CN118256467B - Terminal deoxynucleotidyl transferase variants, nucleic acids and uses - Google Patents

Abstract

The present invention relates to terminal deoxynucleotidyl transferase variants, nucleic acids and applications, and relates to the field of genetic engineering. It includes wild-type or mutant terminal deoxynucleotidyl transferase (TdT), single-stranded DNA stabilizing factor (sSF) and flexible linker; the single-stranded DNA stabilizing factor is fused to the N-terminus or/and C-terminus of the terminal deoxynucleotidyl transferase through the flexible linker, or inserted into the middle of the sequence of the terminal deoxynucleotidyl transferase. The present invention obtains terminal deoxynucleotidyl transferase mutants that are independent of templates, and these TdT mutants can significantly improve their efficiency of coupling natural nucleotides and 3'-OH modified nucleotides, while enhancing the synthesis efficiency of 3'-OH modified nucleotides incorporated into oligonucleotides with hairpin secondary structures, which can be used for efficient and controllable synthetic oligonucleotides, and these TdT mutants can be used for the synthesis of enzymatic oligonucleotides, or for other cyclic synthesis of functional DNA fragments.

Description

Terminal deoxynucleotidyl transferase variants, nucleic acids and applications

Technical Field

The present invention relates to the field of genetic engineering, and in particular to terminal deoxynucleotidyl transferase variants, nucleic acids and applications.

Background Art

DNA is a super information storage carrier evolved by nature, and artificial DNA is an important foundation for future information storage technology. The key methods for synthesizing DNA from scratch include chemical synthesis and biosynthesis. Although the traditional chemical synthesis method is widely used in the industrial preparation of oligonucleotide chains, the chemical method uses a large amount of toxic and flammable organic reagents, which causes great pollution. And due to the limitations of chemical methods, the synthesis length of disposable oligonucleotides is limited. Therefore, how to use biosynthesis to achieve the controllable synthesis of mild and environmentally friendly oligonucleotides has gradually become the research core of DNA synthesis and storage.

Terminal deoxyribonucleoside transferase (TdT) is independent of the template DNA sequence, has a small preference for the four nucleotides and high coupling efficiency, can continuously synthesize and extend single-stranded DNA, and produce homopolymers up to 8000 nt, making it the most promising DNA synthesis tool enzyme. In order to use TdT to achieve controllable single-stranded DNA synthesis, the modification direction of TdT synthetase is focused on improving the activity of TdT in incorporating nucleoside triphosphates carrying blocking groups at the non-natural 3'-OH end into polynucleotides. The nucleotides carrying blocking groups need to be further cleaved of the reversible group before entering the next synthesis cycle, thereby achieving efficient and controllable operation of the enzymatic reaction, such as patents CN110331136B, CN113302289A, and CN114207140A.

In addition, Keasling’s team developed a method for reversibly covalently linking TdT to a single nucleoside triphosphate to prevent further extension of the DNA chain synthesized by TdT. When the reversible covalent bond is broken, the DNA chain can enter a new synthesis cycle. Controllable DNA synthesis based on innovative TdT has received widespread attention from academia and industry. However, as the length of the DNA chain increases, single-stranded DNA is prone to form various hairpin structures. Once the bases near the 3’ end participate in the formation of secondary structures, they will directly hinder TdT’s recognition of the DNA chain, thereby impairing the extension efficiency of TdT. Due to the increasingly significant application of TdT in DNA synthesis, it is necessary to develop highly active TdT that can tolerate long-chain secondary structures.

Summary of the invention

The technical problem to be solved by the present invention is to provide terminal deoxynucleotidyl transferase variants, nucleic acids and applications. The purpose is to achieve wrapping of single-stranded DNA with high binding force to prevent the formation of secondary structures by fusing some single-stranded DNA stabilizing factors to existing TdT, thereby improving the efficiency of TdT in long-chain DNA synthesis.

The technical solution of the present invention to solve the above technical problems is as follows:

In the first aspect, a terminal deoxynucleotidyl transferase variant comprises a wild-type or mutant terminal deoxynucleotidyl transferase (TdT), a single-stranded DNA stabilizing factor (sSF) and a flexible linker; the single-stranded DNA stabilizing factor is fused to the N-terminus and/or C-terminus of the terminal deoxynucleotidyl transferase via the flexible linker, or the single-stranded DNA stabilizing factor is inserted into the middle of the sequence of the terminal deoxynucleotidyl transferase via the flexible linker.

Furthermore, the single-stranded DNA stabilizing factor has a protein domain or subdomain that binds to or wraps single-stranded DNA with a binding energy at a level of mM or below mM.

Furthermore, the single-stranded DNA stabilizing factor is derived from an organism; the organism includes any one of a virus (such as T4 phage, T7 phage), a prokaryotic organism, a bacterium (such as Escherichia coli), an archaeon (such as hyperthermophilic archaea, Thermus aquaticus), and a eukaryotic organism.

The eukaryotic organism may be a lower eukaryotic organism, such as yeast (e.g., Pichia pastoris) or fungi (e.g., Aspergillus), or a higher eukaryotic organism, such as a plant (e.g., Arabidopsis), a reptile (e.g., a bird), or a mammal (e.g., a human).

Furthermore, the sequence of the single-stranded DNA stabilizing factor is shown in any one of SEQ ID NO:1 to SEQ ID NO:6; the sequence of the terminal deoxynucleotidyl transferase is shown in SEQ ID NO:7 or SEQ ID NO:8.

