deoxynucleotidyl transferase

[0106] In this embodiment, metagenomic sequencing and sequence analysis were performed on samples from the Somali Trench. Based on the published sequence structure of MuTdT (GenBank: AYJ71526.1), two novel proteins were identified and named BaTdT and AcTdT, respectively. Their amino acid sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0107] Using MEGA7 sequence alignment software, BaTdT (SEQ ID NO: 5) and AcTdT (SEQ ID NO: 6) were sequence aligned with MuTdT. The results showed that BaTdT and MuTdT had a sequence identity of 54.24%, AcTdT and MuTdT had a sequence identity of 51.8%, and BaTdT and AcTdT had a sequence identity of 59.82%. This indicates that BaTdT and AcTdT discovered in this embodiment are novel proteins with a completely new sequence backbone that is different from traditional terminal transferases.

[0108] Example 2: Three-dimensional structure prediction and analysis of BaTdT and AcTdT

[0109] Alphafold was used to predict the three-dimensional structural models of BaTdT and AcTdT, and the results are shown in Figure 1A. Figure 1B shows the superposition of the three-dimensional structural models of BaTdT, AcTdT, and MuTdT (PDB login number: 4qz9) for comparison. As shown in Figure 1B, comparative analysis with the crystal structure model of MuTdT revealed that the catalytic domains of the two novel proteins have a high degree of similarity to those of MuTdT. This indicates that although the sequence identity of the two novel proteins with existing terminal transferases of the same type is less than 60%, their three-dimensional structures are highly homologous, suggesting that these two novel proteins are highly likely to be terminal deoxynucleotidyl transferases and possess similar terminal transferase functional activities.

[0110] Example 3: Expression and purification of BaTdT and AcTdT

[0111] By observing the three-dimensional structural models of BaTdT and AcTdT in Figure 1A, it was found that both sequences have a free peptide chain far from the catalytic domain at their N-terminus, which might affect the in vitro soluble expression of the protein. Therefore, in this embodiment, the BaTdT and AcTdT sequences were truncated, i.e., their free peptide chains were removed (SEQ ID NO: 3 and SEQ ID NO: 4), and only the peptide chains containing the catalytic domain (SEQ ID NO: 5 and SEQ ID NO: 6) were retained for heterologous expression in *E. coli*. Based on the truncated position, the truncated sequences are referred to as BaTdT. (P118-A480) and AcTdT (T74-A438) .

[0112] 3.1 BaTdT (P118-A480) and AcTdT (T74-A438) expression

[0113] For BaTdT (P118-A480) and AcTdT (T74-A438) The amino acid sequence was codon optimized, and the optimized coding sequences are shown in SEQ ID NO: 7 and 8, respectively. SEQ ID NO: 7 and 8 were artificially synthesized, and NdeI and HindIII were used as restriction enzyme sites. The two sequences were cloned into the pGS-21a expression vector to obtain the recombinant expression vector pGS-21a / BaTdT. (P118— A480) and pGS-21a / AcTdT (T74—A438) and in BaTdT (P118—A480) and AcTdT (T74—A438)6×His tag was introduced before each step for subsequent purification.

[0114] The recombinant expression vector pGS-21a / BaTdT (P118—A480) and pGS-21a / AcTdT (T74—A438) Recombinant *E. coli* cells for recombinant protein expression were obtained by heat shock transformation into *E. coli* BL21(DE3) competent cells (Tiangen Biotech Co., Ltd.) and incubated overnight at 37°C. The recombinant *E. coli* cells were then inoculated into LB medium and cultured at 37°C for 8 hours. Finally, they were transferred to 300 mL of TB fermentation medium at a 5% inoculation rate. The inoculated TB medium was then incubated at 37°C and 200 rpm for 2-3 hours. The bacterial OD values ​​were then measured. 600 When the concentration was between 0.5 and 0.7, IPTG was added to a final concentration of 200 μM and the mixture was transferred to 16°C and 200 rpm for 24 h to induce fermentation. After fermentation, the fermentation broth was homogenized under high pressure (under conditions of 4°C and 80 MPa), and then centrifuged (12000 rpm, 30 min, 4°C). The supernatant was the crude TdT enzyme solution produced by the recombinant bacteria.

