Bst dna polymerase mutants with xna synthesis and reverse transcription activity

Abstract

The application discloses Bst DNA polymerase mutant strains with XNA synthesis and reverse transcription activity. The Bst DNA polymerase mutant strains comprise Sso7d-Bst, Bst DNA polymerase mutant strain Bst(I659E, E660G) and Bst DNA polymerase mutant strain Sso7d-Bst(I659E, E660G); the Bst DNA polymerase mutant strains can recognize and synthesize natural DNA, and can also recognize, synthesize and reverse transcribe RNA and 2'-F, 2'-OMe modified nucleic acid Bst DNA polymerase mutant strains. The three Bst DNA polymerase mutant strains provided by the application can not only efficiently transcribe RNA and 2'-OMe-RNA, but also efficiently reverse transcribe 2'-OMe-RNA compared with the wild type.

Description

Technical Field

[0001] This invention belongs to the field of enzyme molecular modification, specifically involving mutant strains of Bst DNA polymerase and their applications and the gene encoding the mutant. Background Technology

[0002] Natural deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the carriers of genetic information in all life. DNA polymerase, RNA polymerase, and reverse transcriptase are the most crucial enzymes in living organisms, playing key roles in the storage, use, and transmission of genetic information. Non-natural nucleic acids (XNAs) are DNA / RNA analogs with non-natural structural units, and their preparation methods involve modifying the backbone, sugar rings, and bases of natural nucleic acids. In recent years, XNAs have gradually become a novel carrier of genetic information, and their development has promoted the rapid development of the field of xenobiology.

[0003] Among xenobiotic aptamers (XNAs), those with glycosyl modifications are the most extensively studied, including threonine nucleic acid (TNA), hexitol nucleic acid (HNA), d-glycotriol nucleic acid (AtNA), cyclohexenyl nucleic acid (CeNA), locked nucleic acid (LNA), arabinonucleotide (ANA), 2'-fluoroarabinonucleotide (FANA), 2'-F-DNA, and 2'-OMe-DNA. Some of these XNAs can be recognized by various natural and engineered polymerases. Some XNA double strands are more stable than DNA, RNA, or DNA / RNA hybrid double strands, and some XNAs also exhibit high resistance to nucleases. Therefore, XNAs demonstrate significant advantages in applications such as developing nucleic acid aptamers and biomaterials with excellent physiological stability.

[0004] However, due to their high substrate specificity, natural polymerases struggle to efficiently recognize non-natural nucleic acids, hindering the transfer of genetic information between DNA and XNA, such as XNA transcription and reverse transcription. To address this issue, researchers have designed and modified nucleic acid polymerases through directed evolution, rational design, or semi-rational design, enabling them to recognize and synthesize non-natural nucleic acid molecules. For example, the Holliger group evolved a series of Tgo polymerase mutants capable of synthesizing and reverse transcribing various XNAs, such as HNA, TNA, FANA, and ANA. The Chaput group obtained the KOD polymerase mutant RSGA, which showed significantly enhanced specificity for TNA substrates. The Romesberg group performed directed evolution on the Stoffel fragment of Taq DNA polymerase using phage display technology, obtaining polymerase mutants SFM4-3, SFM4-6, and SFM4-9. Among them, SFM4-3 can not only transcribe fully 2'-OMe modified non-natural nucleic acids but also amplify partially 2'-OMe or 2'-F modified nucleic acid chains via PCR.

