A method for terminal transferase mutant, recombinant protein, nucleic acid molecule and oligonucleotide single base extension

Abstract

The application discloses a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and a method for single-base extension of oligonucleotide, and relates to the technical field of oligonucleotide synthesis. The terminal transferase mutant has at least 80% identity with the amino acid sequence shown in SEQ ID NO:1, and has amino acid mutations in at least one of the following positions: R323, W427 and R438. Compared with a wild-type terminal transferase, the catalytic domain amino acid of the terminal transferase mutant is changed, and the catalytic capacity for a 3'-O-closed group triphosphate nucleoside is significantly improved, so that the 3'-O-closed group triphosphate nucleoside is efficiently coupled to a single-stranded oligonucleotide chain. On this basis, the application also develops a method for single-base extension of oligonucleotide. The method has technical advantages such as no need to control humidity, less use of chemical reagents, higher product purity, less chemical synthesis byproducts and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oligonucleotide synthesis, in particular, to a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and a method for single base extension of oligonucleotide. BACKGROUND

[0002] Synthetic nucleic acids (such as oligonucleotides) play an important role in many fields such as molecular biology, forensic science and medical diagnosis. A large number of researchers have tried to synthesize DNA by chemical and enzymatic methods. After years of optimization and improvement, DNA chemical synthesis has undergone a revolutionary development from column synthesis to microchip synthesis and has been widely applied in the market. However, there are still some deficiencies in the chemical synthesis of DNA. On the one hand, the coupling efficiency and reaction by-products of chemical synthesis limit the length of oligonucleotide synthesis to within 300 nt; on the other hand, chemical synthesis also has problems such as long reaction cycle time (6-8 minutes), high consumption of chemical reagents, high cost, large use of toxic and flammable organic reagents, and large pollution.

[0003] Previous studies have shown that terminal transferase can transfer nucleotide triphosphates to single-stranded oligonucleotides, and through the modification of enzymes and substrates and the optimization of coupling process, precise single base extension of single-stranded oligonucleotides can be achieved. Terminal transferase derived from the immune system of vertebrates can catalyze a non-template-dependent polymerization reaction and can randomly add nucleotide triphosphates to the free 3' end of single-stranded oligonucleotides. This property is consistent with the requirements of chemical solid-phase oligonucleotide synthesis technology for catalysts. Previous studies have shown that under optimal conditions, terminal transferase catalyzes the coupling of oligonucleotides with nucleotide triphosphates faster than the chemical synthesis of phosphoramidites, and has excellent advantages in single-stranded oligonucleotide synthesis.

[0004] Currently, several strategies have been reported to control the incorporation of nucleotides into oligonucleotide sequences. For example, Kern Systems uses a modification-free strategy to synthesize oligonucleotides by means of the competition between terminal transferase and adenosine triphosphatase. Terminal transferase integrates substrate triphosphate nucleotides into the end of the oligonucleotide chain, while adenosine triphosphatase hydrolyzes the phosphate group of triphosphate nucleotides to reduce the concentration of triphosphate nucleotides in the system, thereby blocking a new round of synthesis (Lee, H. H., Kalhor, R., Goela, N. et al. Terminator-free template-independent enzymatic DNA synthesis for digital information storage. Nat Commun 10, 2383 (2019)). Ansa Biotechnologies uses a terminal transferase-triphosphate nucleotide conjugate-mediated oligonucleotide synthesis scheme, which couples triphosphate nucleotides and terminal transferase to the 3' end of the oligonucleotide. At this time, the 3' end is protected by terminal transferase and cannot be coupled with the next triphosphate nucleotide. After that, the terminal transferase is removed by a specific method, and a new terminal transferase-triphosphate nucleotide conjugate is coupled to the end of the oligonucleotide, and the above steps are repeated several times to achieve the extension of single-stranded oligonucleotides (Palluk, S., Arlow, D., de Rond, T. et al. De novo DNA synthesis using polymerase-nucleotide conjugates. Nat Biotechnol 36, 645-650 (2018).). Compared with Kern Systems, Ansa Biotechnologies technology only needs two steps to complete a cycle, greatly improving the efficiency of a single cycle, and the coupling process is also simpler, but the coupling of triphosphate nucleotides and terminal transferase in the early stage is more complex, which also limits the application of this method. In order to ensure that the extension of oligonucleotides can be accurately realized, the 3' end hydroxyl group of triphosphate nucleotides must be protected. At present, protective groups such as photolabile groups, amino groups, and phosphate group-modified nucleotides have been developed, which can convert the modified 3' of nucleotides to 3'-OH under the action of chemical reagents or enzymes, realizing the protection and deprotection in the coupling process. However, the catalytic efficiency of common commercial terminal transferase for modified triphosphate nucleotides is low, and the reaction speed is slow, which to some extent limits the development of enzymatic oligonucleotide synthesis.