[0115] 3.2 BaTdT (P118-A480) and AcTdT (T74-A438) Purification

[0116] The two crude TdT enzyme solutions obtained in 3.1 were purified by nickel ion affinity chromatography. The specific steps are as follows.

[0117] First, the two crude TdT enzyme solutions were loaded onto nickel ion affinity chromatography columns (nickel columns, packing material purchased from Tiandi Renhe Biotechnology Co., Ltd.). Then, a gradient elution method was used to purify the recombinant protein: elution buffers 1 (containing 0% buffer B), 2 (containing 5% buffer B), 3 (containing 10% buffer B), 4 (containing 20% ​​buffer B), 5 (containing 50% buffer B), and 6 (containing 100% buffer B) were prepared using buffers A and B. The protein bound to the nickel column was then eluted stepwise with these elution buffers in ascending order of imidazole concentration. All elution buffers were pre-cooled to 4°C during purification to ensure the recombinant protein retained its biological activity. Elution buffer 4, containing the target protein, was concentrated by ultrafiltration using an ultrafiltration tube capable of retaining 30 kDa protein, and the purified elution buffer was replaced with 2× enzyme storage buffer. Finally, an equal volume of glycerol was added to the concentrated protein, mixed well, and stored at -20°C. The purity of the recombinant protein was analyzed by SDS-PAGE, and the results are shown in Figure 2.

[0118] Purification buffer A (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole;

[0119] Purification buffer B (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole;

[0120] 2× Enzyme Storage Buffer (pH 7.2): 40 mM Tris-HCl, 400 mM NaCl, 5% glycerol.

[0121] Figure 2 shows the purified BaTdT in this embodiment. (P118-A480) and AcTdT (T74-A438) The image shows an SDS-PAGE assay, where M represents the protein standard and lane 1 contains purified BaTdT. (P118-A480) Lane 2 contains purified AcTdT. (T74- A438) As shown in Figure 2, corresponding purified protein bands are displayed at the theoretical molecular size of each protein, and no other impurities are present in the lanes, indicating that the purified target protein, BaTdT, was obtained in this embodiment. (P118-A480) and AcTdT (T74-A438) .

[0122] Example 4: Polymerization activity test of BaTdT and AcTdT on natural nucleotides

[0123] Single-chain Oligo(dT) with a degree of polymerization of 18 was used. 18 As oligonucleotide substrates, four natural nucleotides (dATP, dTTP, dCTP, and dGTP) were used as substrates for the purification of BaTdT. (P118-A480) and AcTdT (T74-A438) Template-free polymerization activity was determined. Terminal deoxyribonucleoside transferase (Terminal deoxyribonucleoside transferase from calf thymus, NEB commercial enzyme, M0315S) was used as an experimental control, and Oligo(dT)18 standard was used as a negative control. Oligo(dT) oligonucleotide substrates were subjected to TdT. 18 Multiple dNTPs can be irregularly extended at the 3' end, resulting in products with a length greater than 18 nt. The reaction system for TdT activity testing is shown in Table 1. The reaction program was 37℃ for 10 min, followed by heating at 95℃ for 10 min to terminate the reaction. The reaction products were semi-quantitatively analyzed using urea-polyacrylamide gel electrophoresis (Urea-PAGE, 20% denaturing gel), and the results are shown in Figure 3.

[0124] Table 1

[0125] The 5×Reaction buffer formulation is: 0.5M Na-Cacodylate, 5mM CoCl2, pH 7.2.