Furthermore, the flexible linker has a sequence of glycine (G) and serine (S) as a repeating module: (GS) _m , wherein m=0-20, m is zero or an integer, for example, 1, 2, 4, 6, 8, 10; or the flexible linker has a sequence of glycine (G) as a repeating module: (G) _n , wherein n=0-40, n is zero or an integer.

Furthermore, the sequence of the terminal deoxynucleotidyl transferase variant is shown in any one of SEQ ID NO 9 to SEQ ID NO 15.

The second aspect provides a nucleic acid encoding the terminal deoxynucleotidyl transferase variant.

In a third aspect, a method for synthesizing a nucleic acid molecule in the absence of a template chain comprises the following steps: contacting a starting nucleotide chain with at least one nucleotide in the presence of the terminal deoxynucleotidyl transferase variant.

Furthermore, the terminal deoxynucleotidyl transferase variant is coupled with a nucleotide; the nucleotide is a natural nucleotide or a nucleotide with a modified 3'-OH end, and the nucleotide with a modified 3'-OH end is a nucleotide with a blocking group added to the 3'-OH end.

In a fourth aspect, a kit is provided, comprising the terminal deoxynucleotidyl transferase variant, one or more nucleotides, and at least one starting nucleotide chain.

The beneficial effects of the present invention are as follows: the present invention obtains terminal transferase (TdT) mutants that are independent of templates, and these mutants can significantly improve their efficiency in coupling natural nucleotides and 3'-OH modified nucleotides, while enhancing the synthesis efficiency of 3'-OH modified nucleotides incorporated into DNA with a hairpin secondary structure, and can be used for efficient and controllable synthesis of oligonucleotides. These TdT mutants can be used for the synthesis of enzymatic oligonucleotides, or for other cyclic synthesis of functional DNA fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the construction of TdT variants in the method of the present invention;

Figure 2 is an SDS-PAGE diagram of the expression and purification of the TdT protein of the present invention; wherein, WT-Ms (Lane 2): TdT from mouse species; Sso-Ms (Lane 3): a new variant of mouse TdT fused with the hyperthermophilic archaeon SSB; WT-Za (Lane 5): TdT from the white-throated sparrow species; Ec-Za (Lane 6): a new variant of the white-throated sparrow fused with the Escherichia coli SSB; Sso-Za (Lane 7): a new variant of the white-throated sparrow fused with the hyperthermophilic archaeon SSB; T4-Za (Lane 8): a new variant of the white-throated sparrow fused with the T4 phage SSB; T7-Za (Lane 9): a new variant of the white-throated sparrow fused with the T7 phage SSB; Hs-Za (Lane 10): a new variant of the white-throated sparrow fused with the human SSB; Taq-Za (Lane 11): a new variant of the white-throated sparrow fused with the aquatic Thermus Taq A new variant of the White-throated Sparrow of SSB.

Figure 3 is a natural nucleotide reaction diagram of the present invention; wherein, NC: negative control, only starting nucleotide; Za: adding TdT of white-throated sparrow species; Sso-Za: a new variant of white-throated sparrow fused with hyperthermophilic archaeon SSB; Ec-Za: a new variant of white-throated sparrow fused with Escherichia coli SSB; Ms: adding mouse TdT; SSo-Ms: a new variant of mouse TdT fused with hyperthermophilic archaeon SSB.

Figure 4 shows the incorporation efficiency of nucleotides modified at the 3'-OH end of the present invention; wherein, the bar graph represents the incorporation efficiency of four nucleotides modified with amino groups at the 3' end catalyzed by wild-type white-throated sparrow TdT (gray), white-throated sparrow TdT fused with hyperthermophilic archaea SSB (red), and white-throated sparrow TdT fused with Escherichia coli SSB (red).

FIG5 is a reaction diagram of the incorporation of non-natural nucleotides into a single-stranded hairpin structure DNA of the present invention; wherein four single-stranded DNAs containing a hairpin secondary structure were tested; L14S9: 14 bases in total at 5' and 3' are complementary to form a loop consisting of 9 bases; L14S9+2: two more bases than at the 3' end of L14S9; L14S9+4: four more bases than at the 3' end of L14S9; L14S9+6: six more bases than at the 3' end of L14S9;

FIG6 is a reaction application of the solid phase catalytic oligonucleotide polymerization of the terminal deoxynucleotidyl transferase variant of the present invention; solid phase synthesis was performed using the white-throated finches species variant Ec-Za TdT, for a total of ten cycles; NC: no TdT was added.

DETAILED DESCRIPTION

The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

Definition and Explanation

In the present invention, the single-stranded DNA stabilizing factor is defined as: a protein domain or subdomain that can bind and wrap single-stranded DNA with high affinity to protect the single-stranded DNA from degradation and prevent the formation of secondary structure. The term comes from the early functional research on single-stranded DNA binding protein (SSB). SSB was found to have the function of accelerating the opening of DNA helical structure, binding to free single-stranded DNA to stabilize the single-stranded structure, and ultimately promoting DNA replication. The terminology of the present invention continues to use this concept. However, it should be noted that the DNA stabilizing factor of the present invention includes but is not limited to the known SSB, and other biological structures (such as protein domains) or chemical structures (such as cyclic peptides, modified polypeptides, nucleic acid aptamers, etc.) with similar properties should belong to the category of single-stranded DNA stabilizing factors defined in the present invention.

In the present invention, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); 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).

In the present invention, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of a gene caused by the introduction of exogenous DNA into a cell. The term "terminal deoxyribonucleoside transferase produced by a transformant" refers to a product obtained by culturing a transformant according to a known method for culturing a microorganism.