[0005] In addition, since terminal transferase cannot synthesize oligonucleotides from scratch, more than 7 oligonucleotide sequences are required as priming chains in enzymatic reactions. In enzymatic oligonucleotide synthesis, immobilized priming chains are more advantageous than free priming chains in modifying substrates under reaction systems, and improve the accuracy of oligonucleotide synthesis. Through the immobilization of oligonucleotides, the current method mainly uses two steps. First, the 5' end of the oligonucleotide is modified and combined with the carrier under the action of chemistry or enzyme. For example, Professor Jiang Huifeng et al. modified the 5' end of the oligonucleotide with biotin and combined it with a carrier containing streptavidin, thereby realizing the immobilized synthesis of oligonucleotides (Xiaoyun Lu, Jinlong Li, Congyu Li, et al. Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase. ACS Catalysis 12(5), 2988-2997 (2022).). This method requires the connection of other sequences to the 5' end of the target sequence, and this oligonucleotide modification method may cause the result that the target sequence cannot be matched when the oligonucleotide is harvested.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The present application aims to provide a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and a method for oligonucleotide single base extension to solve the above technical problems.

[0008] The present application is implemented as follows:

[0009] In a first aspect, the present application provides a terminal transferase mutant, which has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, and has at least one amino acid mutation in the following positions: R323, W427 and R438, compared with the amino acid sequence shown in SEQ ID NO: 1.

[0010] In a second aspect, the present application further provides a recombinant protein comprising the terminal transferase mutant described above and a functional sequence.

[0011] In a third aspect, the present application further provides a nucleic acid molecule encoding the terminal transferase mutant described above or encoding the recombinant protein described above.

[0012] In a fourth aspect, the present application further provides a recombinant vector comprising the nucleic acid molecule described above.

[0013] In a fifth aspect, the present application further provides a recombinant cell comprising the nucleic acid molecule described above or the recombinant vector described above.

[0014] In a sixth aspect, the present application further provides an application of the terminal transferase mutant in oligonucleotide single base extension.

[0015] In a seventh aspect, the present application further provides a method for oligonucleotide single base extension, comprising the following steps:

[0016] S1: contacting the single-stranded oligonucleotide with a free 3'-hydroxyl group, the terminal transferase mutant and the 3'-O-blocked nucleoside triphosphate, so as to extend the single-stranded oligonucleotide by combining the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extension fragment;

[0017] S2: deblocking the 3'-O-blocked extension fragment to form an extended fragment with a free 3'-hydroxyl group;

[0018] S3: repeating steps S1 and S2 to form the polynucleotide of interest.

[0019] In an eighth aspect, the present application further provides a reagent or kit, comprising: the terminal transferase mutant, the single-stranded oligonucleotide and the nucleoside triphosphate; and the nucleoside triphosphate is the 3'-O-blocked nucleoside triphosphate.

[0020] The present application has the following advantages:

[0021] The present application provides a terminal transferase mutant, which has an altered catalytic domain amino acid compared with the wild-type terminal transferase, and can significantly improve the catalytic ability of the 3'-O-blocked nucleoside triphosphate, so as to efficiently couple the 3'-O-blocked nucleoside triphosphate to the single-stranded oligonucleotide.

[0022] On this basis, the present application further develops an oligonucleotide single base extension method based on the terminal transferase mutant. The method can efficiently couple the 3'-O-blocked nucleoside triphosphate to the oligonucleotide, and after deblocking, an oligonucleotide chain extended by one base is obtained; after repeating the oligonucleotide single base extension reaction and deblocking reaction, the polynucleotide of interest is obtained. The method has many technical advantages such as no need to control humidity, less amount of chemical reagents used, higher purity of product, less chemical synthesis byproducts, etc.

[0023] In addition, compared with other enzymatic oligonucleotide synthesis schemes, there is no chemical modification group (i.e. blocking group) left after the oligonucleotide is dissociated, and longer chain oligonucleotides can be directly synthesized, which can be widely applied in the field of efficient and controllable oligonucleotide extension or synthesis. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 Technical path and principle diagram for application of terminal transferase variant in oligonucleotide single base extension;

[0026] Figure 2 Terminal transferase SDS-PAGE gel map; lane 1: cell broken protein; lane 2: nickel column purified protein; lane 3: protein after dialysis;

[0027] Figure 3 Mass spectrum of reaction product of wild type terminal transferase (ZF-N0);

[0028] Figure 4 Mass spectrum of reaction product of terminal transferase mutant (ZF-N1);

[0029] Figure 5 Polyacrylamide TBE-urea gel electrophoresis map of 4-cycle product (n+1, n+2, n+3, n+4) of terminal transferase mutant. DETAILED DESCRIPTION

[0030] The embodiments of the present application will now be described in detail with reference to the drawings, one or more examples of which are illustrated below. Each example is provided as an explanation and not a limitation of the present application. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the scope or spirit of the present application. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0031] Unless otherwise indicated, the practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0032] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If no specific conditions are indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If no manufacturers of the reagents or instruments are indicated, the reagents or instruments are all conventional products that can be purchased on the market.