[0126] In Figure 3, lane 1 is the negative control Oligo(dT). 18 Lane 2 is BaTdT (P118-A480) The polymerization product obtained by catalysis; lane 3 is AcTdT (T74-A438) The polymerization product obtained by catalysis; lane 4 is the polymerization product obtained by catalysis of terminal deoxyribonucleoside transferase (NEB commercial enzyme). As shown in Figure 3, BaTdT (P118-A480) and AcTdT (T74-A438) Both methods catalyze the polymerization of natural dNTPs to Oligo(dT) in the absence of a template. 18 The polymer was synthesized on the starting strand, yielding a product longer than 18 nt, indicating that both proteins are terminal deoxyribonucleoside transferases with template-free DNA polymerization activity. Furthermore, compared to the polymer synthesized by the NEB commercial enzyme shown in lane 4, BaTdT… (P118-A480) and AcTdT (T74-A438) The catalytic synthesis yielded longer polymer products, and these longer products also exhibited extremely high concentrations, indicating that the terminal deoxyribonucleoside transferase BaTdT proposed in this embodiment demonstrates its effectiveness. (P118-A480) and AcTdT (T74-A438) It has better polymerization effect and higher polymerization efficiency.

[0127] Example 5: Polymerization activity test of BaTdT and AcTdT on modified nucleotides

[0128] Single-chain Oligo(dT) with a degree of polymerization of 18 was used. 18 As oligonucleotide substrates, four non-natural nucleotides (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, and 3'-O-Azidomethyl-dGTP, whose chemical structures are shown in Figure 5) with an "azidomethyl" modification group at the 3'-OH end were used as nucleotide substrates for the purification of BaTdT. (P118-A480) and AcTdT (T74-A438) Template-free polymerization activity was determined. The oligonucleotide substrate was Oligo(dT). 18 A single nucleotide can be extended under the influence of TdT to obtain the product Oligo(dT). 18 -dNTP-3'-O-Azidomethyl. The reaction system and conditions for the polymerization reaction are the same as in Example 4, using Oligo(dT). 18 The standard was used as a negative control, using Oligo(dT). 19 The standard was used as a positive control. The reaction products were semi-quantitatively analyzed using Urea-PAGE (20% denaturing gel), and the results are shown in Figure 4.

[0129] In Figure 4, lane 1 is the negative control Oligo(dT). 18 Lane 2 is BaTdT (P118-A480) The polymerization product obtained by catalysis; lane 3 is AcTdT (T74-A438) The polymerization product obtained by catalysis; lane 4 is the positive control Oligo(dT). 19 As can be seen from Figure 4, BaTdT (P118-A480) and AcTdT (T74-A438) Both methods catalyzed the polymerization of modified dNTPs into Oligo(dT) in the absence of a template. 18 The two proteins were synthesized on the starting chain and a polymer product of 19 nt in length was obtained, indicating that both proteins, as novel terminal deoxyribonucleoside transferases, have the activity of catalyzing the polymerization of modified nucleotides and can be applied to many fields such as sequencing and de novo DNA synthesis.

[0130] Example 6: Expression and purification of BaTdT and AcTdT mutants

[0131] By analyzing the three-dimensional structural models of BaTdT and AcTdT in Figure 1A, this embodiment further mutated the catalytic active sites of the two novel terminal deoxyribonucleoside transferases at two sites. The mutation sites are BaTdT as shown in SEQ ID NO: 1. (P118-A480) The D310 and R311 sites correspond to D427 and R428 of the full-length BaTdT sequence shown in SEQ ID NO: 5, with the specific mutation being BaTdT. (P118-A480) -D310R / R311E corresponds to BaTdT-D427R / R428E. For AcTdT, the two mutation sites are AcTdT as shown in SEQ ID NO: 2. (T74-A438) The E312 and R313 sites correspond to E385 and R386 of the full-length AcTdT sequence as shown in SEQ ID NO: 6, with the specific mutation form being AcTdT. (T74-A438) -E312R / R313E corresponds to AcTdT-E385R / R386E. The specific steps are as follows.