In the present invention, "synthesis of nucleic acid molecules" includes methods for synthesizing lengths of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), wherein the chain of nucleic acid (n) is extended by adding additional nucleotides (n+1). In one embodiment, the nucleic acid is DNA. In alternative embodiments, the nucleic acid is RNA. For example, a DNA chain synthesis method in which DNA chain (n) is extended by adding additional nucleotides (n+1). The methods described herein provide new uses of the terminal deoxynucleotidyl transferase of the present invention and 3' reversibly modified nucleotides for sequentially adding nucleotides in de novo DNA chain synthesis.

In the present invention, the term "nucleotide" may be a deoxyribonucleotide (dNTPs) or a dideoxyribonucleotide (ddNTPs); in one embodiment, the nucleotide may be a nucleotide triphosphate, which refers to a molecule containing a nucleoside (i.e., a base linked to a deoxyribose or ribose molecule) bound to three phosphate groups. Examples of nucleotide triphosphates containing deoxyribose are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), or deoxythymidine triphosphate (dTTP). Examples of nucleotide triphosphates containing ribose are: adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), or uridine triphosphate (UTP), and other nucleosides may be bound to three phosphates to form nucleotide triphosphates, such as naturally occurring modified nucleosides and artificial nucleosides. In the present invention, "3'-OH terminal modified nucleotides" refer to nucleotides with additional groups at the 3'-OH terminal (e.g., dATP, dGTP, dCTP or dTTP) that prevent further addition of nucleotides during the reaction, i.e., by replacing the 3'-OH group with a blocking group. It should be understood that the present method uses a reversible 3'-terminal blocking group that can be removed by cleavage (e.g., a cleaving agent) to allow the addition of additional nucleotides.

"TdT" in the present invention refers to terminal deoxynucleotidyl transferase (TdT), and includes references to purified and recombinant forms of the enzyme. TdT is also commonly referred to as DNTT (DNA nucleotidyl transferase), and any such terms should be used interchangeably. The terminal deoxynucleotidyl transferase (TdT) of the present invention is added in the presence of an extension solution comprising one or more buffers (e.g., Tris, Hepes, Mops, phosphate, carbonate or dimethyl arsenate, etc.), one or more salts (e.g., Na ⁺ , K ⁺ , Mg2 ⁺ , Mn2 ⁺ , Cu2 ⁺ , Zn2 ⁺ , Co2 ^+, etc., all with appropriate counterions, such as ^Cl- ), one or more solutions of stabilizing protein structures (e.g., glycerol, PMSF or Triton, etc.) and an inorganic pyrophosphatase (e.g., a Saccharomyces cerevisiae homolog). It should be understood that the selection of buffers and salts depends on optimal enzyme activity and stability.

This embodiment relates to a terminal deoxynucleotidyl transferase variant, which includes a wild-type or mutant terminal deoxynucleotidyl transferase (TdT), a single-stranded DNA stabilizing factor (sSF) and a flexible linker; the single-stranded DNA stabilizing factor is fused to the N-terminus and/or C-terminus of the terminal deoxynucleotidyl transferase through the flexible linker, or the single-stranded DNA stabilizing factor is inserted into the middle of the sequence of the terminal deoxynucleotidyl transferase through the flexible linker.

The specific connection mode of the terminal deoxynucleotidyl transferase variant of the present invention is Formula I, Formula II or Formula III, etc.: In formulas Ⅰ to Ⅲ, sSF represents a single-stranded DNA stabilizing factor, TdT represents terminal deoxynucleotidyl transferase, TdT-A and TdT-B represent two parts of terminal deoxynucleotidyl transferase; formula Ⅰ indicates that the single-stranded DNA stabilizing factor is fused to the N-terminus of TdT; formula Ⅱ indicates that the single-stranded DNA stabilizing factor is fused to the C-terminus of TdT; formula Ⅲ indicates that the single-stranded DNA stabilizing factor is fused in the middle of the TdT sequence.

In this embodiment, the single-stranded DNA stabilizing factor has a binding energy at a level of mM or below, such as mM, μM, nM, pM, etc., to bind or wrap the protein domain or subdomain of the single-stranded DNA. The single-stranded DNA stabilizing factor can protect the DNA single strand from degradation and prevent the formation of secondary structures.

In this embodiment, the single-stranded DNA stabilizing factor is preferably derived from an organism; the organism includes any one of a virus (such as T4 phage, T7 phage), a prokaryotic organism, a bacterium (such as Escherichia coli), an archaeon (such as hyperthermophilic archaea, Thermus aquaticus), and a eukaryotic organism.

The eukaryotic organism may be a lower eukaryotic organism, such as yeast (e.g., Pichia pastoris) or fungi (e.g., Aspergillus), or a higher eukaryotic organism, such as a plant (e.g., Arabidopsis), a reptile (e.g., a bird), or a mammal (e.g., a human).

In this embodiment, preferably, sSF can be selected from the amino acid sequence of single-stranded nucleic acid binding protein domain (SSB), including one or more SSB amino acid regions or homologous regions of other species or homologous regions of SSB of any species or amino acid regions of nucleic acid binding proteins of any species. Preferably, the sSF sequence can be shown by any one of SEQ ID NO:1 to SEQ ID NO:6. sSF can also be derived from linear polypeptides, cyclic peptides, or other structural compositions with nucleic acid binding ability (mM and below). Single-stranded nucleic acid binding proteins are ubiquitous in nature and exist in many species. A large number of known single-stranded nucleic acid binding proteins have been reported in the Uniprot database, as shown in Table 1.

