Terminal transferase mutant, recombinant protein, nucleic acid molecule and oligonucleotide single base extension method

Abstract

The invention discloses a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and an oligonucleotide single base extension method, and relates to the technical field of oligonucleotide synthesis. The terminal transferase mutant has at least 80% of identity with an amino acid sequence as shown in SEQ ID NO: 1, and has amino acid mutation at at least one of the following positions: R323, W427 and R438. Compared with wild-type terminal transferase, the terminal transferase mutant has the advantages that after amino acid in a catalytic structural domain of the terminal transferase mutant is changed, the catalytic capability of the terminal transferase mutant on nucleoside triphosphate with a 3 '-O-blocking group can be remarkably improved, so that the nucleoside triphosphate with the 3'-O-blocking group is efficiently coupled to a single-stranded oligonucleotide chain. On the basis, the invention further develops an oligonucleotide single base extension method. The method has the technical advantages that the humidity does not need to be controlled, the usage amount of chemical reagents is small, the product purity is high, and few chemical synthesis byproducts are generated.

Description

Technical Field

[0001] The present invention relates to the technical field of oligonucleotide synthesis, and in particular to a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and a method for extending a single base of an oligonucleotide. Background Art

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

[0003] Studies have shown that terminal transferase can transfer nucleotide triphosphates to single-stranded oligonucleotides, and accurate single-base extension of single-stranded oligonucleotides can be achieved through the modification of enzymes and substrates and the optimization of coupling processes. Terminal transferases derived from the vertebrate immune system can catalyze non-template-dependent polymerization reactions and can randomly add nucleotide triphosphates to the free 3' end of single-stranded oligonucleotides. This characteristic is consistent with the requirements of chemical solid-phase oligonucleotide synthesis technology for catalysts. Studies have shown that under optimal conditions, the time for terminal transferase to catalyze nucleotide triphosphate coupling of oligonucleotides is faster than that of phosphoramidite chemical synthesis, which has excellent advantages in the synthesis of single-stranded oligonucleotides.

[0004] At present, multiple studies have reported a variety of strategies for controlling the incorporation of triphosphate nucleotides into oligonucleotide sequences. For example, Kern Systems uses a modification-free strategy to synthesize oligonucleotides, which relies on the competition between terminal transferase and apyrase. Terminal transferase integrates the substrate triphosphate nucleotide into the end of the oligonucleotide chain, while apyrase hydrolyzes the phosphate group of the triphosphate nucleotide to reduce the concentration of the triphosphate nucleotide in the system, thereby blocking a new round of synthesis (Lee, HH, 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 crosslinker-mediated oligonucleotide synthesis scheme, which couples the triphosphate nucleotide and terminal transferase together to the 3' end of the oligonucleotide. At this time, the position of the 3' end is protected by the terminal transferase and cannot be coupled with the next triphosphate nucleotide. After that, the terminal transferase is removed by a specific method, and the new terminal transferase-triphosphate nucleotide cross-link is coupled to the end of the oligonucleotide. The above steps are repeated many times to achieve the extension of the single-stranded oligonucleotide (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, which greatly improves the efficiency of a single cycle, and the coupling process is also simpler. However, the coupling of triphosphate nucleotides and terminal transferase in the early stage is relatively complicated, which also limits the application of this method. In order to ensure that the extension of oligonucleotides can be accurately achieved, the 3' terminal hydroxyl group of triphosphate nucleotides must be protected. At present, nucleotides modified with protecting groups such as photocleavable groups, amino groups, and phosphate groups have been developed. Under the action of chemical reagents or enzymes, the 3' modified nucleotides can be converted to 3'-OH to achieve protection and deprotection during the coupling process. However, the catalytic efficiency of common commercial terminal transferases for modifying triphosphate nucleotides is low and the reaction rate is slow, which to some extent limits the development of enzymatic oligonucleotide synthesis.