[0132] Using the recombinant vector pGS-21a / BaTdT from Example 2 (P118—A480) Using primers F1 (SEQ ID NO: 9) and R1 (SEQ ID NO: 10) as templates, mutations were introduced at the two sites mentioned above using rapid PCR technology to obtain the mutant BaTdT. (P118-A480) -D310R / R311E; Similarly, the recombinant vector pGS-21a / AcTdT from Example 2 is used. (T74—A438)Using primers F2 (SEQ ID NO: 11) and R2 (SEQ ID NO: 12) as templates, mutations were introduced at the two sites mentioned above using rapid PCR technology to obtain the mutant AcTdT. (T74-A438) -E312R / R313E. The rapid PCR reaction system is shown in Table 2. The reaction program is as follows: pre-denaturation at 94℃ for 5 min; then 21 cycles (denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 7 min 50 s); finally, extension at 72℃ for 7 min, and hold at 4℃.

[0133] Table 2

[0134] F1: CCCAATTTCGCGAAGATCTGCGCCGC (SEQ ID NO: 9)

[0135] R1:CGCAGATCTTCGCGAAATTGGGTGCTG (SEQ ID NO: 10)

[0136] F2: GTCAATTTCGCGAAGATCTGCGCCGC (SEQ ID NO: 11)

[0137] R2:CGCAGATCTTCGCGAAATTGACGGC (SEQ ID NO: 12)

[0138] Dpn I was added to the PCR product, and the mixture was incubated at 37°C for 2 hours to degrade the recombinant vector template. The vector containing the mutant sequence was then transformed into *E. coli* JM109 competent cells (Tiangen Biotech Co., Ltd.). The transformation product was plated on LB solid medium containing 100 mg / L ampicillin and incubated at 37°C for 10-12 hours. Clones were picked and inoculated onto LB liquid medium and incubated at 37°C for 8-10 hours. Sanger sequencing was performed on the bacterial culture to verify the correct introduction of the mutant site. Plasmids were extracted from the correctly sequenced clones and transformed into *E. coli* BL21(DE3) competent cells (Tiangen Biotech Co., Ltd.) to obtain cells expressing BaTdT. (P118-A480) -D310R / R311E and ActdT (T74-A438) A recombinant *E. coli* strain with the -E312R / R313E mutant was successfully expressed and purified using the expression and purification method described in Example 3 to obtain the mutant protein BaTdT with good purity. (P118-A480) -D310R / R311E and ActdT (T74-A438) -E312R / R313E. Figure 6 shows the purified BaTdT from this embodiment. (P118-A480) -D310R / R311E and ActdT (T74-A438)SDS-PAGE image of -E312R / R313E, where M is the protein standard and lane 1 is the purified BaTdT. (P118-A480) -D310R / R311E, lane 2 contains purified AcTdT. (T74-A438) -E312R / R313E.

[0139] Example 7: Polymerization activity test of BaTdT and AcTdT mutants on modified nucleotides

[0140] BaTdT was treated according to the method in Example 5. (P118-A480) -D310R / R311E and ActdT (T74-A438) The template-free DNA polymerization activity of modified nucleotides as substrates was determined using E312R / R313E. The modified nucleotides used included dNTPs with an azidomethyl group at the 3'-OH end (i.e., 3'-O-azidomethyl-dNTP), dNTPs with an amide group at the 3'-OH end (i.e., 3'-O-amide-dNTP), and dTTPs with allyl, methyl, and oxime groups at the 3'-OH end, respectively, corresponding to 3'-O-allyl-dTTP, 3'-O-methy-dTTP, and 3'-O-oxime-dTTP, as shown in Figure 4. The polymerization products were semi-quantitatively analyzed according to the method in Example 5, and the results are shown in Figure 7.

