CN116904420A

CN116904420A - Mutant strain of Stofel fragment with XNA recognition and synthesis activity and application thereof

Info

Publication number: CN116904420A
Application number: CN202310690904.0A
Authority: CN
Inventors: 陈庭坚; 覃彦嘉; 马星雲; 陶睿
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2023-06-12
Filing date: 2023-06-12
Publication date: 2023-10-20

Abstract

The invention discloses a mutant strain of Stofel fragments with XNA recognition and synthesis activity and application thereof. The Stofel fragment mutant of Taq DNA polymerase comprises SFM5-7 and SFM5-46, and the two sites are saturated and mutated based on the Stofel fragment mutant SFM4-3 of Taq DNA polymerase. Compared with SFM4-3, SFM5-7 and SFM5-46 can synthesize long fragment fully modified 2'-OMe-DNA more efficiently, and the activity of SFM5-7 and SFM5-46 is improved when DNA is synthesized by using 2' -OMe-DNA as a template. When 2'-OMe-DNA is synthesized by taking the 2' -OMe-DNA as a template, the activity of SFM5-7 is greatly improved.

Description

Mutant strain of Stofel fragment with XNA recognition and synthesis activity and application thereof

Technical Field

The invention belongs to the field of enzyme molecular transformation, and particularly relates to a mutant strain of Stofel fragments with XNA recognition and synthesis activity and application thereof.

Background

DNA or RNA is a biological macromolecule necessary for the development and functioning of all organisms, its role is to store, retrieve and transfer genetic information, and has also found wide application in biotechnology and biological medicine. However, DNA and RNA are poorly biochemically stable, greatly limiting their use in many applications. Accordingly, researchers have developed various DNA and RNA analogs, modified on the sugar, base, phosphate backbone of natural nucleic acids, respectively, which are referred to as non-natural nucleic acids (XNAs). Among them, the most widely studied is XNA modified on sugar rings. For example, threose Nucleic Acid (TNA), hexanol Nucleic Acid (HNA), cyclohexene nucleic acid (CeNA), locked Nucleic Acid (LNA), arabinose Nucleic Acid (ANA), 2' -fluoro-arabinose nucleic acid (FANA), and the like. Among them, 2' -position modification of ribose or deoxyribose is of interest, such as 2' -azide (2 ' -Az), 2' -amino (2 ' -Am), 2' -fluoro (2 ' -F) and 2' -methoxy (2 ' -OMe) modification. Among them, 2'-F or 2' -OMe modified nucleic acids have better performance in terms of nuclease resistance. The effect of resistance to nuclease degradation makes this XNA a great advantage in evolution based on an exponential enrichment ligand System (SELEX).

The recognition and processing of unnatural substrates by polymerases is closely related to XNA coding and decoding information. However, natural polymerases have difficulty in efficiently recognizing non-natural nucleotides due to higher substrate specificity. Thus, the transfer of genetic information between XNAs, such as transcription, reverse transcription and replication of XNA, is difficult to achieve. Researchers have engineered polymerases through enzyme engineering to enable their identification and utilization of non-natural nucleic acids. The Ellington group evolved a series of Tgo polymerase mutants that were able to synthesize and reverse transcribe various XNAs, such as HNA, TN a, FANA, ANA, and the like. The KOD polymerase mutant RSGA obtained in the Chaput group has enhanced specificity of the TNA substrate. The Romesberg group performs directed evolution on Stoffel fragments based on phage display technology to obtain Stoffel fragment mutants SFM4-3, SFM4-6 and SFM4-9 of Taq DNA polymerase, wherein SFM4-3 not only can synthesize all 2' -OMe modified non-natural nucleic acid, but also can obtain partial 2' -OMe or 2' -F modified nucleic acid chains through PCR amplification. At present, stoffel fragment mutant strains of Taq DNA polymerase (hereinafter referred to as Stoffel fragment mutant strains) still have the defect in the activity of identifying and synthesizing longer all 2'-OMe modified non-natural nucleic acid, and Stoffel fragment mutant strains capable of synthesizing long-fragment all 2' -O Me modified nucleic acid and having the activity of synthesizing DNA, 2'-F-DNA, RNA and 2' -OMe-DNA improved by taking RNA or all modified XNA as templates are obtained through saturated mutation screening.