[0033] As used herein, "nucleotides" include a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. They are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e., the sugar lacks the hydroxyl group present in ribose. The nitrogen-containing heterocyclic base can be a purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to the N-1 of a pyrimidine or the N-9 of a purine.

[0034] As used herein, "nucleosides" are structurally similar to nucleotides, but lack the phosphate moiety. One example of a nucleoside analog is a nucleoside analog in which a label is attached to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" as used herein is understood in its general meaning by one of skill in the art. Examples include, but are not limited to, ribonucleosides comprising a ribose moiety and deoxyribonucleosides comprising a deoxyribose moiety. A modified pentose moiety is one in which an oxygen atom has been replaced by a carbon atom and / or a carbon atom has been replaced by a sulfur or oxygen atom. "Nucleosides" are monomers that can have a substituted base and / or sugar moiety. Furthermore, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0035] In the present invention, "nucleoside with a 3' modification group" is synonymous with "nucleoside with a 3'-0-blocking group".

[0036] In a first aspect, the present invention provides a terminal transferase mutant having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 1 and having an amino acid mutation at at least one of the following positions: R323, W427, and R438, as compared to the amino acid sequence set forth in SEQ ID NO: 1.

[0037] The terminal transferase mutant has, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an amino acid mutation at at least one of the following positions: R323, W427, and R438, as compared to the amino acid sequence set forth in SEQ ID NO: 1.

[0038] The amino acid sequence of SEQ ID NO: 1 is derived from a wild-type terminal transferase mutant from zebrafish.

[0039] The inventors performed homology modeling on wild-type terminal transferase ZF-N0 (amino acid sequence of SEQ ID NO: 1, encoding nucleotide sequence of SEQ ID NO: 2). The homology modeled protein was docked with 3' modified nucleosides (i.e. nucleosides with 3'-O-closed groups) using Autodock software, and the docking results were subjected to alanine virtual screening using Discover studio, obtaining hot amino acids R323, W427 and R438. These three amino acids are located in the catalytic domain of the terminal transferase mutant. After mutating at least one of them, the nucleosides with 3'-O-closed groups can be efficiently coupled to oligonucleotides, significantly improving the substrate conversion rate. Therefore, the terminal transferase mutant provided by the present application can be widely used in the field of efficient and controllable oligonucleotide extension or synthesis, including but not limited to synthesizing nucleic acid molecules without templates, amplifying cDNA ends, adding labeled radioisotopes, adding dideoxynucleotides to the 3' end in gene sequencing, etc.

[0040] R323, W427 and R438 are key catalytic domains of terminal transferase, and those skilled in the art can mutate the amino acids at this position as long as the catalytic ability to modified oligonucleotides (i.e. nucleosides with 3'-O-closed groups) is improved, which is within the protection scope of the present application.

[0041] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0042] MLPSRKRVRAEVSTSQGGEHVKFSDVTLFLVEKRMGKSRRTFLSSLARSKGFCVDD

[0043] ALSGAVTHVVSEGLSAQDLWLWLEDQGFQETHSKHVLNISWFTESMSAGRPLPVEDTHC

[0044] IQNPAADQRSCVHLSATPESAVSPYACQRRTTLENHNKIFTEALEVLALNSEFSGNQGSCL

[0045] GFRRAASVLKSLPAALRSPEEALRLPCLGDNSRAVLEEICECGSSSRVEEIQNDQRYRTMK

[0046] LFCSVFGVGPKTAESWFCRGLKTFEQVLTEPSIRLNRMQTAGFLFYEDISVPVSRAEAAAL

[0047] KMMMEEALLFINPSATVTITGGFRRGKEFGHDVDFIIKAPEGQEDRILPAVIKRFKSQNVLL

[0048] YSDFQKSTFDLRQLPNHRFEAMDRFSKCFLLVKLQMQESRTGRNWRAVRVDLVAPPLER

[0049] FPYALLGWTGSTLFERDLRRFARLERGKLLDNHTLYDKTTKTFLPANTEEDIFQHLGLEYI

[0050] EPWQRNA.

[0051] In a preferred embodiment of the application, the amino acid sequence of the terminal transferase mutant has at least one mutation of R323L, W427A and R438N compared with the amino acid sequence shown in SEQ ID NO: 1. Among them, R323L refers to the mutation of the 323rd arginine in SEQ ID NO: 1 to L (leucine), W427A refers to the mutation of the 427th tryptophan in SEQ ID NO: 1 to alanine (A). R438N refers to the mutation of the 438th arginine in SEQ ID NO: 1 or SEQ ID NO: 3 to asparagine (N).