Table 1

In this embodiment, the terminal deoxynucleotidyl transferase (TdT) sequence can be selected from any known natural or mutant sequence of a wild-type sequence or a truncated form or a known amino acid mutation form having non-template-dependent catalytic nucleotide polymerization activity. Specifically, the sequence of the terminal deoxynucleotidyl transferase is shown in SEQ ID NO: 7 or SEQ ID NO: 8. The terminal deoxyribonucleoside transferase can be derived from: gi768 cattle (Bostaurus); gi16923262 Homo sapiens; gi460163 Gallus gallus; gi494987 African clawed frog (Xenopus laevis); gi1354475 rainbow trout (Oncorhynchus mykiss); gi2149634 short-tailed opossum (Monodelphis domestica); gi1280244 house mouse (Mus musculus); gi28852989 Mexican tiger axolotl (Ambystoma mexicanum); gi1491390057 White-throated sparrow; gi101919206 peregrine falcon; gi1708926270 golden eagle (Aquila chrysaetos); gi 975102570 Gecko (Gekko); gi38603668 Redfin pufferfish (Takifugu rubripes); gi40037389 Crystalline ray (Raiaeglanteria); gi40218593 Nurse shark (Ginglymostoma cirratum); gi46369889 Zebrafish (Danioreri); gi73998101 Domestic dog (Canis lupus familiaris); gi139001476 Ring-tailed lemur (Lemurcatta); gi507537868 African jerboa (Jaculus iaculus); gi507572662 African jerboa; gi507622751 Chilean degu (Octodon degus); gi507640406 Pony Island hedgehog (Echinops telfairi); gi507669049 Pony Island hedgehog; gi507930719 Star-nosed mole (Condylura cristata); gi507940587 Star-nosed mole; gi511850623 Mustela putorius furo; gi512856623 Clawed frog; gi512952456 Naked mole rat; gi524918754 Golden hamster (Mesocricetus auratus); gi527251632 Budgie (Melopsittacus undulatus); gi528493137 Zebrafish; gi528493139 Zebrafish; gis29438486 Peregrine falcon (Falco peregrinus); gi530565557 Western painted turtle (Chrysemys picta bellii；gi532017142Prairie vole (Microtus ochrogaster)；gi 532099471Thirteen-lined ground squirrel (Ictidomys tridecemlineatus)；gi533166077Chinchilla lanigera；gi533189443Chinchilla；gi537205041Chinese ground slug (Cricetulus griseus)；gi537263119Chinese ground slug；gi543247043Chinese ground finch (Geospizafortis)；gi543351492Ground tit (Pseudopodoces humilis)；gi543731985Columbalivia；gi544420267Macaca fascicularis fascicularis); gi545193630 domestic horse; gi548384565 Pundamilia nyererei; gi551487466 Xiphophorus maculatus; gi551523268 Xiphophorus maculatus; gi554582962 Brandt's myotis brandtii; gi554588252 Brandt's myotis; gi556778822 Tibetan antelope (Pantholops hodgsonii); gi556990133 Latimeria chalumnae; gi557297894 Alligator sinensis; gi558116760 Chinese soft-shell turtle (Pelodiscus sinensis); gi558207237 Little brown bat (Myotis lucifugus；gi560895997Wild camel (Camelusferus);gi139001490Dwarf lemur (Microcebus murinus);gi139001511Small-eared monkey (Otolemurgarnettii);gi148708614House mouse;gi149040157Rattus norvegicus;gi149704611Domestic horse (Equus caballus);gi164451472Domestic cattle;gi169642654Tropical clawed frog (Xenopus (Silurana) tropicalis);gi291394899European rabbit (Oryctolagus cuniculus);gi291404551European rabbit;gi301763246Giant panda (Ailuropoda melanoleuca); gi311271684 wild boar (Sus scrofa); gi327280070 Anolis carolinensis; gi334313404 short-tailed opossum; gi 344274915 African elephant (Loxodonta africana); gi345330196 platypus (Ornithorhynchus anatinus); gi348588114 guinea pig (Cavia porcellus); gi351697151 naked mole rat (Heterocephalus glaber); gi355562663 macaque (Macaca mulatta); gi395501816 bag jar (Sarcophilus harrisii); gi395508711 bagged; gi395850042 small-eared monkey; gi397467153 bonobo (Pan paniscus); gi403278452 Saimiriboliviensis boliviensis; gi410903980 red-scaled pufferfish; gi410975770 domestic cat (Felis catus); gi432092624 David's mouse-eared bat (Myotis davidii); gi432113117 David's mouse-eared bat; gi444708211 tree shrew (Tupaia chinensis); gi460417122 European and African ribbed newt (Pleurodeles waltl); gi466001476 killer whale (Orcinus orca); gi471358897 Florida manatee (Trichechus manatus latirostris); gi478507321 White rhinoceros (Ceratotherium simum simum); gi478528402 White rhinoceros; gi488530524 Nine-banded armadillo (Dasypus novemcinctus); gi499037612 Zebra cichlid (Maylandia zebra); gi504135178 North American pika (Ochotona princeps); gi505844004 Common shrew (Sorex araneus); gi505845913 Common shrew; gi560897502 Wild camel; gi562857949 Tree shrew; gi562876575 Tree shrew; gi564229057 Mississippi alligator (Alligator mississippiensis); gi564236372 Mississippi alligator; gi564384286 Rattus norvegicus, etc.