[0141] In Figure 7, lane 1 is BaTdT (P118-A480) The polymerization product obtained by -D310R / R311E catalysis, lane 2 is AcTdT (T74-A438) The polymerization product obtained by E312R / R313E catalysis, with NC serving as the negative control Oligo(dT). 18 As shown in Figure 7, both mutants, TdT and Oligo(dT), can catalyze the polymerization of modified single nucleotides into Oligo(dT) in the absence of a template. 18The starting strand shows that both mutant proteins can also act as novel terminal deoxyribonucleoside transferases, exhibiting catalytic activity for the polymerization of modified nucleotides, such as 3'-O-Amide-dNTP, 3'-O-Allyl-dTTP, 3'-O-Methy-dTTP, and 3'-O-Oxime-dTTP. Furthermore, comparing the yields of polymerization products in Figures 4 and 7 reveals that the TdT mutant shown in Figure 7 synthesized more polymerization products, with a significantly thicker and brighter target band. This indicates that the TdT mutant proposed in this embodiment exhibits higher polymerization activity compared to wild-type BaTdT and AcTdT, thus demonstrating significant application value in various fields such as nucleic acid biosynthesis and sequencing.

[0142] Example 8: Biosynthesis of DNA catalyzed by BaTdT and AcTdT mutants

[0143] In this embodiment, magnetic beads are used as a solid-phase carrier, and terminal deoxynucleotidyl transferase mutant BaTdT is respectively placed on them. (P118-A480) -D310R / R311E and ActdT (T74-A438) Oligonucleotides were synthesized in solid phase under the catalysis of -E312R / R313E, and the specific steps are as follows.

[0144] 8.1 Pretreatment of magnetic beads

[0145] Dynabeads TM After mixing with M-270 Streptavidin, transfer 6.6 μL to a centrifuge tube and wash the magnetic beads with 6.6 μL of 2× Binding and Washing (B&W) Buffer. Place the centrifuge tube on a magnetic rack. Once the liquid is clear, discard the supernatant, then add 6.6 μL of 1× B&W Buffer to resuspend the magnetic beads and wash them again. After washing 1 to 2 times, discard the supernatant and resuspend the magnetic beads with 6.6 μL of 2× B&W Buffer.

[0146] The preparation method of B&W Buffer (2×) is as follows: 10mM Tris-HCl (pH 7.5), 1mM EDTA, 2M NaCl, 0.01–0.1% Tween-20.

[0147] 8.2 Fixing the starting sequence

[0148] Add 0.4 μL of 100 μM starting sequence (Oligo(dT)) to the cleaned magnetic bead system. 18 Then add 6.2 μL of water, mix well, and place on a shaker to incubate for 15 minutes.

[0149] After incubation, place the centrifuge tubes on a magnetic rack and discard the supernatant using the rack. Then, wash the magnetic beads three times with 6.6 μL 1×B&W Buffer, followed by one wash with Reaction Buffer. Discard the supernatant for later use.

[0150] The 5×Reaction buffer formulation is: 0.5M Na-Cacodylate, 5mM CoCl2, pH 7.2.

[0151] 8.3 Modified nucleotide coupling

[0152] The polymerization reaction system was prepared on ice (the system is shown in Table 3 below), in which the terminal deoxynucleotidyl transferase (TdT) was BaTdT. (P118-A480) -D310R / R311E and ActdT (T74-A438) -E312R / R313E, the modified nucleotide used is 3'-O-Oxime-dCTP, the reaction system is thoroughly mixed, and then placed in a PCR instrument at 37℃ for 1 minute.

[0153] Table 3

[0154] 8.4 Cleaning

[0155] Remove the coupling reaction solution, and use a magnetic rack to wash the magnetic bead system three times with 1×B&W Buffer. Then completely remove the washing buffer.

[0156] 8.5 Deprotection

[0157] Add sodium nitrite buffer (700mM, pH 5) to the cleaned magnetic beads and incubate for 1 minute. Repeat the deprotection step twice to remove the protecting group of the 3'-O modified mononucleotide polymerized at the end of the start sequence.

[0158] 8.6 Cleaning

[0159] After cleaning the magnetic bead system three times using a magnetic rack and 1×B&W Buffer, the cleaning buffer was completely removed.