[0052] In a preferred embodiment of the application, the amino acid sequence of the terminal transferase mutant has the following mutations of R323L, W427A and R438N compared with the amino acid sequence shown in SEQ ID NO: 1. The single amino acid mutation has higher conversion efficiency than the original mutation. The triple amino acid mutation has higher conversion efficiency and better stability than the single amino acid mutation.

[0053] The amino acid sequence with mutations at the above three positions is shown in SEQ ID NO: 3.

[0054] The amino acid sequence of SEQ ID NO: 3 is as follows:

[0055] MLPSRKRVRAEVSTSQGGEHVKFSDVTLFLVEKRMGKSRRTFLSSLARSKGFCVDD

[0056] ALSGAVTHVVSEGLSAQDLWLWLEDQGFQETHSKHVLNISWFTESMSAGRPLPVEDTHC

[0057] IQNPAADQRSCVHLSATPESAVSPYACQRRTTLENHNKIFTEALEVLALNSEFSGNQGSCL

[0058] GFRRAASVLKSLPAALRSPEEALRLPCLGDNSRAVLEEICECGSSSRVEEIQNDQRYRTMK

[0059] LFCSVFGVGPKTAESWFCRGLKTFEQVLTEPSIRLNRMQTAGFLFYEDISVPVSRAEAAAL

[0060] KMMMEEALLFINPSATVTITGGFRLGKEFGHDVDFIIKAPEGQEDRILPAVIKRFKSQNVLL

[0061] YSDFQKSTFDLRQLPNHRFEAMDRFSKCFLLVKLQMQESRTGRNWRAVRVDLVAPPLER

[0062] FPYALLGWTGSTLFERDLNRFARLERGKLLDNHTLYDKTTKTFLPANTEEDIFQHLGLEYI

[0063] EPWQRNA.

[0064] In a second aspect, the present application provides a recombinant protein comprising the terminal transferase mutant and a functional sequence as described above.

[0065] In a preferred embodiment of the present application, the functional sequence is selected from at least one of a protein tag, a signal peptide, a scaffold protein, and a protein linker sequence.

[0066] The protein tag includes, but is not limited to, glutathione transferase (GST), poly-His, V5 tag, MBP tag, mCherry tag, Flag polypeptide, HA tag, c-Myc tag, Fc fragment of immunoglobulin, green fluorescent protein, and the like.

[0067] The signal peptide sequence includes, but is not limited to, a signal peptide for protein secretion, a signal peptide for protein cleavage, and the like.

[0068] such as PEP4 signal sequence, CPY+4 signal sequence, DAP2 signal sequence and MFa1 signal sequence, PelB, OmpA, OmpF, OmpC, OmpT, Lpp, StII, PhoA, PhoE, MalE, LamB, LTB, FimH, SPA, RsaA, Endoxylanase, Ice nucleation protein, a-Agglutinin and IgA β-domain, one or several combinations thereof.

[0069] The scaffold protein is selected from any one or a combination of at least two of BCAM, PTGFRN or PLXNA.

[0070] The protein linker sequence is selected from (GS)n, n is 1-6; (GGGGS)n, n is 1-5; (G)n, n is 1-6; or selected from SGGGGG, GGGGG, SGGGSGGGGSGGGG, GGGGGSGGGG, GGGSAAGG or GGGGSGGGGSGGGGSA.

[0071] In a preferred embodiment of the application, the functional sequence is located at the nitrogen terminal and / or carbon terminal of the terminal transferase mutant.

[0072] In a third aspect, the present application provides a nucleic acid molecule encoding the terminal transferase mutant or the recombinant protein as described above.

[0073] In consideration of the degeneracy of codons, the gene sequence encoding the terminal transferase mutant or the recombinant protein as described above can be modified in its coding region without changing the amino acid sequence, so as to obtain a gene encoding the same amino acid sequence of the terminal transferase mutant or the recombinant protein; or the gene can be artificially synthesized and modified according to the codon bias of the host expressing the terminal transferase mutant, so as to improve the expression efficiency of the terminal transferase mutant.

[0074] In a fourth aspect, the present application provides a recombinant vector comprising the nucleic acid molecule as described above.

[0075] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can be used to introduce a DNA fragment of interest into a host cell directly or indirectly (e.g., packaged into a virus) by transformation, transfection or transduction, so as to express the gene of interest.

[0076] One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, to which a DNA fragment of interest can be ligated into the plasmid circle, including but not limited to pET series plasmids, pUC series plasmids, pMD series plasmids, and the like, which can be expressed in strains of enterobacterium or yeast and the like. Another type of vector is a viral vector, which can ligate a DNA fragment of interest into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). These vectors, after entering a host cell, can express the gene of interest.

[0077] In a fifth aspect, the present application also provides a recombinant cell comprising the nucleic acid molecule described above or the recombinant vector described above.