Disclosure of Invention

All mutation sites of Stofel fragment mutant SFM4-3, including V518A, N583S, I614E, E615G, D655N, E681K, E742Q and M747R (starting with the first amino acid of Taq DNA polymerase).

In order to improve the capability of the Stofel fragment mutant strain SFM4-3 to synthesize the full 2'-OMe modified non-natural nucleic acid and enable the Stofel fragment mutant strain SFM4-3 to synthesize the longer full 2' -OMe modified non-natural nucleic acid, the invention carries out saturation mutation on thumb and finger key site residues 520 and 681 on the basis of the Stofel fragment mutant strain SFM4-3 so as to obtain the Stofel fragment mutant strain with higher activity.

The first object of the present invention is to provide Stofel fragment mutants SFM5-7 and SFM5-46 of Taq DNA polymerase.

Stoffel fragment mutant SFM5-7 is obtained by performing double-site saturation mutation on the basis of SFM4-3, wherein the newly added mutation is E520P and K681R; stoffel fragment mutant SFM5-46 was subjected to double site saturation mutagenesis based on SFM4-3, and contained the newly added mutations E520P and K681L, as well as the additional amino acid fragment ALRLSQRLAIPYAPPPLR.

A Stoffel fragment mutant SFM5-7 has an amino acid sequence shown in SEQ ID NO. 1.

A Stoffel fragment mutant SFM5-46 has an amino acid sequence shown in SEQ ID NO. 2.

The second object of the present invention is to provide a gene sequence of the Stofel fragment mutant strain.

The gene for coding the Stoffel fragment mutant strain SFM5-7 has a gene sequence shown in SEQ ID NO. 3.

The gene for coding the Stoffel fragment mutant strain SFM5-46 has a gene sequence shown in SEQ ID NO. 4.

Further, the Stofel fragment mutants SFM5-7 and SFM5-46 are novel Stofel fragment mutants obtained by subjecting Stofel fragment mutant SFM4-3 to enzyme molecular mutation and screening.

Furthermore, a saturated mutation library of E520 and K681 locus codons in the sequence of the Stoffel fragment mutant strain SFM4-3 gene is constructed by oligonucleotide primers.

Preferably, the Stofel fragment mutant strain SFM5-7 and SFM5-46 are obtained by screening a codon saturated mutation library of E520 and K681 sites in the Stofel fragment mutant strain SFM4-3 gene sequence.

Further, the synthetic activities of each Stoffel fragment mutant SFM4-3, SFM5-7 and SFM5-46 on the substrates of 2' -OMe-dNTPs using DNA as a template were measured, and the synthetic activities of each mutant on the substrates of dNTPs, 2' -F-dNTPs, rNTPs and 2' -OMe-dNTPs using 2' -F-DNA, RNA and 2' -OMe-DNA templates, respectively, were measured.

A third object of the present invention is to provide the use of the Stofel fragment mutant strain of the first object for preparing a nucleic acid preparation product.

Further, the nucleic acid preparation comprises synthesizing 2' -OMe-DNA using DNA as a template; synthesizing DNA, 2' -F-DNA, RNA or 2' -OMe-DNA by using the 2' -F-DNA as a template; synthesizing DNA, 2'-F-DNA, RNA or 2' -OMe-DNA by taking RNA as a template; DNA, 2' -F-DNA, RNA or 2' -OMe-DNA was synthesized using 2' -OMe-DNA as a template.

Further, the substrates for nucleic acid preparation include ribonucleotides, deoxyribonucleotides and unnatural nucleotides.

Still further, the unnatural nucleotides include 2'-F-dNTPs, 2' -OMe-dNTPs.

It is a fourth object of the present invention to provide a nucleic acid preparation product comprising the Stofel fragment mutant strain of the first object.

The fifth object of the present invention is to provide a recombinant plasmid comprising the vector and the mutant strain gene encoding Stofel fragment of the second object.

Further, the vector comprises plasmid pET23b.

The sixth object of the present invention is to provide a genetically engineered bacterium comprising the recombinant plasmid of the fifth object.

Further, the genetically engineered bacteria include escherichia coli.

The amino acid sequence and the gene sequence of the Stofel fragment mutant strain SFM4-3 are shown as SEQ NO.5 and SEQ NO.6 respectively.