[0160] 8.7 Repeat steps 6.3-6.6—coupling, washing, deprotection, and washing—four times in total to synthesize the target sequence. After all steps are completed, terminate the reaction by heating at 95°C for 10 minutes.

[0161] 8.8 Separation of the target sequence

[0162] After the reaction was terminated, the supernatant was discarded using a magnetic rack. The magnetic beads were washed three times with 6.6 μL of 1×B&W Buffer. Then, 10 μL of 0.1% SDS solution was added to the magnetic beads, and the mixture was heated at 98 degrees Celsius for 5 minutes to separate the synthesized target sequence. The supernatant was then transferred to a new EP tube.

[0163] 8.9 Detection of Synthetic Sequences

[0164] The target sequences synthesized in each cycle were detected using 20% ​​Urea-PAGE, and the results are shown in Figure 8. The -A portion represents the sequence synthesized in BaTdT. (P118-A480) The detection results of DNA synthesis products obtained under the catalysis of the -D310R / R311E mutant, Part B is the result of AcTdT (T74-A438) The detection results of DNA synthesis products obtained under the catalysis of the -E312R / R313E mutant are shown in lanes 1 to 8, respectively, at the starting sequence Oligo(dT). 18 The synthetic sequence of adding 1 to 8 single nucleotides is shown in Figure 8. As can be seen from Figure 8, the mutant TdT proposed in this embodiment can be used for de novo synthesis of oligonucleotides carrying protecting groups under template-free conditions based on a solid-phase support.

[0165] Example 9: Biosynthesis of Fluorescently Labeled DNA

[0166] This embodiment follows the synthesis steps of Example 8, and separately synthesizes the terminal deoxynucleotidyl transferase mutant BaTdT. (P118- A480) -D310R / R311E and ActdT (T74-A438) Solid-phase synthesis of fluorescently modified oligonucleotides was carried out under the catalysis of -E312R / R313E, using the modified base 3'-O-Oxime-dCTP-CY3 with CY3 fluorescent group on the base group.

[0167] The synthesized target sequence was detected by 20% Urea-PAGE, and the results are shown in Figure 9. In Figure 9, part A represents the sequence obtained in BaTdT... (P118-A480) The detection results of DNA synthesis products obtained under the catalysis of the -D310R / R311E mutant are shown in Figure 9, part B, which represents the products synthesized using AcTdT. (T74-A438) The detection results of DNA synthesis products obtained under the catalysis of the -E312R / R313E mutant. Lane 1 represents the starting sequence Oligo(dT). 18 Lanes 2 to 4 represent fluorescently labeled products with 1 to 3 single nucleotides added to the starting sequence, respectively. Figure 9 shows that in the mutant BaTdT (P118-A480) -D310R / R311E and ActdT (T74-A438)Catalyzed by -E312R / R313E, polymerized products of modified nucleotides were successfully generated, and each nucleotide in the product contained fluorescent groups. This indicates that the mutant proposed in this embodiment can be used for the biosynthesis of oligonucleotides modified with fluorescently labeled protecting groups under template-free conditions based on a solid-phase support.

[0168] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0169] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A terminal deoxynucleotidyl transferase or a biologically active fragment thereof, comprising a catalytic domain, said catalytic domain: a. Has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; b. An amino acid sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the catalytic domain is functional in catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain, wherein the mutation includes substitution, deletion, and / or addition; or c. An amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the catalytic domain having the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

2. The terminal deoxynucleotidyl transferase or its bioactive fragment according to claim 1, wherein the catalytic domain has the amino acid sequence shown in SEQ ID NO:

1. Optionally, the terminal deoxynucleotidyl transferase or its bioactive fragment is derived from the giant yarrelli (Bagarius yarrelli).

3. The terminal deoxynucleotidyl transferase or its bioactive fragment according to claim 1 or 2, wherein the catalytic domain has an amino acid mutation at at least one of positions 310 and 311 of SEQ ID NO: 1, wherein the mutation is preferably a substitution.