[0078] In a preferred embodiment of the present application, the recombinant cell is a bacterium, a fungus, a 293 cell, a 293T cell, a 293FT cell, a CHO cell, a COS cell, a Per6 cell, a 293 series cell, a Per6 cell, or a CHO cell.

[0079] In a preferred embodiment of the present application, the bacterium is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli, or Bacillus subtilis.

[0080] In a preferred embodiment of the present application, the fungus is Trichoderma reesei or a yeast.

[0081] The host cell described above includes transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny can not be completely identical to the parent cell in nucleic acid content, but can contain mutations.

[0082] In a preferred embodiment of the present application, the yeast is selected from at least one of the following genera: Dekkera, Brettanomyces, Hanseniaspora, Kluyveromyces, Pichia, Candida, Kluyveromyces, Debaryomyces, Kazachstania, Wickerhamomyces, Lindnera, Zygotorulaspora, Zygosaccharomyces, Rhodosporidium, and Schizosaccharomyces.

[0083] The recombinant cell described above is prepared by transforming a recombinant expression vector into a host cell (e.g., a microorganism) by conventional methods in the art; the host microorganism can be various host microorganisms conventionally used in the art, as long as the recombinant expression vector can be stably replicated by itself and the exogenous gene carried thereby can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0084] In a preferred embodiment of the present application, the recombinant cell refers to at least one of a resting cell of a recombinant bacterium, a living cell of a recombinant bacterium, a dead bacterium of a recombinant bacterium, and a cell breakage of a recombinant bacterium.

[0085] The resting cell, also known as the quiescent cell, is a special cell state. In this state, the cell does not grow and reproduce, but still contains various enzyme systems and has oxidation and fermentation ability. Under suitable conditions, the resting cell can restore growth. The characteristics of the resting cell include: a. The cell maintains growth potential: although in a dormant state, when given appropriate stimulation, these cells can re-enter the cell cycle and restore the ability to proliferate. b. Strong specificity: the resting cell has strong specificity in the reaction, which can improve the substrate conversion rate. c. Less susceptible to contamination: due to its characteristics, the resting cell can reduce the inhibition of product on bacterial growth and enzyme synthesis during use.

[0086] The dead bacterium of the recombinant bacterium includes, but is not limited to, the bacterium obtained by inactivation through heat, pressure, radiation, etc.

[0087] The cell breakage refers to: including but not limited to the cell breakage obtained by changing the permeability of the cell membrane through ultrasonic, mechanical, chemical, biological, etc., causing the contents of the cell to leak out.

[0088] In a preferred embodiment of the present application, the dead bacterium is selected from at least one of the precipitate of the dead bacterium and the cell-free supernatant of the dead bacterium. The cell-free supernatant of the dead bacterium refers to the "exudation contents" remaining after removing the outer shell of the dead bacterium.

[0089] In a sixth aspect, the present application also provides the use of the terminal transferase mutant in the single-base extension of oligonucleotides. The use is not for the purpose of disease diagnosis.

[0090] In a preferred embodiment of the present application, the use includes at least one of the following application modes:

[0091] (1) Synthesis of nucleic acid molecules without template;

[0092] (2) Addition of nucleotides in cDNA end amplification reaction;

[0093] (3) Addition of nucleotides labeled with radioisotopes;

[0094] (4) In gene sequencing, it is used for adding dideoxynucleotides at the 3' end.

[0095] The application (2) is the addition of nucleotides in cDNA end amplification reaction, such as the addition of nucleotides in cDNA end amplification reaction (RACE).

[0096] The radioisotope includes but is not limited to212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0097] In other embodiments, the application also includes a nucleotide for adding a labeled chemiluminescent molecule.

[0098] In a seventh aspect, the present application provides a method for single base extension of an oligonucleotide, comprising the steps of:

[0099] S1: contacting an oligonucleotide single strand having a free 3'-hydroxyl group, the terminal transferase mutant and a 3'-O-blocked nucleoside triphosphate, so that the oligonucleotide single strand is extended by incorporating the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extension fragment;

[0100] S2: deblocking the 3'-O-blocked extension fragment to form an extended fragment having a free 3'-hydroxyl group;

[0101] S3: repeating steps S1 and S2 to form a polynucleotide of interest.

[0102] In preferred embodiments of the application, prior to step S1, the 5' end of the oligonucleotide single strand is first coupled to a solid support.

[0103] The solid support includes, but is not limited to, a membrane, a plate, a nanoparticle, a microwell plate, glass, etc.

[0104] The conditions of the contacting in step S1 are: incubation at room temperature for 15-40 min; room temperature refers to 15-30℃.