[0005] In addition, since the terminal transferase cannot synthesize oligonucleotides from scratch, more than 7 oligonucleotide sequences are required as initiator chains in the enzymatic reaction. In enzymatic oligonucleotide synthesis, fixed initiator chains are more conducive to changing the substrate under the reaction system than free initiator chains, thereby improving the accuracy of oligonucleotide synthesis. Through the study of oligonucleotide immobilization, a two-step method is currently mainly used. First, the 5' end of the oligonucleotide is modified and bound to a carrier under chemical or enzymatic action. For example, Professor Jiang Huifeng et al. biotinylated the 5' end of the oligonucleotide and bound it to 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 DeoxynucleotidylTransferase. ACS Catalysis 12 (5), 2988-2997 (2022).). This method requires connecting other sequences to the 5' end of the target sequence, and this oligonucleotide modification method may result in a result that cannot match the target sequence when the oligonucleotide is harvested.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is 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 invention is achieved in that:

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

[0010] In a second aspect, the present invention further provides a recombinant protein, which includes the terminal transferase mutant and the functional sequence mentioned above.

[0011] In a third aspect, the present invention also provides a nucleic acid molecule encoding the terminal transferase mutant or the recombinant protein.

[0012] In a fourth aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0013] In a fifth aspect, the present invention further provides a recombinant cell, which comprises the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0014] In a sixth aspect, the present invention also provides the use of terminal transferase mutants in oligonucleotide single-base extension.

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

[0016] S1: contacting a single-stranded oligonucleotide having a free 3'-hydroxyl group, the terminal transferase mutant and a triphosphate nucleoside with a 3'-O-blocking group, so that the single-stranded oligonucleotide is extended by binding to the 3'-O-blocked triphosphate nucleoside to form a 3'-O-blocked extended segment;

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

[0018] S3: Repeat steps S1 and S2 to form a target polynucleotide.

[0019] In an eighth aspect, the present invention further provides a reagent or a kit, comprising: the terminal transferase mutant mentioned above, a single-stranded oligonucleotide and nucleoside triphosphates; the nucleoside triphosphates are nucleoside triphosphates with a 3'-O-blocking group.

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a terminal transferase mutant. Compared with a wild-type terminal transferase, the terminal transferase mutant has a catalytic domain amino acid that is changed, thereby significantly improving the catalytic ability of nucleoside triphosphates with 3'-O-blocking groups, so that the nucleoside triphosphates with 3'-O-blocking groups can be efficiently coupled to a single-stranded oligonucleotide chain.

[0022] On this basis, the present invention also developed an oligonucleotide single base extension method based on a terminal transferase mutant. This method can efficiently couple a nucleoside triphosphate with a 3'-O-blocking group to an oligonucleotide, and after deblocking, an oligonucleotide chain with one base extended is obtained; after repeating the oligonucleotide single base extension reaction and deblocking reaction, the target polynucleotide is obtained. This method has many technical advantages such as no need to control humidity, less chemical reagent usage, higher product purity, and less chemical synthesis by-products.

[0023] In addition, compared with other enzymatic oligonucleotide synthesis schemes, no chemical modification groups (i.e., blocking groups) will remain after the dissociation of the oligonucleotides, and longer-chain oligonucleotides can be directly synthesized, which can be widely used in the field of efficient and controllable oligonucleotide extension or synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 Provide technical pathways and schematics for the application of terminal transferase variants in single-base extension of oligonucleotides;

[0026] Figure 2 Terminal transferase SDS-PAGE gel image; Lane 1: Broken bacterial protein; Lane 2: Nickel column purified protein; Lane 3: Dialyzed protein;

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

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

[0029] Figure 5 Polyacrylamide TBE-urea gel electrophoresis of the products (n+1, n+2, n+3, n+4) after the terminal transferase mutant completes four cycles. DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0032] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0033] As used herein, "nucleotide" includes nitrogenous heterocyclic bases, sugars, and one or more phosphate groups. They are monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, that is, the sugar lacks the hydroxyl group present in ribose. Nitrogenous heterocyclic bases can be purine or pyrimidine bases. 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 pyrimidine or the N-9 of purine.