[0005] Bacillus thermophilus DNA polymerase (Bst DNA polymerase) is a member of the DNA polymerase A family. Due to its strong thermostability, strand displacement activity, and polymerase activity, it is widely used in various isothermal amplification techniques. Among these, loop-mediated isothermal amplification (LAMP) uses four specific primers designed for six regions of the target gene. Under the action of Bst DNA polymerase, it can achieve exponential amplification of DNA in a short time. It is characterized by its simplicity, high specificity, and easily detectable products, making it a powerful and widely used diagnostic method, such as for the detection of SARS-CoV-2. Rolling circle amplification (RCA) uses a short circular oligonucleotide as a template, which is amplified by Bst DNA polymerase to produce a long repeating single-stranded DNA. It is also widely used in genomics, proteomics, molecular diagnostics, biosensing, and drug development. Developing Bst polymerase mutants capable of recognizing XNA will hopefully enable isothermal and rolling circle amplification of XNA, thus greatly expanding the application range of XNA.

[0006] Sso7d, derived from the thermophilic archaea *Sulfolobus solfataricus*, is a positively charged DNA-binding protein with a molecular weight of only 7 kDa. Upon binding to DNA, it increases the negative supercoiling of DNA and raises its melting temperature. Sso7d can act as a splicing protein to connect DNA to other organic molecules, or as a scaffold protein to link multiple proteins into a functional complex and promote their interactions. The fusion of Sso7d with many DNA polymerases (such as Pfu) can significantly improve the synthesis rate and continuity of these polymerases.

[0007] The site where DNA polymerase interacts with nucleotides contains amino acid residues known as spatial gates. The side chains of these amino acid residues spatially block the 2'-OH group of the ribonucleotide, thus preventing erroneous insertion of the ribonucleotide into the DNA. Studies have shown that mutating the spatial gate amino acid residues of DNA polymerase can enable it to synthesize RNA and various nucleic acids with sugar ring modifications. For example, the I614 and E615 sites of the Stoffel fragment are close to the sugar group of the bound nucleotide, and these two amino acids are located at the N-terminus of the O-helix, which is crucial for the interaction between the polymerase and the substrate nucleotide. These functional features suggest that the amino acids at positions 614 and 615 in the Stoffel fragment may act as spatial gates to restrict the integration of nucleotides modified at the 2' position of the sugar group. The superior activity of the Stoffel fragment mutant SFM19 (I614E, E615G) for 2' modifications demonstrates that replacing these amino acids with smaller ones can expand the space of the active site, thereby enabling the recognition of both ribonucleotides and nucleotides modified at the 2' position.

[0008] Bst DNA polymerase and Taq polymerase are both members of the DNA polymerase family A, and their amino acid sequences share certain homology. Therefore, we homologously transplanted the I614E and E615G sites on the Taq polymerase mutant strain SFM4-3 into Bst DNA polymerase, and simultaneously fused the Sso7d protein to the N-terminus of the Bst mutant strain, hoping to develop a Bst DNA polymerase mutant strain capable of efficiently transcribing XNA. Summary of the Invention

[0009] In order to modify the substrate specificity of wild-type Bst DNA polymerase so that it can recognize, synthesize and reverse transcribe nucleic acids such as RNA and 2'-F-RNA, the primary objective of this invention is to provide three Bst DNA polymerase mutants that can recognize and synthesize natural DNA, as well as recognize, synthesize and reverse transcribe RNA and 2'-F and 2'-OMe modified nucleic acids.

[0010] Another object of the present invention is to provide the gene sequence and amino acid sequence of the above-mentioned Bst DNA polymerase mutant.

[0011] Another object of the present invention is to provide the application of the above-mentioned Bst DNA polymerase mutant strain.

[0012] The objective of this invention is achieved through the following technical solutions.

[0013] A Bst DNA polymerase mutant strain, Sso7d-Bst, has the amino acid sequence shown in SEQ ID NO.1.

[0014] A Bst DNA polymerase mutant strain, Bst(I659E,E660G), has the amino acid sequence shown in SEQ ID NO.2.

[0015] A Bst DNA polymerase mutant strain, Sso7d-Bst (I659E, E660G), has the amino acid sequence shown in SEQ ID NO.3.

[0016] The present invention also provides a gene encoding the above-mentioned Bst DNA polymerase mutant strain Sso7d-Bst, the gene sequence of which is shown in SEQ ID NO.4.