The invention has the following advantages and beneficial effects: compared with the Stoffel fragment mutant SFM4-3, the Stoffel fragment mutant SFM5-7 and SFM5-46 provided by the invention can synthesize full 2' -OMe modified nucleic acid with longer fragments by taking DNA as a template. Stoffel fragment mutants SFM5-7 and SFM5-46 simultaneously retain the basic activity of SFM4-3, i.e., DNA, 2'-F-DNA, RNA or 2' -OMe-DNA was synthesized using 2'-F-DNA, RNA or 2' -OMe-DNA as templates. On the basis, compared with the Stofel fragment mutant SF M4-3, the Stofel fragment mutant SFM5-7 and SFM5-46 have improved activity of synthesizing DNA by taking 2' -OMe-DNA as a template, and the SFM5-7 has greatly improved activity of synthesizing 2' -OMe-DNA by taking 2' -OMe-DNA as a template.

Drawings

FIG. 1 is a schematic representation of the structure of DNA, RNA, 2'-F modifications and 2' -OMe modified nucleic acids.

FIG. 2 is an activity test chart of the Stofel fragment mutant SFM4-3, SFM5-7 and SFM5-46 for synthesizing the fully modified 2'-OMe-DNA, wherein A in FIG. 2 is an activity test chart of the DNA T95 as a template for synthesizing the fully modified 2' -OMe-DNA; b in FIG. 2 is an activity test chart of the synthesis of the fully modified 2' -OMe-DNA using the DNA T120 as a template.

FIG. 3 is a diagram showing activity of Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 recognizing 2'-F-DNA template synthetic DNA, 2' -F-DNA, RNA and 2'-OMe-DNA, wherein A in FIG. 3 is a diagram showing activity of 2' -F-DNA template synthetic DNA; b in FIG. 3 is an activity test chart for synthesizing fully modified 2'-F-DNA by using 2' -F-DNA as a template; FIG. 3C is a graph showing activity of RNA synthesis using 2' -F-DNA as a template; d in FIG. 3 is an activity test chart of the synthesis of fully modified 2'-OMe-DN A using 2' -F-DNA as template.

FIG. 4 is a diagram showing activity of Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 recognizing RNA template synthetic DNA, 2'-F-DNA, RNA and 2' -OMe-DNA, wherein A in FIG. 4 is a diagram showing activity of DNA synthesis using RNA as a template; FIG. 4B is an activity test chart of synthesizing fully modified 2' -F-DNA by using RNA as a template; FIG. 4C is a graph showing activity test of RNA synthesis using RNA as a template; FIG. 4D is a diagram showing activity test of synthesizing fully modified 2' -OMe-DNA using RNA as template

FIG. 5 is a diagram showing activity tests of Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 for DNA synthesis using 2'-OMe-DNA as a template, 2' -F-DNA, RNA and 2'-OMe-DNA, wherein A in FIG. 5 is a diagram showing activity test of DNA synthesis using 2' -OMe-DNA as a template; FIG. 5B is an activity test chart of synthesizing fully modified 2'-F-DNA by using 2' -OMe-DNA as a template; FIG. 5C is a graph showing activity of RNA synthesis using 2' -OMe-DNA as a template; a in FIG. 5 is an activity test chart of the synthesis of the fully modified 2'-OMe-DNA using the 2' -OMe-DNA as a template.

FIG. 6 is a schematic representation of amino acid sequence alignments of Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46.

FIG. 7 is a graph showing the structural homology modeling and mutation site distribution of Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. It should be noted that the following descriptions are not provided for the purpose of detailed description, and those skilled in the art can realize or understand the description with reference to the prior art.

In the present invention, 2'-F-dNTPs refer to deoxyribonucleotides in which the H atom of the 2' site is substituted with a fluorine atom;

2'-OMe-dNTPs refer to deoxyribonucleotides in which the H atom at the 2' -position is replaced by methoxy;

2'-F-DNA is a DNA analogue synthesized by taking deoxyribonucleotide with H atom of 2' site replaced by fluorine atom as substrate;

2'-OMe-DNA refers to DNA analogues synthesized from deoxyribonucleotides substituted with a methoxy group at the H atom at the 2' position.

Example 1

Stoffel fragment mutant SFM4-3, E.coli DH5 a/pET 23b (+) -SFM4-3 and competent cells were all derived from laboratory preservation. pET23b (+) -SFM4-3 plasmid was extracted from E.coli DH 5. Alpha./pET 23b (+) -SFM4-3 using a high purity plasmid miniextraction medium kit (Tiangen Biochemical technology (Beijing)) Co.