[0034] As used herein, "nucleoside" is similar in structure to nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is a nucleoside analog in which a label is connected to a base and there is no phosphate group connected to a sugar molecule. The term "nucleoside" as used herein is a general meaning understood by those skilled in the art. Examples include, but are not limited to, ribonucleosides comprising a ribose moiety and deoxyribonucleosides comprising a deoxyribose moiety. The modified pentose moiety is a pentose moiety 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. "Nucleoside" is a monomer that can have a substituted base and / or sugar moiety. In addition, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0035] In the present invention, "nucleoside with a 3'-modified group" is synonymous with "nucleoside with a 3'-O-blocked group".

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

[0037] The terminal transferase mutant, for example, has 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 with the amino acid sequence shown in SEQ ID NO: 1, and has an amino acid mutation at at least one of the following positions compared to the amino acid sequence of SEQ ID NO: 1: R323, W427 and R438.

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

[0039] The inventors conducted homology modeling on wild-type terminal transferase ZF-N0 (amino acid sequence is SEQ ID NO: 1, and encoding nucleotide sequence is SEQ ID NO: 2). Autodock software was used to perform docking simulation on homology modeling protein and 3' modified nucleosides (i.e. nucleosides with 3'-O-blocking groups), and the docking results were virtual screened for alanine using Discover studio to obtain hotspot amino acids R323, W427 and R438. These three amino acids are located in the catalytic domain of terminal transferase mutants. After at least one of them is mutated, nucleosides with 3'-O-blocking groups can be efficiently coupled to oligonucleotides, significantly improving substrate conversion. Therefore, the terminal transferase mutants provided by the present invention can be widely used in the field of efficient and controllable oligonucleotide extension or synthesis, including but not limited to the synthesis of nucleic acid molecules, amplification reactions of cDNA ends, addition of nucleotides labeled with radioactive isotopes, and addition of dideoxynucleotide capping at the 3' end in gene sequencing.

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

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

[0042] MLPSRKRVRAEVSTSQGGEHVKFSDVTLFLVEKRMGKSRRTFLSSLARSKGFCVDD

[0043] ALSGAVTHVVSEGLSAQDLWLWLEDQGFQETHSKHVLNISWFTESMSAGRPLPVEDTHC

[0044] IQNPAADQRSCVHLSATPESAVSPYACQRRTTLENHNKIFTEALEVLALNSEFSGNQGSCL

[0045] GFRRAASVLKSLPAALRSPEEALRLPCCLGDNSRAVLEEICECGSSSRVEEIQNDQRYRTMK

[0046] LFCSVFGVGPKTAESWFCRGLKTFEQVLTEPSIRLNRMQTAGFLFYEDISVPVSRAEAAAL

[0047] KMMMEEALLFINPSATVTITGGFRRGKEFGHDVDFIIKAPEGQEDRILPAVIKRFKSQNVLL

[0048] YSDFQKSTFDLRQLPNHRFEAMDRFSKCFLLVKLQMQESRTGRNWRAVRVDLVAPPLER

[0049] FPYALLGWTGSTLFERDLLRRFARLERGKLLDNHTLYDKTTKTFLPANTEEDIFQHLGLEYI

[0050] EPWQRNA.

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

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

[0053] The amino acid sequence having mutations in 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] GFRRAASVLKSLPAALRSPEEALRLPCCLGDNSRAVLEEICECGSSSRVEEIQNDQRYRTMK

[0059] LFCSVFGVGPKTAESWFCRGLKTFEQVLTEPSIRLNRMQTAGFLFYEDISVPVSRAEAAAL

[0060] KMMMEEALLFINPSATVTITGGFRLGKEFGHDVDFIIKAPEGQEDRILPAVIKRFKSQNVLL

[0061] YSDFQKSTFDLRQLPNHRFEAMDRFSKCFLLVKLQMQESRTGRNWRAVRVDLVAPPLER

[0062] FPYALLGWTGSTLFERDLNRFARLERGKLLDNHTLYDKTTKTFLPANTEEDIFQHLGLEYI

[0063] EPWQRNA.