[0017] The present invention provides a gene encoding the above-mentioned Bst DNA polymerase mutant strain Bst(I659E,E660G), the gene sequence of which is shown in SEQ ID NO.5.

[0018] The present invention provides a gene encoding the above-mentioned Bst DNA polymerase mutant strain Sso7d-Bst(I659E,E660G), the gene sequence of which is shown in SEQ ID NO.6.

[0019] Furthermore, the Sso7d-Bst mutant strain was obtained by introducing the Sso7d sequence into the wild-type Bst DNA gene using overlap extension PCR technology.

[0020] Furthermore, the aforementioned Bst(I659E,E660G) mutant strain was obtained by transplanting the mutation sites I614E and E615G of SFM-4-3 polymerase into Bst DNA polymerase through site-directed mutagenesis mediated by overlap extension PCR.

[0021] Furthermore, the Sso7d-Bst (I659E, E660G) mutant strain was obtained by transplanting the mutation sites I614E and E615G of SFM-4-3 polymerase into the Sso7d-Bst mutant strain through site-directed mutagenesis mediated by overlap extension PCR.

[0022] Furthermore, the transcriptional activities of each Bst DNA polymerase mutant strain on RNA, 2'-F-NTPs, and 2'-OMe-NTPs substrates were determined using DNA as a template.

[0023] Compared with wild-type Bst DNA polymerase, the present invention has the following advantages and beneficial effects: The three Bst DNA polymerase mutants provided by the present invention can efficiently transcribe both RNA and 2'-OMe-RNA, and can also efficiently reverse transcribe 2'-OMe-RNA, compared with wild-type. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structures of DNA, RNA, 2'-F-RNA, and 2'-OMe-RNA.

[0025] Figure 2 This is a schematic diagram showing the amino acid sequence alignment of SF polymerase, SFM4-3 polymerase, Bst DNA polymerase, and the mutant Bst (I659E, E660G) polymerase. The dashed lines indicate the original amino acids at residues 614 and 615 of SF polymerase and residues 659 and 660 of Bst DNA polymerase. The solid lines indicate the amino acids at residues 614 and 615 of SFM4-3 polymerase and the mutated amino acids at residues 659 and 660 of the mutant Bst (I659E, E660G).

[0026] Figure 3The diagrams show the fusion of the Sso7d protein with Bst DNA polymerase and the mutant strain Bst (I659E, E660G) polymerase, respectively. From top to bottom, they are the mutant strain Sso7d-Bst and the mutant strain Sso7d-Bst (I659E, E660G).

[0027] Figure 4 It is wild-type Bst DNA polymerase and its various mutant strains in the presence of 1mM Mn 2+ The results of the RNA transcription activity assay using DNA as a template in the reaction buffer and in the Mn-free buffer. 2+ The results of the RNA transcription activity assay using DNA as a template in the reaction buffer are shown. From left to right, lanes 1 and 6 are controls; lanes 2-5 are wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a solution containing 1 mM Mn. 2+ RNA transcription products with DNA as a template were placed in the reaction buffer; lanes 7-10, in turn, contained wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a buffer without Mn. 2+ RNA transcription products using DNA as a template are in the reaction buffer. P: 18nt primer; F: 40nt full-length product.

[0028] Figure 5 It is wild-type Bst DNA polymerase and its various mutant strains in the presence of 1mM Mn 2+ The results of the activity test for transcribing 2'-F-RNA using DNA as a template in the reaction buffer and in the presence of Mn 2+ The results of the activity assay for transcribing 2'-F-RNA using DNA as a template in the reaction buffer are shown. From left to right, lanes 1 and 6 are controls; lanes 2-5 are wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a solution containing 1 mM Mn. 2+ The reaction buffer contained 2'-F-RNA transcripts with DNA as a template; lanes 7-10, in turn, contained wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a buffer without Mn. 2+ The 2'-F-RNA transcription product with DNA as a template is in the reaction buffer. P: 18nt primer; F: 40nt full-length product.