[0105] The conditions of the deblocking in step S2 are: addition of a strong oxidant, incubation at room temperature for 15-40 min, and then centrifugal recovery of the solid powder;

[0106] The method has the advantages of mild reaction conditions, no need to control humidity, less use of chemical reagents, higher product purity, and less chemical synthesis byproducts. In addition, compared with other enzymatic oligonucleotide synthesis schemes, no chemical modification group remains after oligonucleotide dissociation, and longer chain oligonucleotides can be directly synthesized, which can be widely applied in the field of efficient and controllable oligonucleotide extension or synthesis.

[0107] The 5' end of the oligonucleotide single strand is connected to a solid support (such as controllable microporous glass), and the 3' end has a 3'-hydroxyl functional group; under specific reaction conditions, the 3'-O-blocked nucleoside triphosphate is contacted and connected with the oligonucleotide single strand under the action of the terminal transferase variant, so that the short-chain oligonucleotide with N bases fixed on the solid support is extended by one base at the 3' end, forming an oligonucleotide chain with N+1 bases, and the 3' end of the new chain has a blocking group. Further, the N+1 base fragment obtained in the above step is reacted with a deblocking reagent to form an N+1 base fragment with a free 3'-hydroxyl group, and the above steps are repeated multiple times to form a polynucleotide. Finally, the polynucleotide is reacted with a termination reagent to obtain a polynucleotide with a hydroxyl group at the 3'.

[0108] In a preferred embodiment of the application, the blocking group is selected from 3'-O-azidomethyl, 3'-aminooxy, 3'-O-(N-oxime), 3'-O-allyl, 3'-O-cyanoethyl, 3'-O-acetyl, 3'-O-nitrate, 3'-phosphate, 3'-O-acetylacetylpropionate, 3'-O-tert-butyldimethylsilane, 3'-O-trimethyl(silyl)ethoxymethyl, 3'-O-o-nitrobenzyl or 3'-O-p-nitrobenzyl, 3'-O-amino, 3'-O-phosphate groups;

[0109] The blocking group is selected from 3'-O-amino. The amino group has smaller molecular weight and smaller steric hindrance in the enzymatic reaction process compared to other groups, and on the other hand, in the relevant literature reports, the position has better catalytic effect when the position is a polar group, and the amino group has better polar effect compared to other groups.

[0110] In an eighth aspect, the application also provides a reagent or kit, which comprises: the terminal transferase mutant, the oligonucleotide single strand and the nucleoside triphosphate described above; and the nucleoside triphosphate is a 3'-O-blocked nucleoside triphosphate.

[0111] In preferred embodiments of the application, the blocking group is selected from an amino, phosphoric acid or azido group.

[0112] In preferred embodiments of the application, the blocking group is selected from a 3'-0-azidomethyl, 3'-aminooxy, 3'-0-(N-oxime), 3'-0-allyl, 3'-0-cyanoethyl, 3'-0-acetyl, 3'-0-nitrate, 3'-phosphate, 3'-0-acetylenic acid, 3'-0-tert-butyldimethylsilyl, 3'-0-trimethyl(silyl)ethoxymethyl, 3'-0-o-nitrobenzyl or 3'-0-p-nitrobenzyl, 3'-0-amino, 3'-0-phosphate group.

[0113] In preferred embodiments of the application, the blocking group is selected from an amino group.

[0114] In other embodiments, the reagent or kit further comprises a deblocking agent, a termination reagent.

[0115] The deblocking agent is, for example, selected from a strong oxidizing agent, including but not limited to sodium nitrite.

[0116] The features and nature of the present application will appear more fully with regard to the accompanying examples which follow.

[0117] Strains and vectors: E. coli DH5a competent cells, E. coli BL21 competent cells and ampicillin were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0118] Kit: Plasmid extraction kit and gel purification and recovery kit were also purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0119] E. coli culture medium (LB medium): composed of 0.5% yeast extract, 1% peptone, 1% NaCl.

[0120] Plasmid construction, whole gene synthesis and point mutation were entrusted to Shengong Bioengineering (Shanghai) Co., Ltd.

[0121] Example 1

[0122] In this example, wild-type terminal transferase was expressed and purified by E. coli.

[0123] All the gene sequences of the amino acid sequences shown in the synthetic sequence list can be used for the construction of expression vectors. The gene sequence is constructed into the restriction sites Ndel and Xhol of the expression vector pET21a containing a His tag, so as to obtain a recombinant plasmid, named pET 21a-HIS-ZF. In order to detect the terminal transferase enzyme activity in vitro, the enzyme is expressed and purified in Escherichia coli. The host bacteria in this embodiment are Escherichia coli BL21(DE3), but the host bacteria are not limited thereto.

[0124] (1) Induction expression of terminal transferase

[0125] The whole genome synthesized Escherichia coli expression type recombinant plasmid pET21a HIS-ZF is transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacteria. The positive clone screening is performed by using ampicillin resistant plate, and a single colony is picked into 5 mL LB liquid medium and cultured at 37°C, 220 r / min until the OD 600 is 0.8-1.0. Then, the temperature is lowered to 16°C, IPTG is added to a final concentration of 0.5 mM, and induction expression is performed for 24 hours.