4. The terminal deoxynucleotidyl transferase or its bioactive fragment according to claim 3, wherein the catalytic domain has the following mutation compared to SEQ ID NO: 1: Replace the D in the 310th position with R; and / or Replace the R in the 311th position with E.

5. The terminal deoxynucleotidyl transferase or its bioactive fragment according to claim 1, wherein the catalytic domain has the amino acid sequence shown in SEQ ID NO:

2. Optionally, the TdT or its bioactive fragments are derived from the cichlid (Amphilophus citrinellus).

6. The terminal deoxynucleotidyl transferase or its bioactive fragment according to claim 1 or 5, wherein the catalytic domain has an amino acid mutation at at least one of positions 312 and 313 of SEQ ID NO: 2, wherein the mutation is preferably a substitution.

7. The terminal deoxynucleotidyl transferase or its bioactive fragment according to claim 6, wherein the catalytic domain has the following mutation compared to SEQ ID NO: 2: Replace the E in the 312th position with R; and / or Replace the R in the 313th position with E.

8. The terminal deoxynucleotidyl transferase or its bioactive fragment according to any one of claims 1 to 7, further comprising an N-terminal or C-terminal non-catalytic domain, said N-terminal or C-terminal non-catalytic domain comprising an α-helix and / or a β-sheet. Optionally, the N-terminal non-catalytic domain has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO:

4.

9. The terminal deoxynucleotidyl transferase or its bioactive fragment according to any one of claims 1 to 4, wherein the terminal deoxynucleotidyl transferase or its bioactive fragment has an amino acid sequence as shown in SEQ ID NO: 5 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5, and wherein the terminal deoxynucleotidyl transferase or its bioactive fragment has the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

10. The terminal deoxynucleotidyl transferase or its bioactive fragment according to any one of claims 1 to 7, wherein the terminal deoxynucleotidyl transferase or its bioactive fragment has an amino acid sequence as shown in SEQ ID NO: 6 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6, and wherein the terminal deoxynucleotidyl transferase or its bioactive fragment has the function of catalyzing the polymerization of a single nucleotide at the 3'-OH end of a polynucleotide chain.

11. A fusion protein comprising a terminal deoxynucleotidyl transferase or a biologically active fragment thereof as described in any one of claims 1 to 10, and an additional portion fused thereto. Optionally, the additional portion is a tag protein. Optionally, the tag protein is at least one selected from Poly His, FLAG, GFP, Strep-Tag II, Poly Arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, Ubiquitin, and NusA. Optionally, the additional portion is located at the N-terminus and / or C-terminus of the terminal deoxynucleotidyltransferase or its bioactive fragment.

12. A polynucleotide encoding a terminal deoxynucleotidyl transferase or a biologically active fragment thereof as described in any one of claims 1 to 10, or a fusion protein or its complementary sequence as described in claim 11. Optionally, the polynucleotide comprises a sequence as shown in SEQ ID NO: 7 or SEQ ID NO:

8.

13. A vector comprising the polynucleotide as described in claim 12.

14. A cell comprising the polynucleotide of claim 12 or the vector of claim 13, or expressing a terminal deoxynucleotidyl transferase or a bioactive fragment thereof as described in any one of claims 1 to 10 or a fusion protein of claim 11.

15. A kit comprising: Terminal deoxynucleotidyl transferase or its bioactive fragment as described in any one of claims 1 to 10, or fusion protein as described in claim 11; Optionally, the kit further includes enzyme buffer and / or nucleotides. Optionally, the enzyme buffer is selected from at least one of the following: phosphate buffer, borate buffer, citrate buffer, Tris buffer, arsenate buffer, and Hepes buffer. Preferably, the nucleotide includes at least one of natural nucleotides and non-natural nucleotides. Preferably, the non-natural nucleotide is a nucleotide modified at the 3'-OH end. Preferably, the 3'-OH modified nucleotide is obtained by modifying the nucleotide by adding a blocking group to the 3'-OH end. The blocking group includes at least one of alkyl, aralkyl, alkenyl, alkynyl, allyl, aryl, heteroaryl, heterocyclic, benzyl, azide, azido, amino, ketone, isocyanate, phosphate, carbonate, thio, acyl, oxime, cyano, alkoxy, aryloxy, heteroaryloxy, and amide.