[0029] Figure 6 It is wild-type Bst DNA polymerase and its various mutant strains in the presence of 1mM Mn 2+ The results of the activity test for transcribing 2'-OMe-RNA using DNA as a template in the reaction buffer and in the Mn-free buffer. 2+ The results of the activity assay for transcribing 2'-OMe-RNA using DNA as a template in the reaction buffer are shown. From left to right, lanes 1 and 6 are controls; lanes 2-5 are wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a solution containing 1 mMMn. 2+ The reaction buffer contains 2'-OMe-RNA transcripts templated with DNA; lanes 7-10 contain, respectively, 2'-OMe-RNA transcripts templated with DNA from wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant. P: 18nt primer; F: 40nt full-length product.

[0030] Figure 7 This is the result of an activity assay for wild-type Bst DNA polymerase and its mutant strains using RNA as a template to reverse transcribe DNA. From left to right, lane 1 is the control; lanes 2-5 are, respectively, wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a solution containing 1 mM Mn. 2+ DNA reverse transcription products using RNA as a template are in the reaction buffer. P: 15nt primer; F: 30nt full-length product.

[0031] Figure 8 This is the result of an activity assay for wild-type Bst DNA polymerase and its mutant strains using 2'-F-RNA as a template for reverse transcription of DNA. From left to right, lane 1 is the control; lanes 2-5 are, respectively, wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a solution containing 1 mM Mn. 2+ DNA reverse transcription products using 2'-F-RNA as a template in the reaction buffer. P: 15nt primer; F: 30nt full-length product.

[0032] Figure 9This is the result of an activity assay for wild-type Bst DNA polymerase and its mutant strains using 2'-OMe-RNA as a template for reverse transcription of DNA. From left to right, lane 1 is the control; lanes 2-5 are, respectively, wild-type Bst DNA polymerase, Bst(I659E,E660G) mutant, Sso7d-Bst mutant, and Sso7d-Bst(I659E,E660G) mutant in a solution containing 1 mM Mn. 2+ DNA reverse transcription products using 2'-OMe-RNA as a template in the reaction buffer. P: 18nt primer; F: 30nt full-length product. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto. It should be noted that any details not specifically described below are for reference by those skilled in the art to implement or understand.

[0034] Example 1

[0035] use The ultra-fidelity DNA polymerase was amplified by PCR, followed by overlap extension PCR to obtain the fusion Sso7d sequence and the Bst DNA polymerase gene containing the mutants I659E and E660G. The primers used for site-directed mutagenesis are shown in the table below.

[0036]

[0037] The PCR amplification system and reaction conditions used for site-directed mutagenesis are as follows: DNA fragment 1:

[0038]

[0039] DNA fragment 2:

[0040]

[0041]

[0042] DNA fragment 3:

[0043]

[0044] DNA fragment 4:

[0045]

[0046]

[0047] DNA fragment 5:

[0048]

[0049] DNA fragment 6:

[0050]

[0051] PCR reaction conditions:

[0052]

[0053] After the PCR reaction was completed, the DNA fragments were recovered using the Tiangen Ultrathin Recovery Kit. The recovered fragments were then used in the next step of overlap extension PCR to obtain the full-length Bst gene containing the mutation.

[0054] The amplification system and reaction conditions used in overlap extension PCR are as follows: Full length of the Sso7d-Bst gene:

[0055]

[0056] Full length of the Bst(I659E,E660G) gene:

[0057]

[0058]

[0059] Full length of the Sso7d-Bst(I659E,E660G) gene:

[0060]

[0061] Overlap extension PCR reaction conditions:

[0062]

[0063] After the PCR reaction was completed, the DNA fragments were recovered using the Tiangen Ultra-Thin Recovery Kit. The recovered fragments and plasmid pET30a were digested with restriction endonucleases XbaI and EcoRI and incubated overnight at 37°C in the reaction system described below.