[0126] The above cultured bacteria liquid is collected into a bacteria collection bottle and centrifuged at 5500 r / min for 15 minutes. The supernatant is discarded, and the obtained bacterial pellet is suspended with 35 mL protein buffer (50 mM TrisHCl, 500 mM NaCl, pH 7.4), poured into a 50 mL centrifuge tube, and stored in a -80°C refrigerator.

[0127] (2) Purification of terminal transferase

[0128] Protein purification and bacteria breaking: the bacterial pellet obtained above is broken twice by using a high-pressure low-temperature disrupter at a pressure of 1000 bar and 4°C. Centrifugation is performed at 4°C and 10000 r / min for 45 minutes.

[0129] Purification: after the supernatant is filtered by a 0.45 μm microporous filter, nickel affinity chromatography purification is performed. The nickel column is incubated at 20 mM imidazole, and eluted at 250 mM imidazole. The target protein components are combined, and dialysis is performed on the target protein in 70 mM (pH 6.0) potassium acetate buffer by using a dialysis membrane, so as to finally obtain the purified protein terminal transferase. After sample preparation, 12% SDS PAGE detection is performed, and the results are shown in Figure 2 .

[0130] Example 2

[0131] In this embodiment, the terminal transferase is screened.

[0132] First, the wild-type terminal transferase ZF-N0 (amino acid sequence SEQ ID NO: 1, encoding nucleotide sequence SEQ ID NO: 2) was subjected to homology modeling. Autodock software was used to dock the homology modeled protein with 3' modified nucleosides, and the docking results were subjected to alanine virtual screening using Discover studio. The hot spot amino acids R323, W427, and R438 were subjected to saturation mutation by GenScript Biotech Corporation, and the terminal transferase mutants R323L, W427A, and R438N (three mutations) were obtained, with the amino acid sequence SEQ ID NO: 3, and the encoding nucleotide sequence SEQ ID NO: 4 was synthesized with reference to the sequence. Compared with the ZF-N0 terminal transferase, 0.1 mg / mL mutant ZF-N1 significantly improved the substrate conversion rate in a reaction system of 0.1 mM 3'-O-NH2 nucleoside triphosphate, 0.25 mM CoCl2, 5 mM oligonucleotide, 70 mM potassium acetate, pH 6.0. After 37°C reaction for 30 minutes, the product was recovered using wine precipitation solution (1.5 M sodium acetate 80 mL, 14 mL acetic acid, anhydrous ethanol to 4 L) and subjected to mass spectrometry analysis. The substrate conversion rate of 3' modified nucleosides was evaluated according to the mass spectrometry detection of substrate molecular weight and product molecular weight signal intensity, and the short chain oligonucleotide synthesized in this embodiment had the specific sequence SEQ ID NO: 5.

[0133] Specifically, the method for recovering the product is as follows: the wine precipitation solution is mixed with the reaction system at a ratio of 7:1, then an equal volume of water is added, and then the detection is carried out under mass spectrometry.

[0134] Figure 3 The mass spectrum of the reaction product of the ZF-N0 terminal transferase catalyzing 3' modified nucleosides is shown in Figure 4 The mass spectrum of the reaction product of the terminal transferase mutant ZF-N1 catalyzing 3' modified nucleosides is shown in the figure. The results show that the substrate conversion rate of the terminal transferase mutant 3' modified nucleosides reaches 100%.

[0135] Example 3

[0136] This embodiment provides a method for single base extension of oligonucleotides, and the extension principle is shown in Figure 1 The method comprises the following steps:

[0137] (1) Oligonucleotide immobilization

[0138] The short chain oligonucleotide synthesized by GenScript Biotech (Shanghai) Co., Ltd. was immobilized on controllable porous glass beads. The short chain oligonucleotide synthesized in this embodiment has the specific sequence SEQ ID NO: 5.

[0139] The method for fixing the oligonucleotide is based on patent No. CN202410212527.

[0140] The oligonucleotide is fixed on the universal CPG particle (particle size: 1000) of the controllable porous glass bead.

[0141] (2) Oligonucleotide extension

[0142] The 0.5 OD of the oligonucleotide-fixed controllable porous glass bead (solid powder) synthesized above is added to a reaction system containing terminal transferase, which comprises a final concentration of 0.1 mg / mL ZF-N1, 0.1 mM 3'-O-NH2 nucleotide triphosphate, 0.25 mM CoCl, 70 mM potassium acetate, the pH of the system is 6.0, the total volume is 1 mL, and the reaction is performed for 30 minutes. The solid powder is recovered by centrifugation at 10,000 rpm for 3 minutes, and an equal volume of 1M NaNO and 0.7M NaAc (pH 5.2) is added, and incubated at room temperature for 30 minutes to remove the blocking group, and the solid powder is recovered again using a centrifuge (to prepare n+1). The product (n+1) is subjected to enzymatic reaction again to prepare the product (n+2), the product (n+3) and the product (n+4) in turn.