UsingAnd (3) performing PCR amplification by using pET23b (+) -SFM4-3 as a template by using the ultra-fidelity DNA polymerase, and performing saturation mutation on codons of 520 and 681 sites by overlapping extension PCR to obtain a double-site saturation mutation library containing SF M4-3 genes. The primers used for the saturation mutation are shown in the following table.

The PCR amplification system and reaction conditions used for saturation mutation are as follows: DNA fragment 1:

DNA fragment 2:

DNA fragment 3:

PCR reaction conditions:

after the PCR reaction is finished, the DNA fragments are recovered by using an ultrathin DNA product purification kit (Tiangen Biochemical technology (Beijing) Co., ltd.) to obtain a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3, and the recovered fragments are used for the next overlapping extension PCR reaction to obtain the full length of the SFM4-3 gene containing the mutation.

The amplification system and reaction conditions used for overlap extension PCR were as follows:

overlap extension PCR reaction conditions:

after the completion of the PCR reaction, the DNA fragment was recovered using an ultra-thin DNA product purification kit (Tiangen Biochemical technology (Beijing) Co., ltd.), and the recovered full-length gene fragment/pET 23b (+) -SFM4-3 was digested with restriction enzymes Xba I and Not I, and placed in the following reaction system and incubated overnight at 37 ℃.

The enzyme digestion reaction system is as follows:

after the completion of the cleavage reaction, the DNA fragment was recovered using an ultra-thin DNA product purification kit (Tiangen Biochemical technology (Beijing) Co., ltd.) to obtain a cleaved DNA fragment. The digested vector was separated by agarose gel electrophoresis, and recovered using an agarose gel DNA recovery kit (Guangzhou Mei Biotechnology Co., ltd.). The full-length gene after cleavage and the vector after cleavage were obtained by ligation using T4 DNA ligase, and incubated overnight at 16℃in the following reaction system.

The enzyme-linked reaction system is as follows:

after the enzyme-linked reaction is finished, the obtained enzyme-linked product is purified by using an ultrathin DNA product purification kit (Tiangen Biochemical technology (Beijing) limited company), the enzyme-linked product is transformed into E.coli clone strain DH5 alpha competent cells by an electric shock transformation method, monoclonal thalli are collected by scraping plates, recombinant plasmids containing SFM4-3 double-site saturated mutation libraries are extracted by using a purity plasmid small-extraction medium-amount kit (Tiangen Biochemical technology (Beijing) limited company), and then the recombinant plasmids are transformed into E.coli clone strain BL21 (DE 3)/pLysS competent cells by an electric shock transformation method, and BL21 (DE 3)/pLysS/pET 23b (+) -SFM5-7 and BL21 (DE 3)/pLysS/pET 23b (+) -SFM5-46 monoclonal strains are obtained after screening.

Example 2

The BL21 (DE 3)/pLysS/pET 23b (+) -SFM5-7 and BL21 (DE 3)/pLysS/pET 23b (+) -SFM5-46 were selected and cultured overnight at 37℃and 220rpm in 20mL of 2 XYT medium containing 100. Mu.g/mL ampicillin and 25. Mu.g/mL chloramphenicol, respectively. The overnight culture was transferred to 800mL of 2 XYT medium containing 100. Mu.g/mL ampicillin and 25. Mu.g/mL chloramphenicol and incubated at 37℃and 220rpm to an absorbance OD ₆₀₀ =0.6-0.8, 0.4mM IPTG (isopropyl thiogalactoside) was added and incubated overnight at 25 ℃. After centrifugation at 6000rpm for 10min at 4℃to collect the cells, the cells were washed with 1 Xwash buffer (50 mM Tris-HCl, 150mM NaCl, 5mM imidozole (imidazole)) pH 8.0) and then crushing the bacterial cells by a high-pressure homogenizer for 15min in a water bath at 70 ℃. After centrifugation at 10000rpm at 4℃for 30min, the supernatant was subjected to removal of cell debris with a 0.45 μm filter, and the filtered supernatant was purified by nickel column affinity chromatography. After incubation of the protein supernatant with the nickel column for 30min, the protein of interest was eluted with 1 Xof buffer (50 mM Tris-HCl, 150mM NaCl, 10-300mM imidozole, pH 8.0) containing different concentrations of imidazole. The resulting product was confirmed by SDS-PAGE (polyacrylamide gel electrophoresis). The target protein was further concentrated using a 50kDa Amicon-Ultra centrifugal ultrafiltration tube to obtain Stofel fragment mutants SFM5-7 and SFM5-46, and finally an equal volume of 100% glycerol was added and stored at-20 ℃.