[0064] The enzyme digestion reaction system is as follows:

[0065]

[0066] After the enzyme digestion reaction, the DNA fragments were recovered using the Tiangen Ultrathin Recovery Kit. The digested vector was separated by agarose gel electrophoresis and recovered using the Meiji Gel Recovery Kit. The digested DNA fragments and vector fragments were ligated using T4 DNA ligase and incubated overnight at 16°C in the reaction system described below.

[0067] The enzyme ligation reaction system is as follows:

[0068]

[0069] The enzyme-linked polymerase chain product was directly transformed into E. coli DH5α. Positive transformants were verified by colony PCR, and plasmids were extracted and sequenced.

[0070] This invention extracts correctly sequenced recombinant plasmids pET30a-Sso7d-Bst, pET30a-Bst(I659E,E660G), and pET30a-Sso7d-Bst(I659E,E660G) from recombinant bacteria DH5α / Sso7d-Bst, DH5α / Bst(I659E,E660G), and transforms them into Escherichia coli expression strain BL21(DE3)pLysS to obtain recombinant expression strains BL21(DE3)pLysS / Sso7d-Bst, BL21(DE3)pLysS / Bst(I659E,E660G), and BL21(DE3)pLysS / Sso7d-Bst(I659E,E660G).

[0071] Example 2

[0072] Select BL21(DE3)pLysS / pET30a-Sso7d-Bst, BL21(DE3)pLysS / pET30a-Bst(I659E,E660G) and

[0073] Monoclonal clones of BL21(DE3)pLysS / pET30a-Sso7d-Bst(I659E,E660G) were cultured overnight at 37°C and 220 rpm in 20 mL of 2×YT medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, respectively. The overnight cultures were then transferred to 1 L of 2×YT medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol and cultured at 37°C and 220 rpm until OD200. 600=0.6-0.8, add IPTG to a final concentration of 0.5 mmol / L, and incubate at 37℃ for 4-5 h. Collect bacterial cells by centrifugation at 6000 rpm for 10 min at 4℃, resuspend in buffer A (50 mM Tris·HCl, 150 mM NaCl, 5 mM imidazole, pH 7.5), and then homogenize by high pressure. Centrifuge at 10000 rpm for 1 h at 4℃. Remove cell debris from the supernatant using a 0.22 μm aqueous filter membrane. Purify the filtered supernatant by nickel column affinity chromatography. The general purification procedure is as follows: incubate the protein supernatant with a nickel column for 30 min, and elute the target protein with 1×Elution buffer (50 mM Tris·HCl, 150 mM NaCl, 10-500 mM Mimidazole, pH 7.5) containing different concentrations of imidazole. Verify the obtained product by SDS-PAGE. The target protein was further concentrated using 50 kDa Amicon-Ultra, and finally an equal volume of 100% glycerol was added and stored at -20°C.

[0074] Example 3

[0075] Transcriptional activity assays of polymerase mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) against NTP substrates were conducted. The RNA transcriptional activity of the mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) according to the present invention was determined by the following assays.

[0076] The transcription system is as follows:

[0077]

[0078] The DNA sequence used is:

[0079]

[0080] The mixture of template T40 and complementary primer T60-FAM-R was denatured at 95°C for 5 min, slowly cooled to room temperature, and incubated on ice for 5 min. Then, the remaining reagents were added. Transcription was performed at 50°C for 2 h. After transcription, the transcription product was added to one volume of 2×TBE-Urea loading buffer (Sangon Biotech: C506046-0005), denatured at 95°C for 10 min, and its activity was verified by 20% denaturing polyacrylamide gel electrophoresis. After electrophoresis, the FAM fluorescence was observed under blue light with a 535 nm filter to determine the size of the transcription product bands. Results are shown below. Figure 4Activity assays were performed on wild-type Bst DNA polymerase and its mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) to transcribe RNA using DNA as a template. Figure 4 It can be seen that the mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) can synthesize full-length products and have good RNA transcription activity.