[0143] The sample is dissociated by using an ammonolysis solution, and the dissociated sample is subjected to electrophoretic separation in a 15% polyacrylamide-urea gel. The gel is stained in SYBR Gold at room temperature for 5-10 minutes, and imaged on a gel imaging system. The fluorescent product before and after staining is observed.

[0144] Figure 5 It is shown that, in the presence of the starting sequence (oligonucleotide), using the terminal transferase mutant provided in Example 2 of the present application, and using the 3'-O-NH2 nucleotide triphosphate provided in the present application as the substrate, the DNA chain can be extended to (n+1, n+2, n+3 or n+4) or more under the condition of the fixed oligonucleotide. Figure 5 In the figure, n refers to the oligonucleotide gel electrophoresis graph before the extension reaction, n+1 refers to the oligonucleotide gel electrophoresis graph after the extension reaction for 1 time, n+2 refers to the oligonucleotide gel electrophoresis graph after the extension reaction for 2 times, n+3 refers to the oligonucleotide gel electrophoresis graph after the extension reaction for 3 times, and n+4 refers to the oligonucleotide gel electrophoresis graph after the extension reaction for 4 times.

[0145] Example 4

[0146] This example provides a terminal transferase mutant, which is different from Example 2 only in that the terminal transferase mutant of R323L (single mutation) is obtained.

[0147] Example 5

[0148] This example provides a terminal transferase mutant, the difference compared with example 2 is only that the terminal transferase mutant with R438N (single mutation) is obtained.

[0149] Example 6

[0150] This example provides a terminal transferase mutant, the difference compared with example 2 is only that the terminal transferase mutant with R438N (single mutation) is obtained.

[0151] Example 7

[0152] This example provides a terminal transferase mutant, the difference compared with example 2 is only that the terminal transferase mutant with R438N (single mutation) is obtained.

[0153] Example 8

[0154] This example provides a terminal transferase mutant, the difference compared with example 2 is only that the terminal transferase mutant with R438N (single mutation) is obtained.

[0155] Example 9

[0156] This example provides a terminal transferase mutant, the difference compared with example 2 is only that the terminal transferase mutant with R438N (single mutation) is obtained.

[0157] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A terminal transferase mutant, characterized in that, R323L, W427A and R438N are made compared to the amino acid sequence shown in SEQ ID NO:

1.

2. A recombinant protein, characterized in that, It comprises the terminal transferase mutant of claim 1 and a functional sequence selected from a protein tag located at the nitrogen terminal and / or carbon terminal of the terminal transferase mutant.

3. A nucleic acid molecule, characterized in that, It encodes the terminal transferase mutant of claim 1 or the recombinant protein of claim 2.

4. A recombinant vector, characterized in that, It comprises the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that, It comprises the nucleic acid molecule of claim 3 or the recombinant vector of claim 4.

6. The terminal transferase mutant of claim 1 for use in oligonucleotide single base extension.

7. Use according to claim 6, characterized in that, The use comprises at least one of the following modes of use: (1) adding nucleotides in the amplification reaction of cDNA ends; (2) adding nucleotides labeled with radioisotopes; (3) adding dideoxynucleotides for 3' end capping in gene sequencing.

8. A method of oligonucleotide single base extension, comprising: It comprises the following steps: S1: contacting an oligonucleotide single strand with a free 3'-hydroxyl group, the terminal transferase mutant of claim 1 and a 3'-O-blocked nucleoside triphosphate, so as to extend the oligonucleotide single strand by binding 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extension fragment; S2: deblocking the 3'-O-blocked extension fragment to form an extended fragment with a free 3'-hydroxyl group; S3: repeating steps S1 and S2 to form a polynucleotide of interest; the blocking group is amino.

9. The method of oligonucleotide single base extension according to claim 8, wherein, Before the step S1, it further comprises coupling the 5' end of the oligonucleotide single strand to a solid support.

10. The method of oligonucleotide single base extension according to claim 8, wherein, The contacting in the step S1 is under the condition of incubation at room temperature for 15-40 min.

11. The method of oligonucleotide single base extension according to claim 8, wherein, The deblocking in the step S2 is under the condition of adding a strong oxidant, incubation at room temperature for 15-40 min, and then centrifugal recovery of solid powder.

12. A reagent or kit characterized in that, It comprises the terminal transferase mutant of claim 1, an oligonucleotide single strand and a nucleoside triphosphate; the nucleoside triphosphate is a 3'-O-blocked nucleoside triphosphate; the blocking group is amino.

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