[0064] In a second aspect, the present invention further provides a recombinant protein, which includes the terminal transferase mutant and the functional sequence mentioned above.

[0065] In a preferred embodiment of the present invention, 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] Protein tags include but are not limited to: glutathione transferase (GST), polyhistidine (poly-His), V5 tag, MBP tag, mCherry tag, Flag peptide, HA tag, c-Myc tag, Fc segment of immunoglobulin, green fluorescent protein, etc.

[0067] The signal peptide sequence includes but is not limited to: signal peptides for protein secretion, signal peptides for protein cleavage, etc.

[0068] Such as PEP4 signal sequence, CPY+4 signal sequence, DAP2 signal sequence, MFα1 signal sequence, PelB, OmpA, OmpF, OmpC, OmpT, Lpp, StII, PhoA, PhoE, MalE, LamB, LTB, FimH, SPA, RsaA, Endoxylanase, Ice nucleation protein, a-Agglutinin and IgAβ-domain, one or a combination of several.

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

[0070] The protein linking sequence is selected from (GS)n, where n is 1-6; (GGGGS)n, where n is 1-5; (G)n, where n is 1-6; or selected from SGGGGG, GGGGG, SGGGGSGGGGSGGGG, GGGGGGSGGGG, GGGGGSAAGG or GGGGGSGGGGSGGGGSA.

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

[0072] In a third aspect, the present invention also provides a nucleic acid molecule encoding the terminal transferase mutant or the recombinant protein.

[0073] Taking into account the degeneracy of codons, the gene sequence encoding the terminal transferase mutant or recombinant protein can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same terminal transferase mutant or recombinant protein amino acid sequence; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the terminal transferase mutant to improve the expression efficiency of the terminal transferase mutant.

[0074] In a fourth aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[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 directly or indirectly (such as packaging into a virus) introduce the target DNA fragment into the host cell through transformation, transfection or transduction to express the target gene.

[0076] One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can connect the target DNA fragment to the plasmid ring, including but not limited to pET series plasmids, pUC series plasmids, pMD series plasmids, etc., which can be expressed in strains such as Enterobacteriaceae or yeast. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, the target gene can be expressed.

[0077] In a fifth aspect, the present invention further provides a recombinant cell, which comprises the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0078] In a preferred embodiment of the present invention, 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 invention, the bacteria is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis.

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

[0081] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parental cells in nucleic acid content, but may contain mutations.

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

[0083] The above-mentioned recombinant cells are prepared by transforming the recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and the foreign gene carried can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0084] In a preferred embodiment of the present invention, 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 cell of a recombinant bacterium, and a cell fragment of a recombinant bacterium.

[0085] Dormant cells, also known as resting cells, are a special cell state. In this state, cells do not grow and reproduce, but still contain various enzyme systems and have oxidation and fermentation capabilities. Under suitable conditions, dormant cells can resume growth. The characteristics of dormant cells include: a. Cells maintain growth potential: Although in a dormant state, these cells can re-enter the cell cycle and restore their proliferation ability when given appropriate stimulation. b. Strong specificity: Dormant cells have strong specificity in the reaction and can improve the substrate conversion rate. c. Not easy to be contaminated by foreign bacteria: Due to its characteristics, dormant cells can reduce the inhibition of bacterial growth and enzyme synthesis by products during use.

[0086] Dead bacteria of recombinant bacteria include but are not limited to bacteria obtained by inactivation by heat, pressure, radiation, etc.

[0087] Cell disruption products refer to: including but not limited to, cell disruption products obtained by changing the permeability of cell membranes through ultrasonic, mechanical, chemical, biological and other means, resulting in leakage of cell contents.