[0081] Example 4

[0082] Transcriptional activity assays of polymerase mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) for 2'-F-NTP substrates were conducted. The 2'-F-RNA transcriptional activity of the mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) according to the present invention was determined by the following assays.

[0083] The transcription system is as follows:

[0084]

[0085]

[0086] The DNA sequence used is:

[0087]

[0088] The mixture of template T40 and complementary primer T60-FAM-R was denatured at 95°C for 5 min, slowly cooled to room temperature, and incubated on ice for 5 min. Then, the remaining reagents were added. Transcription was performed at 50°C for 2 h. After transcription, the transcription product was added to one volume of 2×TBE-Urea loading buffer (Sangon Biotech: C506046-0005), denatured at 95°C for 10 min, and its activity was verified by 20% denaturing polyacrylamide gel electrophoresis. After electrophoresis, the FAM fluorescence was observed under blue light with a 535 nm filter to determine the size of the transcription product bands. Results are shown below. Figure 5 The activity of wild-type Bst DNA polymerase and its mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) in transcribing 2'-F-NTPs using DNA as a template was tested. Figure 5 It is known that the mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) can synthesize full-length products and have good 2'-F-RNA transcriptional activity.

[0089] Example 5

[0090] Transcriptional activity assays of polymerase mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) for 2'-OMe-NTPs substrates were conducted. The 2'-OMe-RNA transcriptional activity of the mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) according to the present invention was determined by the following assays.

[0091] The transcription system is as follows:

[0092]

[0093] The DNA sequence used is:

[0094]

[0095] The mixture of template T40 and complementary primer T60-FAM-R was denatured at 95℃ for 5 min, slowly cooled to room temperature, and incubated on ice for 5 min. Then, the remaining reagents were added. Transcription was performed at 50℃ for 4 h. After transcription, the transcription product was added to one volume of 2×TBE-Urea loading buffer (Shanghai Sangon Biotech: C506046-0005), denatured at 95℃ for 10 min, and its activity was verified by 20% denaturing polyacrylamide gel electrophoresis. After electrophoresis, the FAM fluorescence was observed under blue light with a 535 nm filter to determine the size of the transcription product bands. Results are shown below. Figure 6 The activity of wild-type Bst DNA polymerase and its mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) in transcribing 2'-OMe-NTPs using DNA as a template was tested. Figure 6 It can be seen that the mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) have certain 2'-OMe-RNA transcriptional activity.

[0096] Example 6

[0097] The reverse transcription activity of polymerase mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) using RNA as a template was investigated. The reverse transcription activity of the mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) according to the present invention using RNA as a template was determined by the following tests.

[0098] The reverse transcription system is as follows:

[0099]

[0100] The oligonucleotide sequences used are:

[0101]

[0102] The mixture of RNA template and complementary primer FAM-RP was denatured at 65°C for 5 min, slowly cooled to room temperature, and incubated on ice for 5 min. The remaining reagents were then added. Reverse transcription was performed at 50°C for 3 h. After reverse transcription, the reverse transcription product was added to one volume of 2×TBE-Urea loading buffer (Shanghai Sangon Biotech: C506046-0005), denatured at 95°C for 10 min, and its activity was verified by 20% denaturing polyacrylamide gel electrophoresis. After electrophoresis, FAM fluorescence was observed under blue light with a 535 nm filter to determine the size of the transcription product bands. Results are shown below. Figure 7 Activity assays were performed on wild-type Bst DNA polymerase and its mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) for reverse transcription of DNA using RNA as a template. Figure 7 It can be seen that the mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) can reverse transcribe to produce full-length products, and have good reverse transcription activity from RNA to DNA.