Example 3

Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 were tested for activity by synthesizing 2' -O Me-DNA using DNA as a template. The activity of the Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 according to the present invention was determined by the following test, and the structure of the DNA and 2'-OMe-DNA was schematically shown in FIG. 1 using DNA as a template to synthesize the fully modified 2' -OMe-DNA.

The activity of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 for synthesizing 2' -OMe-DNA using DNA as a template was determined using T95 and T120 as DNA templates and FAM-P20 and FAM-P24 as primers, respectively.

The DNA template and the complementary primers (T95/FAM-P20, T120/FAM-P24) are mixed in a ratio of 2:1 in advance, denatured at 95 ℃ for 10min, slowly cooled to room temperature, incubated on ice for 5min and the remaining reagents are replenished. The reaction procedure was (50 ℃ C. For 2h, 70 ℃ C. For 30 min). Times.6, 50 ℃ C. For 2h. The DNA templates and primer sequences and reaction systems are shown in the following table. After the reaction, two volumes of 2 XTBE-Urea loading buffer were added to the product, denatured at 95℃for 10min, and finally the product was verified by 20% denaturing polyacrylamide gel electrophoresis.

In FIG. 2, lane 1 is a control, lanes 2-4 are the products of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, in this order, using DNA T95 as a template to synthesize fully modified 2' -OMe-DNA, wherein P indicates the primer, and F indicates the full-length product. In FIG. 2, lane 1 is a control, lanes 2-4 are the products of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, in this order, using DNA T120 as a template to synthesize fully modified 2' -OMe-DNA, where P indicates the primer, and F indicates the full-length product. As can be seen from FIG. 2A, when the 95-nt template and 20-nt primer were used to synthesize the fully modified 2' -OMe-DNA, the Stofel fragment mutants SFM5-7 and SFM5-46 could both synthesize the majority of the full-length product, whereas SFM4-3 only produced a small portion of the full-length product and the majority of the truncated product. As can be seen from FIG. 2B, when the 120-nt template and 24-nt primer were used to synthesize the fully modified 2'-OMe-DNA, the mutant SFM4-3 had less 2' -OMe-DNA full-length product and more non-full-length product. The Stoffel fragment mutants SFM5-7 and SFM5-46 had more full-length 2' -OMe-DNA product and only a small amount of non-full-length product. Compared with the mutant strain SFM4-3, the mutant strains SFM5-7 and SFM5-46 have obviously improved synthesis activity on long fragment 2' -OMe-DNA.

DNA template and complementary primer sequence

Reaction system

Example 4

Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 identified 2'-F-DNA template activity test studies involving the synthesis of DNA, 2' -F-DNA, RNA and 2'-OMe-DNA products using 2' -F-DNA as template. Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 of the present invention were confirmed by the following test for the recognition of 2'-F-DNA template activity, and the schematic structure of 2' -F-DNA and RNA is shown in FIG. 1.

The XNA template 2' -F-T30 and the complementary primer FAM-P15 are mixed in a ratio of 2:1 in advance, denatured at 95 ℃ for 10min, slowly cooled to room temperature, incubated on ice for 5min, and the remaining reagents are replenished. XNA template and complementary primerSequence, reaction system, reaction program and Mn in reaction ²⁺ The concentrations are shown in the following table. When rNTPs are used as substrates in the mutant strain, 1U/. Mu.L of RNase inhibitor is added into the reaction system. After the reaction, two volumes of 2 XTBE-Urea loading buffer were added to the product, denatured at 95℃for 10min, and finally the product was verified by 20% denaturing polyacrylamide gel electrophoresis.

Lanes 1 in FIG. 3A are primers, and lanes 2-4 are, from left to right, products of DNA synthesis from Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, using 2' -F-DNA as a template. In FIG. 3, lane 1 is a primer, and lanes 2 to 4 are, from left to right, products of Stofel fragment mutants SFM4 to 3, SFM5 to 7 and SFM5 to 46, respectively, for synthesizing fully modified 2'-F-DNA using 2' -F-DNA as a template. In FIG. 3, lane 1 is the primer, and lanes 2-4 are, from left to right, the products of RNA synthesis from Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, using 2' -F-DNA as a template. In FIG. 3, lane 1 is the primer, and lanes 2-4 are, from left to right, the products of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, for synthesizing the fully modified 2'-OMe-DNA using the 2' -F-DNA as a template. A band that is more than full length is a band that results from incomplete denaturation of the product. Wherein P refers to the primer and F refers to the full-length product.