In this embodiment, the flexible linker is preferably allowed to be composed of any type and length of amino acids or other chemical structural groups of similar length as the connecting part. The flexible linker is a sequence of glycine (G) and serine (S) as a repeating module: (GS) _m , where m = 0 to 20, m is zero or an integer, for example, it can be 1, 2, 4, 6, 8, 10; or the flexible linker is a sequence of glycine (G) as a repeating module: (G) _n , where n = 0 to 40, n is zero or an integer. The linker can also be other chemical structural groups of similar length, such as PEG.

When the TdT variant is derived from mice (145-510 amino acid fragment), the N-terminal segment is fused with the 2-117 amino acid fragment of the hyperthermophilic archaeon SSB protein, and the linker is amino acid EF. When the TdT variant is derived from white-throated sparrow (145-513 amino acid fragment), it also contains C187A, C215S, C319S, R335L, K337G, C375A, C417S point mutations, and the N-terminal segment is fused with the 2-115 amino acid fragment of the SSB protein of Escherichia coli, or the 2-117 amino acid fragment of the SSB protein of hyperthermophilic archaea, or the 21-153 amino acid fragment of the SSB protein of T4 bacteriophage, or the 2-186 amino acid fragment of the SSB protein of T7 bacteriophage, or the 2-230 amino acid fragment of the SSB protein of Thermus aquaticus, or the 2-110 amino acid fragment of the SSB protein of human origin; the linker is amino acid EF.

Preferably, in this embodiment, the sequence of the terminal deoxynucleotidyl transferase variant is shown in any one of SEQ ID NO 9 to SEQ ID NO 15.

This embodiment also relates to a nucleic acid, characterized in that the nucleic acid encodes the terminal deoxynucleotidyl transferase variant. The nucleic acid can also be a nucleic acid sequence in which nucleic acid codons are replaced but the amino acid sequence is not changed, or is obtained from a nucleic acid sequence encoding the nucleic acid domain or a precursor thereof, a terminal deoxyribonucleoside transferase or a precursor thereof.

This embodiment also relates to a method for producing the terminal deoxynucleotidyl transferase variant, the method comprising:

Cultivating a host cell expressing the nucleic acid under conditions suitable for expression of the nucleic acid encoding the terminal deoxynucleotidyl transferase variant; and recovering the terminal deoxynucleotidyl transferase variant from the cell culture.

The present embodiment is preferred, the host cell can be a prokaryote, such as Escherichia coli, or a eukaryote. The eukaryote can be a lower eukaryote, such as yeast (e.g., Pichia pastoris or Kluyveromyces lactis) or fungi (e.g., belonging to Aspergillus (Aspergillus)) or higher eukaryotes such as insect cells (e.g., Sf9 or Sf21), mammalian cells or plant cells. The cell can be a mammalian cell, such as COS (green monkey cell line), CHO (Chinese hamster ovary cell line), mouse cell and human cell, etc.

This embodiment also relates to an expression cassette for producing the terminal deoxynucleotidyl transferase variant, comprising all elements for expressing the terminal deoxynucleotidyl transferase variant, including elements necessary for transcription and translation in the host cell, for example, the expression cassette comprises a promoter and a terminator, and the promoter and the terminator are not particularly limited, and can be a promoter and a terminator known in the art that can achieve the expression of the variant. For example, the promoter can be prokaryotic or eukaryotic, and can be selected from, for example, Lacl, LacZ, pLacT, ptac, T3 or T7 phage RNA polymerase promoter, CMV promoter, HSV thymidine kinase promoter, SV40 promoter, mouse metallothionein-L promoter, etc.

The expression cassette of the present invention also includes a vector. The vector may be a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC, a BAC, an Agrobacterium pTi plasmid, etc. The vector may preferably contain one or more elements selected from the following: an origin of replication, a multiple cloning site, and a selectable gene. Preferably, the vector is a plasmid. Some non-exhaustive examples of prokaryotic vectors are as follows: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescriptSK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A; ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, pRIT5, pET-28a. Preferably, the vector is an expression vector, preferably pET-28a.

This embodiment also relates to a method for synthesizing a nucleic acid molecule in the absence of a template chain, comprising the following steps: contacting a starting nucleotide chain with at least one nucleotide in the presence of the terminal deoxynucleotidyl transferase variant, wherein the terminal deoxynucleotidyl transferase variant can be efficiently coupled with nucleotides, including being used for the synthesis of enzymatic oligonucleotides, or for other cyclic synthesis of functional DNA and/or RNA fragments, or for the cyclic iteration of synthesizing DNA/RNA in an enzymatic DNA synthesizer.

In this embodiment, the terminal deoxynucleotidyl transferase variant is coupled with a nucleotide; the nucleotide is a natural nucleotide or a nucleotide with a modified 3'-OH end, and the nucleotide with a modified 3'-OH end is a nucleotide with a modified blocking group added to the 3'-OH end. Specifically, the blocking group may also include, but is not limited to, a phosphate group, an azide, and a 2-nitrophenyl group.

In this embodiment, the starting nucleotide chain is a single-stranded DNA with a secondary hairpin structure. The terminal deoxynucleotidyl transferase variant of the present invention can incorporate nucleotides with modified 3'-OH ends into a variety of hairpin structure DNAs in the absence of a template chain, and can enhance the chain extension ability of the single-stranded DNA with a secondary structure to synthesize nucleic acid molecules compared to the original TdT sequence.

This embodiment also relates to a kit, which includes the terminal deoxynucleotidyl transferase variant, one or more nucleotides, and at least one starting nucleotide chain.

The embodiments of the present invention will be described in detail below with reference to specific examples.