[0103] Example 7

[0104] The reverse transcription activity of polymerase mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) using 2'-F-RNA as a template was investigated. The following tests determined the reverse transcription activity of the mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) according to the present invention using 2'-F-RNA as a template.

[0105] The reverse transcription system is as follows:

[0106]

[0107] The oligonucleotide sequences used are:

[0108]

[0109] The mixture of 2'-F-RNA template and complementary primer FAM-FP was denatured at 95°C for 5 min, slowly cooled to room temperature, and incubated on ice for 5 min. Then, the remaining reagents were added. Reverse transcription was performed at 50°C for 1 h. After reverse transcription, the reverse transcription product was added to one volume of 2×TBE-Urea loading buffer (Shanghai Sangon Biotech: C506046-0005), denatured at 95°C for 10 min, and its activity was verified by 20% denaturing polyacrylamide gel electrophoresis. After electrophoresis, the FAM fluorescence was observed under blue light with a 535 nm filter to determine the size of the transcription product bands. Results are shown below. Figure 8 The activity of wild-type Bst DNA polymerase and its mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) in reverse transcription of DNA using 2'-F-RNA as a template was tested. Figure 8 It can be seen that the mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) can reverse transcribe to produce full-length products, and have good reverse transcription activity from 2'-F-RNA to DNA.

[0110] Example 8

[0111] The reverse transcription activity of polymerase mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) using 2'-OMe-RNA as a template was investigated. The following tests determined the reverse transcription activity of the mutants Bst(I659E,E660G), Sso7d-Bst, and Sso7d-Bst(I659E,E660G) according to the present invention using 2'-OMe-RNA as a template.

[0112] The reverse transcription system is as follows:

[0113]

[0114]

[0115] The oligonucleotide sequences used are:

[0116]

[0117] The mixture of 2'-OMe-RNA template and complementary primer FAM-OMe-P was denatured at 95°C for 5 min, slowly cooled to room temperature, and incubated on ice for 5 min. The remaining reagents were then added. Reverse transcription was performed at 50°C for 1 h. After reverse transcription, the reverse transcription product was added to one volume of 2×TBE-Urea loading buffer (Shanghai Sangon Biotech: C506046-0005), denatured at 95°C for 10 min, and its activity was verified by 20% denaturing polyacrylamide gel electrophoresis. After electrophoresis, the FAM fluorescence was observed under blue light with a 535 nm filter to determine the size of the transcription product bands. Results are shown below. Figure 9 The activity of wild-type Bst DNA polymerase and its mutant strains Sso7d-Bst, Bst(I659E,E660G), and Sso7d-Bst(I659E,E660G) in reverse transcription of DNA using 2'-OMe-RNA as a template was tested. Figure 9 It is known that the mutant strain Sso7d-Bst (I659E, E660G) can reverse transcribe the full-length product and has good reverse transcription activity from 2'-OMe-RNA to DNA.

Claims

1. A Bst DNA polymerase mutant strain with XNA synthesis and reverse transcription activity, characterized in that, The mutant strains are selected from Bst DNA polymerase mutant strain Bst and Bst DNA polymerase mutant strain Sso7d-Bst; wherein, the mutant strains are capable of transcribing RNA, 2'-F-RNA and 2'-OMe-RNA using DNA as a template, and are capable of reverse transcribing DNA using RNA, 2'-F-RNA and 2'-OMe-RNA as templates; the amino acid sequence of the Bst DNA polymerase mutant strain Bst is shown in SEQ ID NO.2; the amino acid sequence of the Bst DNA polymerase mutant strain Sso7d-Bst is shown in SEQ ID NO.

3.

2. The Bst DNA polymerase mutant strain with XNA synthesis and reverse transcription activity according to claim 1, characterized in that, The gene sequence encoding the Bst DNA polymerase mutant strain Bst is shown in SEQ ID NO.5; the gene sequence encoding the Bst DNA polymerase mutant strain Sso7d-Bst is shown in SEQ ID NO.6.

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