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

[0089] In a sixth aspect, the present invention also provides the use of the terminal transferase mutant in oligonucleotide single base extension. The purpose of the use is not to diagnose a disease.

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

[0091] (1) Synthesizing nucleic acid molecules in the absence of a template;

[0092] (2) Addition of nucleotides to the amplification reaction at the ends of the cDNA;

[0093] (3) adding nucleotides labeled with radioactive isotopes;

[0094] (4) Used to add dideoxynucleotides to the 3' end in gene sequencing.

[0095] Application (2) is to add nucleotides in a cDNA end amplification reaction, such as adding nucleotides in amplification reaction of cDNA ends (RACE).

[0096] Radioisotopes include but are 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 Such as 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 use further comprises nucleotides for adding labeled chemiluminescent molecules.

[0098] In a seventh aspect, the present invention further provides a method for oligonucleotide single base extension, comprising the following steps:

[0099] S1: contacting a single-stranded oligonucleotide having a free 3'-hydroxyl group, the terminal transferase mutant and a triphosphate nucleoside with a 3'-O-blocking group, so that the single-stranded oligonucleotide is extended by binding to the 3'-O-blocked triphosphate nucleoside to form a 3'-O-blocked extended segment;

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

[0101] S3: Repeat steps S1 and S2 to form a target polynucleotide.

[0102] In a preferred embodiment of the present invention, before step S1, the method further includes coupling the 5' end of the single-stranded oligonucleotide to a solid phase carrier;

[0103] Solid phase supports include but are not limited to: membranes, plates, nanoparticles, microplates, glass, etc.

[0104] The contacting conditions in step S1 are: incubation at room temperature for 15 to 40 minutes; room temperature refers to 15 to 30°C.

[0105] The deblocking conditions in step S2 are: adding a strong oxidant, incubating at room temperature for 15 to 40 minutes, and then centrifuging to recover the solid powder;

[0106] This method has the advantages of mild reaction conditions, no need for humidity control, low use of chemical reagents, high product purity, and few chemical synthesis by-products. In addition, compared with other enzymatic oligonucleotide synthesis schemes, no chemical modification groups remain after oligonucleotide dissociation, and longer chain oligonucleotides can be directly synthesized, which can be widely used in the field of efficient and controllable oligonucleotide extension or synthesis.

[0107] The 5' end of the single-stranded oligonucleotide is connected to a solid phase carrier (such as a controllable microporous glass), and the 3' end has a 3'-hydroxyl functional group; under specific reaction conditions, the 3'-O-blocked nucleoside triphosphate contacts and connects with the single-stranded oligonucleotide under the action of a terminal transferase variant, so that the 3' end of the short-chain oligonucleotide of N bases fixed on the solid phase carrier is extended by one base to form an oligonucleotide chain with N+1 bases, and the 3' end of the new chain has a blocking group. Further, the fragment of N+1 bases obtained in the previous 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 poly-oligonucleotide. Finally, the poly-oligonucleotide reacts with a termination reagent to obtain a poly-oligonucleotide with a hydroxyl group at 3'.

[0108] In a preferred embodiment of the present invention, 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-acetyllevulinate, 3'-O-tert-butyldimethylsilane, 3'-O-trimethyl(silyl)ethoxymethyl, 3'-O-o-nitrobenzyl or 3'-O-p-nitrobenzyl, 3'-O-amino, 3'-O-phosphate group;

[0109] The blocking group is selected from 3'-O-amino. Compared with other groups, the amino group has a smaller molecular weight and less steric hindrance during the enzymatic reaction. On the other hand, in relevant literature reports, the position has a better catalytic effect when it is a polar group. The amino group has a better polar effect than other groups.

[0110] In an eighth aspect, the present invention further provides a reagent or a kit, comprising: the terminal transferase mutant mentioned above, a single-stranded oligonucleotide and nucleoside triphosphates; the nucleoside triphosphates are nucleoside triphosphates with a 3'-O-blocking group.