Example 1: Construction of TdT variants

1. Amino acid sequence of single-stranded DNA stabilizing factor

The amino acid sequence of the single-stranded DNA stabilizing factor derived from hyperthermophilic archaea is SEQ ID NO: 1 (EcSSB (P0AGE0, Escherichia coli), 2-115); the amino acid sequence of the single-stranded DNA stabilizing factor derived from Escherichia coli is SEQ ID NO: 2 (SsoSSB (Q97W73, Sulfolobus solfataricus), 2-117); the amino acid sequence of the single-stranded DNA stabilizing factor derived from T4 phage is SEQ ID NO: 3 (T4SSB (P03695, Enterobacteria phage T4), 21-253); the amino acid sequence of the single-stranded DNA stabilizing factor derived from T7 phage is SEQ ID NO: 4 (T7SSB (P03696, Escherichia phage T7), 2-186); the amino acid sequence of the single-stranded DNA stabilizing factor derived from aquatic Thermus is SEQ ID NO: 5 (TaqSSB (Q9KH06, Thermus aquaticus), 2-230); the amino acid sequence of human single-stranded DNA stabilizing factor is SEQ ID NO: 6 (HsSSB (Q9BQ15, Homo sapiens) 2-110).

2. Original amino acid sequence of TdT protein

The amino acid sequence of mouse TdT is SEQ ID NO:7 (MsTdT (P09838, Mus musculus) 145-510); the amino acid sequence of white-throated sparrow TdT is SEQ ID NO:8 (ZaTdT (A0A8C5JUL5, Zonotrichia albicollis) 145-513 (including mutations C187A, C215S, C319S, R335L, K337G, C375A, C417S)).

3. Construction of TdT variants

The TdT variants are combined according to the method of Figure 1. Any gene sequence that can encode the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 8 can be used to construct the expression vector. The gene sequences encoding the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:6 were amplified by PCR, and the gene sequences were constructed into the expression vector pET-28a (Novagen, Kan+) containing the original TdT sequence SEQ ID NO:7 or SEQ ID NO:8 using the ligation method of restriction sites and T4 ligase to obtain the recombinant plasmid of the variant. The amino acid sequences of the constructed TdT variants were SEQ ID NO:9 (His-SsoSSB-MsTdT), SEQ ID NO:10 (His-EcSSB-ZaTdT), SEQ ID NO:11 (His-SsoSSB-ZaTdT), SEQ ID NO:12 (His-T4SSB-ZaTdT), SEQ ID NO:13 (His-T7SSB-ZaTdT), SEQ ID NO:14 (His-HsSSB-ZaTdT), and SEQ ID NO:15 (His-TaqSSB-ZaTdT). pET-28a is described in the examples, but the expression vector of the present invention is not limited thereto.

Example 2: Preparation of wild-type and variant TdT proteins

1. Gene expression

In order to detect the activity of TdT enzyme in vitro, the enzyme was exogenously expressed and purified in E. coli. The host bacteria described in the embodiment is E. coli BL21 (DE3) (purchased from Quanshi Gene, catalog number CD601-02). The following steps are included:

(1) The E. coli expression recombinant plasmid pET-28a-TdT was transformed into E. coli BL21 (DE3) to obtain recombinant bacteria. Positive clones were screened using kanamycin resistance plates (Kan+, 50 μg/mL) and cultured overnight at 37°C;

(2) Pick a single clone into 5 mL LB liquid medium (Kan+, 50 μg/mL) and culture overnight at 37°C and 220 rpm. Transfer the bacterial solution in 5 mL LB medium to 800 mL LB medium (Kan+, 50 μg/mL) and culture at 37°C and 220 rpm until OD600 is 0.6-0.8, then cool to 18°C, add IPTG to a final concentration of 0.5 mM, and induce expression for 16 h;

(3) Collect the above culture solution into a collection bottle and centrifuge at 4000 rpm for 30 min;

(4) Discard the supernatant, suspend the resulting bacterial pellet with 35 mL of protein buffer (50 mM Tris-HCl, 500 mM NaCl, pH 7.5), pour into a 50 mL centrifuge tube, and store in a -80°C refrigerator.

2. Protein purification

The steps include:

(1) Bacterial disruption: The bacterial precipitate obtained in step 3 above was disrupted twice using a high-pressure low-temperature disruptor at a pressure of 700 bar and 4°C. The precipitate and supernatant were collected after centrifugation at 4°C and 18,000 r/min for 60 min and sampled;

(2) Purification: The supernatant was filtered through a 0.22 μm microporous filter membrane and purified by nickel affinity chromatography (HisTrap HP5 ml). The specific steps are as follows:

a: Column balancing: Before loading the supernatant, wash the Ni affinity column with ddH ₂ O for 2 column volumes, and then balance the Ni affinity column with protein buffer for 2 column volumes;

b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 5 mL/min, repeat once;

c: Elution of impurities: Use protein buffer to wash 1 column volume, then use 50 mL of protein buffer containing 50 mM imidazole to elute strongly bound impurities and prepare samples;

d: Elution of target protein: 50 mL of 50-500 mM linear imidazole gradient buffer was used to elute the target protein and prepare samples, and 12% SDS-PAGE was used for detection. The results are shown in Figure 2. Lane 1 corresponds to protein standard Marker, Lane 2 corresponds to SEQ ID NO: 7 (WT-MS), Lane 3 corresponds to SEQ ID NO: 9 (Sso-MS), Lane 4 corresponds to protein standard Marker, Lane 5 corresponds to SEQ ID NO: 8 (WT-Za), Lane 6 corresponds to SEQ ID NO: 10 (Ec-Za), Lane 7 corresponds to SEQ ID NO: 11 (Sso-Za), Lane 8 corresponds to SEQ ID NO: 12 (T4-Za), Lane 9 corresponds to SEQ ID NO: 13 (T7-Za), Lane 10 corresponds to SEQ ID NO: 14 (Hs-Za), and Lane 11 corresponds to SEQ ID NO: 15 (Taq-Za).