As can be seen from FIG. 3, stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 can synthesize DNA, RNA, fully modified 2' -F-DNA or fully modified full-length 2' -OMe-D NA products using 2' -F-DNA as a template.

XNA template and complementary primer sequences

f:2' -F modified nucleotides

Reaction system

Reaction procedure and Mn in the reaction ²⁺ Concentration of

Example 5

Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 identified RNA templates, including DNA, 2'-F-DNA, RNA and 2' -OMe-DNA products were synthesized using RNA as templates. The Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 of the present invention were verified for their activity to recognize RNA templates by the following test.

RNA template RNA-T30 and the complementary primer FAM-P15 are mixed in a ratio of 2:1 in advance, denatured at 65 ℃ for 10min, and the rest reagent is replenished after rapid incubation on ice for 5min. RNA template and complementary primer sequence, reaction system, reaction program and Mn in reaction ²⁺ The concentrations are shown in the following table. After the reaction, two volumes of 2 XTBE-Urea loading buffer were added to the product, denatured at 95℃for 10min, and finally the product was verified by 20% denaturing polyacrylamide gel electrophoresis.

In FIG. 4, lane 1 is the primer, and lanes 2-4 are, from left to right, the products of DNA synthesis from Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, using RNA as a template. In FIG. 4, lane 1 is a primer, and lanes 2 to 4 are, from left to right, products of Stofel fragment mutants SFM4 to 3, SFM5 to 7 and SFM5 to 46, respectively, for synthesizing fully modified 2' -F-DNA using RNA as a template. In FIG. 4, lane 1 is the primer and lanes 2-4 are, from left to right, the products of RNA synthesis using RNA as template from Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively. In FIG. 4, lane 1 is the primer and lanes 2-4 are, from left to right, the products of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, for the synthesis of fully modified 2' -OMe-DNA using RNA as template. Wherein P refers to the primer and F refers to the full-length product.

As can be seen from FIG. 4, stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 were able to synthesize full-length products of DNA, 2' -F-DNA and RNA using RNA as a template. Using 2' -OMe-dNTPs as substrates, stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 all produced partial full-length products.

RNA templates and complementary primer sequences

r: ribonucleotides

Reaction system

Reaction procedure

Example 6

Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 recognize 2' -OMe-DNA template activity test study, which involved synthesizing DNA, 2' -F-DNA, RNA and 2' -OMe-DNA products using 2' -OMe-DNA as templates, the activity of Stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 recognizing 2' -OMe-DNA templates in the present invention was verified by the following test.

The XNA template 2' -OMe-T30 and the complementary primer FAM-P18 are mixed in a ratio of 2:1 in advance, denatured at 95 ℃ for 10min, slowly cooled to room temperature, incubated on ice for 5min, and the remaining reagents are filled up. XNA template and complementary primer sequence, reaction system, reaction program and Mn in reaction ²⁺ The concentrations are shown in the following table. When rNTPs are used as substrates in the mutant strain, 1U/. Mu.L of RNase inhibitor is added into the reaction system. After the reaction was completed, twice the volume of 2 XTBE-Urea loading buffer was added to the product, denatured at 95℃for 10min, and finally the product was verified by 20% denaturing polyacrylamide gel electrophoresis.

Lanes 1 in FIG. 5A are primers, and lanes 2-4 are, from left to right, products of DNA synthesis from Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, using 2' -OMe-DNA as a template. In FIG. 5, lane 1 is the primer, and lanes 2-4 are, from left to right, the products of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, for synthesizing the fully modified 2'-F-DNA using the 2' -OMe-DNA as a template. In FIG. 5, lane 1 is the primer and lanes 2-4 are, from left to right, the products of RNA synthesis from Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, using 2' -OMe-DNA as template. In FIG. 5, lane 1 is the primer and lanes 2-4 are, from left to right, the products of the Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46, respectively, synthesizing fully modified 2'-OMe-DNA using 2' -OMe-DNA as a template. A P guide object; f refers to the full length product.