[0111] In a preferred embodiment of the present invention, the blocking group is selected from amino, phosphoric acid or azidomethyl;

[0112] In a preferred embodiment of the present invention, 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-acetyllevulinate, 3'-O-tert-butyldimethylsilane, 3'-O-trimethyl(silyl)ethoxymethyl, 3'-O-o-nitrobenzyl or 3'-O-p-nitrobenzyl, 3'-O-amino, 3'-O-phosphate group.

[0113] In a preferred embodiment of the present invention, the blocking group is selected from amino groups.

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

[0115] The deblocking agent is selected from strong oxidants, including but not limited to sodium nitrite.

[0116] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0117] Strains and vectors: Escherichia coli DH5α competent bacteria, Escherichia coli BL21 competent bacteria and ampicillin were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0118] Kits: Plasmid extraction kit and gel purification recovery kit were also purchased from Sangon Biotech (Shanghai) Co., Ltd.

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

[0120] Plasmid construction, whole gene synthesis and point mutagenesis were commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0121] Example 1

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

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

[0124] (1) Terminal transferase induced expression

[0125] The whole genome synthesized E. coli expression recombinant plasmid pET21a HIS-ZF was transferred into E. coli BL21 (DE3) to obtain recombinant bacteria. Positive clones were screened using ampicillin resistance plates, and single clones were picked into 5 mL LB liquid culture medium and cultured at 37°C and 220 r / min until OD 600 The temperature was then lowered to 16°C, and IPTG was added to a final concentration of 0.5 mM to induce expression for 24 hours.

[0126] The above culture solution was collected into a collection bottle and centrifuged at 5500 r / min for 15 minutes. The supernatant was discarded, and the obtained bacterial precipitate was 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) Terminal transferase purification

[0128] Protein purification and bacterial disruption: The bacterial precipitate obtained above was disrupted twice using a high-pressure low-temperature disruptor at a pressure of 1000 bar and 4° C. Centrifugation was performed at 4° C. and 10,000 r / min for 45 minutes.

[0129] Purification: The supernatant was filtered through a 0.45 μm microporous filter membrane and then purified by nickel affinity chromatography. The nickel column was incubated under 20 mM imidazole and eluted under 250 mM imidazole. The target protein components were combined and dialyzed in 70 mM (pH 6.0) potassium acetate buffer using a dialysis membrane to obtain purified terminal transferase. After sample preparation, 12% SDS PAGE was performed. The results are as follows: Figure 2 shown.

[0130] Example 2

[0131] This example screens terminal transferase.

[0132] First, homology modeling was performed on the wild-type terminal transferase ZF-N0 (amino acid sequence is SEQ ID NO:1, encoding nucleotide sequence is SEQ ID NO:2). Autodock software was used to perform docking simulation between the homology modeling protein and the 3' modified nucleoside, and the docking results were used for alanine virtual screening using Discover studio. The hot amino acids R323, W427, and R438 were handed over to Sangon Biotech for saturation mutation, and the terminal transferase mutant ZF-N1 with R323L, W427A, and R438N (triple mutation) was obtained, and its amino acid sequence is SEQ ID NO:3. The encoding nucleotide sequence synthesized with reference to this sequence is SEQ ID NO:4. Compared with ZF-N0 terminal transferase, the substrate conversion rate of 0.1 mg / mL mutant ZF-N1 was significantly improved in the reaction system of 0.1 mM 3'-O-NH2 nucleoside triphosphate, 0.25 mM CoCl2, 5 mM oligonucleotide, 70 mM potassium acetate, and pH 6.0. After reacting at 37° C. for 30 minutes, the product was recovered using an alcohol precipitation solution (80 mL of 1.5 M sodium acetate, 14 mL of acetic acid, and anhydrous ethanol was fixed to 4 L) and subjected to mass spectrometry analysis. The substrate molecular weight detected by mass spectrometry and the mass spectrometry signal intensity of the product molecular weight were evaluated to calculate the substrate conversion rate of the 3'-modified nucleoside; the specific sequence of the short-chain oligonucleotide synthesized in this example is: SEQ ID NO: 5.