(3) Gel filtration chromatography: The collected target protein was concentrated by centrifugation (4°C, 3400 r/min) using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) to 1 mL, loaded with Cytiva Hiload Superdex 200 16/60 molecular sieve, and the protein peak was collected and concentrated.

(4) The concentration of the concentrated protein was detected using a Nondrop 2000 micro-spectrophotometer to obtain the purified and concentrated TdT protein.

Example 3: Natural nucleotide reactivity test

The starting nucleotide chain used in this example is a single-stranded DNA with a FAM fluorescent label at the 5' end, and the inserted nucleotide is dATP.

TdT reaction system: 50 mM Tris-Ac pH 7.2, 100 mM KAc, 0.25 mM CoCl _2. Substrate: 1 μM 5'-FAM labeled single-stranded DNA (14 bp), 100 μM dATP. Enzyme: 2 μM TdT wild type and mutant.

Reaction conditions: incubate at 30°C for 20 min, inactivate at 95°C for 5 min (to denature the protein and release the oligo chains), centrifuge and collect the supernatant for urea denatured PAGE gel.

The results are shown in Figure 3, which shows that the efficiency of template-free extension of polynucleotides can be improved compared with the original sequence.

Example 4: 3'-OH terminal modified nucleotide incorporation efficiency test

The starting nucleotide chain used in this example is a single-stranded DNA with a FAM fluorescent label at the 5' end, and the inserted nucleotide is a nucleotide with an amino group added to the 3'-OH end as a blocking group (3'-O-NH2-2'-dNTP).

TdT reaction system: 50mM Tris-Ac pH 7.2, 100mM KAc, 0.25mM CoCl _2. Substrate: 1μM 5'-FAM labeled single-stranded DNA oligo (14bp), 20μM nucleotides with modified groups at the 3' end. Enzyme: 2μM TDT wild type and mutants.

Reaction conditions: incubate at 30°C for 10 min, inactivate at 95°C for 5 min (to denature the protein and release the oligo chains), centrifuge and collect the supernatant for urea denatured PAGE gel.

TdT wild type and variant activity detection: 20% denaturing polyacrylamide gel analysis was used. The gel was poured in advance and allowed to stand until polymerization. It was then placed in an electrophoresis tank of appropriate size filled with 1xTBE buffer. The sample was loaded onto the gel, and then the gel was subjected to a potential difference of 180V for 80 minutes. After satisfactory migration, the gel was released, and the gel electrophoresis image was obtained using the ChemiDoc (BioRad) gel imager. The brightness of each DNA band in the image was further analyzed using Biorad's Gel Image analysis software to obtain the brightness value, and the brightness value of the DNA band after the incorporation of nucleotides (the brightness value of the DNA band corresponding to 15bp) was divided by the brightness value of the total DNA band (the sum of the brightness values of the DNA bands corresponding to 14bp and 15bp) to obtain the nucleotide incorporation efficiency, as shown in Figure 4.

According to the results in Figure 4, it can be seen that the single nucleotide incorporation efficiency of the two mutants fused with single-stranded DNA stabilizing factors (Ec-SSB and Sso-SSB) in the embodiment is better than that of wild-type TdT, showing a higher catalytic efficiency, and nucleic acid molecules can be synthesized efficiently and controllably in the absence of a template chain.

Example 5: Single-stranded DNA extension activity test containing hairpin structure

This example uses single-stranded DNA containing a hairpin structure as the starting nucleotide chain and nucleotides with an amino group as a blocking group at the 3'-OH end (3'-O-NH ₂ -2'-dATP) as substrates to test the effect of the secondary structure of the starting nucleotide chain on the extension reaction.

TdT reaction system: 50mM Tris-Ac pH 7.2, 100mM KAc, 0.25mM CoCl _2. Substrate: 1μM single-stranded DNA oligo (32, 34, 36 and 38bp), 20μM nucleotides with modified groups at the 3' end. Enzyme: 2μM TDT wild type and mutants.

Reaction conditions: incubate at 30°C for 10 min, inactivate at 95°C for 5 min (to denature the protein and release the oligo chains), centrifuge and collect the supernatant for urea denatured PAGE gel.

TDT wild-type and variant activity detection: 15% denaturing polyacrylamide gel analysis was used. The gel was poured in advance and allowed to stand until polymerization. It was then placed in an electrophoresis tank of appropriate size filled with 1xTBE buffer. The sample was loaded onto the gel, which was then subjected to a potential difference of 180V for 80 minutes. After satisfactory migration, the gel was released and stained with SybrGold for 10 minutes, and then the gel electrophoresis image (Figure 5) was obtained using a ChemiDoc (BioRad) gel imager.

According to the results in Figure 5, it can be seen that the starting nucleotide chain contains a secondary structure, especially the 3' end is involved in the hairpin pairing TdT activity, and the variant of the present invention fused with the single-stranded DNA stabilizing factor has a stronger nucleotide incorporation efficiency than the wild type, especially the variant fused with Sso SSB greatly improves the efficiency of inserting nucleotides as substrates. With the increase of the 3' terminal free base, such as L14S9+2, L14S9+4 and L14S9+6, the hairpin structure becomes loose, and the efficiency of WT-Za begins to gradually increase, but the terminal deoxynucleotidyl transferase variant of the present invention, especially Sso-Za, still maintains a higher incorporation efficiency than wild WT-Za.