As can be seen from FIG. 5, stoffel fragment mutants SFM4-3, SFM5-7 and SFM5-46 can synthesize DNA, 2'-F-DNA or RNA full-length products using 2' -OMe-DNA as a template. SFM5-7 and SFM5-46 produced more of the full length DNA product using 2' -OMe-DNA as template than Stofel fragment mutant SFM 4-3. In comparison with the Stoffel fragment mutants SFM4-3 and SFM5-46, SFM5-7 was able to synthesize a large portion of the full-length product of the fully modified 2'-OMe-DNA and a very small number of truncations using the 2' -OMe-DNA as template.

The amino acid sequence alignment results of Stofel fragment mutants SFM4-3, SFM5-7 and SFM5-46 are shown in FIG. 6, wherein the amino acids at 520 and 681 sites of SFM4-3 are E and K, the amino acids at 520 and 681 sites of SFM5-7 are P and R, the amino acids at 520 and 681 sites of SFM5-46 are P and L, and an additional inserted amino acid sequence ALRLSQRLAIPYAPPPLR is arranged behind 520 sites. The distribution of the mutation sites of the three Stofel fragment mutants in the protein structure is shown in FIG. 7, the left graph shows the mutation sites contained in Stofel fragment mutant SFM4-3 and the distribution in the structure, the middle is the mutation of Stofel fragment mutant SFM5-7 at 520 and 681 sites and the distribution in the structure, and the right graph shows the mutation of Stofel fragment mutant SFM5-46 at 520 and 681 sites and the distribution of additional amino acid fragments and the distribution in the structure.

XNA template and complementary primer sequences

m:2' -OMe modified nucleotides

Reaction system

Reaction procedure and Mn in the reaction ²⁺ Concentration of

The Stofel fragment mutants SFM5-7, SFM5-46 and SFM4-3 used in the present invention have the following amino acid sequences and gene sequences.

SEQ NO.1: stoffel fragment mutant SFM5-7 amino acid sequence

MAQPASPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRA

PEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTP

EGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLS

AVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQL

ERVLFDELGLPAIGKTEKTGKRSTSAAALPALREAHPIVEKILQYRELTKLKST

YIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQSIPVRTPLGQRIRRAFI

AEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPR

EAVNPLMRRAAKTINFGVLYGMSAHRLSQRLAIPYEEAQAFIERYFQSFPKVR

AWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRQAAERRAFNMPVQG

TAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKE

VMEGVYPLAVPLEVEVGIGEDWLSAKEAA

SEQ NO.2 Stofel fragment mutant SFM5-46 amino acid sequence MAQPASPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAALPALRLSQRLAIPYAPPPLRALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQSIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVNPLMRRAAKTINFGVLYGMSAHRLSQLLAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRQAAERRAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA

SEQ NO.3 Stofel fragment mutant SFM5-7 Gene sequence

ATGGCCCAGCCGGCCAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCC

GCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGT

GGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGTCGGGTCCACCGG

GCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCT

TCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCC

CGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACA

CCACCCCCGAGGGGGTAGCCCGGCGCTATGGCGGGGAGTGGACGGAGGA

GGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGG

GGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAG

AGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCT

GGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCG

CCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCA

ACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCC

GCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCG

CCCTGCCTGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGT

ACCGGGAGCTCACCAAGCTGAAGAGCACCTATATTGACCCCTTGCCGGACC

TCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCC

ACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAGCATCCC

CGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGG

AGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGGAAGGGCTCAGGGTG

CTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGAGGG

GCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGG

AGGCCGTGAACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGG

GTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGAGGCTAGCCATCCCT

TACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAG

GTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGT

ACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCC

CGGGTGAAGAGCGTGCGGCAGGCGGCCGAGCGCAGGGCCTTCAACATGC

CCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCT

TCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGAC

GAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCACGGC

TGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAG

GTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGCGGCC

SEQ NO.4 Stoffel fragment mutant SFM5-46 Gene sequence ATGGCCCAGCCGGCCAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCC

GCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGT

GGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGTCGGGTCCACCGG

GCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCT

TCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCC

CGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACA

CCACCCCCGAGGGGGTAGCCCGGCGCTATGGCGGGGAGTGGACGGAGGA

GGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGG

GGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAG

AGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCT

GGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCG

CCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCA

ACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCC

GCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCG

CCCTGCCTGCCCTCCGCCTCTCCCAGAGACTAGCCATCCCTTACGCGCCGC

CGCCCCTGAGGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTG

CAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTATATTGACCCCTTGCCG

GACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGAC

GGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAGCA

TCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCG

AGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGGAAGGGCTCAGG

GTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGGA

GGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCC

GGGAGGCCGTGAACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTC

GGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGCTACTAGCCATC

CCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCC

AAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGG

GGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAG

GCCCGGGTGAAGAGCGTGCGGCAGGCGGCCGAGCGCAGGGCCTTCAACA

TGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAG

CTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCA

CGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCA

CGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCT

GGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGGCG

GCC

SEQ NO.5: stoffel fragment mutant strain SFM4-3 amino acid sequence

MAQPASPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRA

PEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPE

GVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAV

LAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERV

LFDELGLPAIGKTEKTGKRSTSAAALEALREAHPIVEKILQYRELTKLKSTYIDP

LPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQSIPVRTPLGQRIRRAFIAEEG

WLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVNP

LMRRAAKTINFGVLYGMSAHRLSQKLAIPYEEAQAFIERYFQSFPKVRAWIEK

TLEEGRRRGYVETLFGRRRYVPDLEARVKSVRQAAERRAFNMPVQGTAADL

MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGV

YPLAVPLEVEVGIGEDWLSAKEAA

SEQ NO.6 Stofel fragment mutant SFM4-3 Gene sequence

ATGGCCCAGCCGGCCAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCC

GCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGT

GGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGTCGGGTCCACCGG

GCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCT

TCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCC

CGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACA

CCACCCCCGAGGGGGTAGCCCGGCGCTATGGCGGGGAGTGGACGGAGGA

GGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGG

GGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGA

GAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCC

TGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATC

GCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTC

AACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCC

CGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCC

GCCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCA

GTACCGGGAGCTCACCAAGCTGAAGAGCACCTATATTGACCCCTTGCCGG

ACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACG

GCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAGCAT

CCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCG

AGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGGAAGGGCTCAG

GGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTCTTCCAGG

AGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCC

CGGGAGGCCGTGAACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTT

CGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGAAGCTAGCCA

TCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCC

CCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCG

GGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAG

AGGCCCGGGTGAAGAGCGTGCGGCAGGCGGCCGAGCGCAGGGCCTTCAA

CATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGA

AGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTC

CACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGG

CACGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCC

CTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGG

CGGCC

Claims

A stoffl fragment mutant, characterized in that the stoffl fragment mutant comprises two stoffl fragment mutants: stoffel fragment mutant SFM5-7, the amino acid sequence of which is shown as SEQ ID NO.1, stoffel fragment mutant SFM5-7 is obtained by performing double-site saturation mutation on the basis of Stoffel fragment mutant SFM4-3, and newly added mutation is E520P and K681R; stoffel fragment mutant SFM5-46, the amino acid sequence of which is shown as SEQ ID NO.2, stoffel fragment mutant SFM5-46 is obtained by performing double-site saturation mutation on the basis of Stoffel fragment mutant SFM4-3, and the new mutation is E520P and K681L, and an additional amino acid sequence.
2. A gene encoding the Stoffel fragment mutant strain according to claim 1, wherein the sequence of the gene encoding the Stoffel fragment mutant strain SFM5-7 is shown in SEQ ID NO. 3; the gene sequence of the Stoffel fragment mutant strain SFM5-46 is shown in SEQ ID NO. 4.
3. Use of a Stofel fragment mutant according to claim 1 for the preparation of a nucleic acid preparation product.
4. The use according to claim 3, wherein the nucleic acid preparation comprises synthesis of 2'-OMe-DNA using DNA as a template, synthesis of DNA, 2' -F-DNA, 2'-OMe-DNA or RNA using 2' -OMe-DNA or RNA as a template; substrates for the preparation of the nucleic acids include ribonucleotides, deoxyribonucleotides and non-natural nucleotides.
5. The use of claim 4, wherein the unnatural nucleotides comprise 2'-F-dNTPs, 2' -OMe-dNTPs.
6. A nucleic acid preparation comprising the Stoffel fragment mutant of claim 1.
7. A recombinant plasmid comprising a vector and the gene of claim 2.
8. The recombinant plasmid of claim 7, wherein the vector comprises plasmid pET23b.
9. A genetically engineered bacterium comprising the recombinant plasmid of claim 7 or 8.
10. The genetically engineered bacterium of claim 9, wherein the genetically engineered bacterium comprises escherichia coli.