[0133] Specifically, the method for recovering the product is as follows: after mixing the wine precipitation solution and the reaction system in a ratio of 7:1, an equal volume of water is added, and then detected under mass spectrometry.

[0134] Figure 3 This is the mass spectrum of the reaction products of 3'-modified nucleosides catalyzed by ZF-N0 terminal transferase. Figure 4 The mass spectrum of the reaction product of 3'-modified nucleosides catalyzed by the terminal transferase mutant ZF-N1 shows that the substrate conversion rate of 3'-modified nucleosides by the terminal transferase mutant reaches 100%.

[0135] Example 3

[0136] This example provides a method for extending a single base of an oligonucleotide. The extension principle is as follows Figure 1 As shown, it includes the following steps:

[0137] (1) Oligonucleotide Immobilization

[0138] The short-chain oligonucleotide synthesized by Shanghai Shenggong Biotechnology Co., Ltd. was fixed on the controllable porous glass beads. The specific sequence of the short-chain oligonucleotide synthesized in this example is: SEQ ID NO:5.

[0139] The oligonucleotide fixation method is carried out according to Patent No.: CN202410212527.

[0140] Oligonucleotides were immobilized on controlled pore glass beads (universal CPG particle size: 1000).

[0141] (2) Oligonucleotide extension

[0142] The above-mentioned synthetic 0.5OD controlled porous glass beads (solid powder) fixed with oligonucleotides are added to a reaction system containing terminal transferase, the system comprising a final concentration of 0.1mg / mL ZF-N1, 0.1mM 3'-O-NH2 nucleoside triphosphate, 0.25mM CoCl, 70mM potassium acetate, the system pH is 6.0, the total volume is 1mL, and the reaction is 30 minutes. The solid powder is recovered by centrifugation at 10000rpm for 3 minutes, an equal volume of 1M NaNO and 0.7M NaAc (pH 5.2) is added, and the blocking group is removed by incubation at room temperature for 30 minutes, and the solid powder is recovered by centrifugation again (preparation of n+1). The product (n+1) is subjected to enzymatic reaction again to prepare product (n+2), product (n+3) and product (n+4) in sequence.

[0143] The samples were dissociated with an aminolysis solution to dissociate the oligonucleotides, and the dissociated samples were separated by electrophoresis in a 15% polyacrylamide-urea gel. The gel was stained with SYBR Gold for 5-10 minutes at room temperature and imaged on a gel imaging system. The fluorescent products before and after staining were observed.

[0144] Figure 5 It is shown that in the presence of a starting sequence (oligonucleotide), by using the terminal transferase mutant provided in Example 2 of the present invention and the 3'-O-NH2 nucleoside triphosphate provided by the present invention as a substrate, it is possible to extend the DNA chain to more than (n+1, n+2, n+3 or n+4) while fixing the oligonucleotide. Figure 5 Where n refers to the oligonucleotide gel electrophoresis image before extension reaction, n+1 refers to the oligonucleotide gel electrophoresis image after one extension reaction, n+2 refers to the oligonucleotide gel electrophoresis image after two extension reactions, n+3 refers to the oligonucleotide gel electrophoresis image after three extension reactions, and n+4 refers to the oligonucleotide gel electrophoresis image after four extension reactions.

[0145] Example 4

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

[0147] Example 5

[0148] This example provides a terminal transferase mutant, which is different from Example 2 in that a terminal transferase mutant of W427A (single mutation) is obtained.

[0149] Example 6

[0150] This example provides a terminal transferase mutant, which is different from Example 2 only in that a terminal transferase mutant of R438N (single mutation) is obtained.

[0151] Example 7

[0152] This example provides a terminal transferase mutant, which is different from Example 2 in that a terminal transferase mutant with W427A and R438N (double mutation) is obtained.