Example 6: Solid-phase catalytic oligonucleotide polymerization

The starting nucleotide chain used in this embodiment is a single-stranded DNA with a biotin label at the 5' end, and the inserted nucleotide is a nucleotide with an amino group as a blocking group added to the 3'-OH end (3'-O-NH2-2'-dATP). The following steps are included:

(1) Incubation and binding of the starting nucleotide chain with the solid phase matrix: 1 μM biotin-labeled starting nucleotide chain (5’-biotin-ACTAGGACGACTCGAATT-3’) was added to Streptavidin Sepharose High Performance (Cytiva) and incubated at 30°C with shaking at 1000 rpm for 30 minutes.

(2) Nucleotide insertion: 50 mM Tris-Ac pH 7.2, 100 mM KAc, 0.25 mM CoCl2, 20 μM nucleotide with a modified group at the 3’ end (3’-O-NH2-2’-dATP), 2 μM Sso-Za, 30°C, 1000 rpm shaking for 10 min.

(3) Washing: Centrifuge at 800 g for 1 minute, aspirate the supernatant reaction solution, and wash the gel beads with buffer (50 mM Tris-Ac pH 7.2, 100 mM KAc).

(4) Deamination: Wash the gel beads with 0.7 M NaNO2 pH 5.4 solution, repeat once.

(5) Washing: Centrifuge at 800 g for 1 min and wash the beads twice with buffer (50 mM Tris-Ac pH 7.2, 100 mM KAc).

(6) Repeat steps (2) to (5) for 9 rounds.

Samples were taken from each round of reaction for 20% denaturing polyacrylamide gel analysis, and the results are shown in Figure 6. The results show that the terminal deoxynucleotidyl transferase variant provided by the present invention can achieve efficient stepwise synthesis of oligonucleotides in solid phase.

In summary, the terminal deoxynucleotidyl transferase (TdT) variant provided by the present invention introduces some related single-stranded DNA stabilizing factors (ssDNAStabilizingFactor) into the original TdT sequence by modifying the main chain backbone of the terminal deoxynucleotidyl transferase, and has the following advantages over the original sequence: (i) it can improve the efficiency of template-free extension of polynucleotides; (ii) it exhibits enhanced stability or enhanced efficiency in incorporating 3'-OH-blocked nucleoside triphosphates into polynucleotides; (iii) and promotes the nucleotide extension ability of a single-stranded DNA with a hairpin secondary structure, which is conducive to achieving efficient and controllable nucleic acid molecule synthesis. The present invention relates to a plurality of terminal deoxynucleotidyl transferase variants modified based on this strategy. The present invention also relates to the use of these TdT variants in the synthesis of DNA of any given sequence.

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

Claims (9)

1. A terminal deoxynucleotidyl transferase variant, characterized in that it comprises a wild-type or mutant terminal deoxynucleotidyl transferase, a single-stranded DNA stabilizing factor and a flexible linker; the single-stranded DNA stabilizing factor is fused to the N-terminus and/or the C-terminus of the terminal deoxynucleotidyl transferase through the flexible linker, or the single-stranded DNA stabilizing factor is inserted into the middle of the sequence of the terminal deoxynucleotidyl transferase through the flexible linker; the sequence of the terminal deoxynucleotidyl transferase variant is shown in any one of SEQ ID NO 9 to SEQ ID NO 15. 2. The terminal deoxynucleotidyl transferase variant according to claim 1, characterized in that the single-stranded DNA stabilizing factor has a protein domain or subdomain that binds to or wraps single-stranded DNA with a binding energy at a level of mM or below mM. 3. The terminal deoxynucleotidyl transferase variant according to claim 2, characterized in that the single-stranded DNA stabilizing factor is derived from an organism; the organism includes any one of a virus, a prokaryote, and a eukaryote. 4. The terminal deoxynucleotidyl transferase variant according to claim 1, characterized in that the sequence of the single-stranded DNA stabilizing factor is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6; the sequence of the terminal deoxynucleotidyl transferase is shown in SEQ ID NO: 7 or SEQ ID NO: 8. 5. The terminal deoxynucleotidyl transferase variant according to claim 1, characterized in that the flexible linker has a sequence of glycine and serine as a repeating module: (GS) _m , wherein m = 0-20, m is zero or an integer; or the flexible linker has a sequence of glycine as a repeating module: (G) _n , wherein n = 0-40, n is zero or an integer. 6. A nucleic acid, characterized in that the nucleic acid encodes the terminal deoxynucleotidyl transferase variant according to any one of claims 1 to 5. 7. A method for synthesizing a nucleic acid molecule in the absence of a template chain, characterized in that it comprises the following steps: contacting a starting nucleotide chain with at least one nucleotide in the presence of the terminal deoxynucleotidyl transferase variant according to any one of claims 1 to 5. 8. A method for synthesizing nucleic acid molecules in the absence of a template chain according to claim 7, characterized in that the terminal deoxynucleotidyl transferase variant is coupled with a nucleotide; the nucleotide is a natural nucleotide or a nucleotide modified at the 3'-OH end, and the nucleotide modified at the 3'-OH end is a nucleotide modified by adding a blocking group to the 3'-OH end. 9. A kit, characterized in that the kit comprises the terminal deoxynucleotidyl transferase variant according to any one of claims 1 to 5, one or more nucleotides, and at least one starting nucleotide chain.