16. The use of the terminal deoxynucleotidyl transferase or its bioactive fragment as described in any one of claims 1 to 10, the fusion protein as described in claim 11, or the kit as described in claim 15 in one or more of the following: i) Catalyzing the polymerization between nucleotides to extend mononucleotides or polynucleotides, preferably under template-free conditions; ii) Synthesize polynucleotides, preferably under template-free conditions; iii) Catalyzes the replication of polynucleotides; iv) Labeling and / or detection of the target nucleic acid; v) Nucleic acid synthesis in bioinformatics storage; and vi) Prepare products for any one of i)-iv). The nucleotides mentioned therein include at least one of natural nucleotides and non-natural nucleotides, optionally dNTPs and / or NTPs, preferably dNTPs.

17. A method for synthesizing polynucleotides, comprising: Under suitable conditions for polymerization, the terminal deoxynucleotidyl transferase or its bioactive fragment as described in any one of claims 1 to 10, or the fusion protein as described in claim 11, is contacted with nucleotides and primers. The terminal deoxynucleotidyl transferase or its bioactive fragment or the fusion protein catalyzes the polymerization between nucleotides to elongate mononucleotides or polynucleotides, thereby synthesizing polynucleotides. The nucleotides mentioned include at least one of natural nucleotides and non-natural nucleotides. Preferably, the non-natural nucleotide is a nucleotide modified at the 3'-OH end. Preferably, the 3'-OH modified nucleotide is obtained by modifying the nucleotide by adding a blocking group to the 3'-OH end. The blocking group includes at least one of O-alkyl, O-amide, O-amino, O-allyl, O-oxime, O-azido, and O-phosphate groups. Preferably, the synthesis is performed in the absence of the template chain. Preferably, the non-natural nucleotide further comprises a detectable marker, which may optionally be a fluorescent marker, biotin, digoxigenin, and / or a radiolabel.

18. A method for labeling nucleic acids, comprising: Under suitable conditions for polymerization, a terminal deoxynucleotidyl transferase or its bioactive fragment or the fusion protein as described in any one of claims 1 to 10 is contacted with a nucleotide and the nucleic acid, wherein the nucleotide is a non-natural nucleotide containing a detectable marker, and the terminal deoxynucleotidyl transferase or its bioactive fragment or the fusion protein catalyzes the polymerization of one or more of the non-natural nucleotides at the 3' end of the nucleic acid to label the nucleic acid.

19. A method for detecting the presence of a target nucleic acid in a sample to be tested, comprising: a) Provide a sample to be tested, wherein the sample contains nucleic acid. b) Contacting a probe specifically targeting a nucleic acid with a sample to be tested for in situ hybridization to obtain an in situ hybridization product, wherein the probe contains a detectable marker, and the probe is prepared by the method for synthesizing polynucleotides according to claim 17. c) Remove probes that do not specifically bind to the target nucleic acid, and d) Detect the probe marker in the in situ hybridization product to determine whether the target nucleic acid is present in the sample to be tested. Optionally, the nucleic acid in the sample to be tested is single-stranded and is directly or indirectly immobilized. Optionally, the probe is a single-stranded nucleic acid probe and contains non-natural nucleotides. Optionally, the detectable marker may be a fluorescent marker, biotin, digoxigenin, and / or a radiolabeled marker.

20. The method of claim 19, further comprising: Based on the presence of the target nucleic acid in the sample to be tested, the target nucleic acid is qualitatively, quantitatively, and / or locally analyzed by the signal intensity of the marker.

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