[0153] Example 8

[0154] This example provides a terminal transferase mutant, which is different from Example 2 in that a terminal transferase mutant with R323L and R438N (double mutation) is obtained.

[0155] Example 9

[0156] This example provides a terminal transferase mutant, which is different from Example 2 in that a terminal transferase mutant of W427A and R323L (double mutation) is obtained.

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A terminal transferase mutant, characterized in that: It has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1, and compared with the amino acid sequence shown in SEQ ID NO: 1, has an amino acid mutation in at least one of the following positions: R323, W427 and R438.

2. The terminal transferase mutant according to claim 1, characterized in that The amino acid sequence of the terminal transferase mutant has at least one of the following mutations compared to the amino acid sequence shown in SEQ ID NO: 1: R323L, W427A and R438N; Preferably, the amino acid sequence of the terminal transferase mutant has the following mutations compared to the amino acid sequence shown in SEQ ID NO: 1: R323L, W427A and R438N.

3. A recombinant protein, characterized in that It comprises the terminal transferase mutant and functional sequence according to any one of claims 1 to 2.

4. The recombinant protein according to claim 3, characterized in that The functional sequence is selected from at least one of a protein tag, a signal peptide, a scaffold protein and a protein connection sequence; Preferably, the functional sequence is located at the nitrogen terminus and / or carbon terminus of the terminal transferase mutant.

5. A nucleic acid molecule, characterized in that It encodes the terminal transferase mutant according to any one of claims 1-2 or the recombinant protein according to any one of claims 3-4.

6. A recombinant vector, characterized in that: It comprises the nucleic acid molecule according to claim 5.

7. A recombinant cell, characterized in that It comprises the nucleic acid molecule according to claim 5 or the recombinant vector according to claim 6.

8. Use of the terminal transferase mutant according to any one of claims 1 to 2 in oligonucleotide single base extension; Preferably, the application includes at least one of the following application modes: (1) Synthesizing nucleic acid molecules in the absence of a template; (2) Addition of nucleotides to the amplification reaction at the ends of the cDNA; (3) adding nucleotides labeled with radioactive isotopes; (4) Used to add dideoxynucleotides to the 3' end in gene sequencing.

9. A method for extending an oligonucleotide single base, characterized in that: It includes the following steps: S1: contacting an oligonucleotide single chain having a free 3'-hydroxyl group, the terminal transferase mutant according to any one of claims 1 to 2, and a nucleoside triphosphate with a 3'-O-blocking group, so that the oligonucleotide single chain is extended by binding to the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extended segment; S2: deblocking the 3'-O-blocked extension fragment to form an extension fragment with a free 3'-hydroxyl group; S3: repeating steps S1 and S2 to form a target polynucleotide; Preferably, before step S1, the method further comprises coupling the 5' end of the single-stranded oligonucleotide to a solid phase carrier; Preferably, the contacting conditions in step S1 are: incubation at room temperature for 15 to 40 minutes; Preferably, the deblocking conditions in step S2 are: adding a strong oxidant, incubating at room temperature for 15 to 40 minutes, and then centrifuging to recover the solid powder; Preferably, 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-acetyllevulinate, 3'-O-tert-butyldimethylsilane, 3'-O-trimethyl(silyl)ethoxymethyl, 3'-O-o-nitrobenzyl or 3'-O-p-nitrobenzyl, 3'-O-amino, 3'-O-phosphate group; Preferably, the blocking group is selected from 3'-O-amino groups.

10. A reagent or a kit, characterized in that: It comprises: the terminal transferase mutant according to any one of claims 1 to 2, a single-stranded oligonucleotide and a nucleoside triphosphate; the nucleoside triphosphate is a nucleoside triphosphate with a 3'-O-blocking group; Preferably, the blocking group is selected from amino, phosphate or azidomethyl; Preferably, the blocking group is selected from amino groups.

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