CN117586984A - Tth DNA polymerase mutant strain with RNA and XNA synthesis activity and application thereof - Google Patents

Tth DNA polymerase mutant strain with RNA and XNA synthesis activity and application thereof Download PDF

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CN117586984A
CN117586984A CN202211297381.5A CN202211297381A CN117586984A CN 117586984 A CN117586984 A CN 117586984A CN 202211297381 A CN202211297381 A CN 202211297381A CN 117586984 A CN117586984 A CN 117586984A
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dna polymerase
mutant
tth
rna
tth dna
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陈庭坚
王光远
马星雲
汪杨铭
向禹铭
刘家韵
柳耘
陈雨晴
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South China University of Technology SCUT
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Abstract

The invention relates to a Tth DNA polymerase mutant strain with RNA and XNA synthesis activity and application thereof. The mutant strain can recognize natural dNTPs substrate and also can recognize NTPs,2'-F-NTPs or non-natural substrate Tth DNA polymerase mutant strain of partial 2' -OMe-NTPs. The mutant strain comprises at least one mutation site consisting of V520A, N585S, I616E, E617G, D657N, L659M, E683V/K, E744Q, M749R. In the series of Tth DNA polymerase mutant strains provided by the invention, the mutant strains ssTth M1-7 to ssTth M1-10 can be used for efficiently transcribing NTPs,2'-F-NTPs and partial 2' -OMe-NTPs, and compared with a wild type, the activity of the mutant strain is obviously improved.

Description

Tth DNA polymerase mutant strain with RNA and XNA synthesis activity and application thereof
Technical Field
The invention belongs to the field of enzyme molecular transformation, and particularly relates to a Tth DNA polymerase mutant strain and application thereof as well as a gene for encoding the mutant.
Background
The central laws of molecular biology were first proposed in 1958 and focused on in 1970, revealing the transfer of genetic information in DNA, RNA and proteins. With the proliferation of synthetic biology, unnatural nucleic acids (XNAs) have attracted attention, which is significant for the enrichment and expansion of the existing central laws. Non-natural nucleic acids include nucleic acids containing non-natural bases, nucleic acids containing non-natural phosphate backbones, and nucleic acids containing non-natural sugar rings, with sugar ring modifications being most widely studied.
Sugar ring modified XNAs, including Threose Nucleic Acid (TNA), hexitol Nucleic Acid (HNA), d-sugar triol nucleic acid (AtNA), cyclohexenyl nucleic acid (CeNA), locked Nucleic Acid (LNA), arabinonucleic acid (ANA), 2-fluoroarabic nucleic acid (FANA), 2'-F-RNA, 2' -OMe-RNA, and the like, are recognized by various natural and engineered polymerases. XNA exhibits a greater function than natural nucleic acids, they possess greater chemical and biological stability, especially in terms of resistance to nuclease degradation. Generally, XNA double strands are more stable than DNA, RNA or DNA/RNA hybrid strands. Therefore, XNA aptamers, such as sugar ring modified nucleic acid aptamers of 2'-F and 2' -OMe et al, show great advantages and potential in the resistance to ribozyme degradation based on exponential enrichment ligand evolution (SELEX).
However, natural polymerases do not recognize sugar ring modified non-natural nucleic acids well. To solve this problem, a great deal of effort has been devoted to the design and engineering of natural polymerases capable of recognizing sugar-modified nucleotides, and several polymerases having unnatural activity have been developed. For example, a 9℃N variant DNA polymerase containing an A485L mutation can synthesize TNA oligonucleotides. Philipp Holliger and colleagues evolved KOD polymerase into reverse transcriptase, which can reverse transcribe HNA and AtNA efficiently and with a relatively high fidelity. Piet Herhewijn and colleagues found that Vent (exo-) polymerase, taq polymerase, HIV reverse transcriptase, all of different sources, were able to recognize CeNA.
Tth polymerase is derived from Thermus thermophilus, has good thermal stability, and Tth DNA polymerase and Taq DNA polymerase both belong to the A polymerase family, and the sequence homology is up to 80%. Compared with Taq DNA polymerase, tth DNA polymerase has better tolerance to inhibitors, and has natural reverse transcription activity, can be used for one-step reverse transcription PCR, and has very wide application. Thus, the modification of Tth DNA polymerase is very valuable. Considering the ultra high homology with Taq DNA polymerase, while Taq DNA polymerase has been widely studied and engineered, we considered the grafting of beneficial amino acid mutations of Taq DNA polymerase to Tth DNA polymerase to obtain an alteration in activity. SFM4-3/4-6/4-9 is a mutant strain which is obtained by evolution from the Stoffel fragment of Taq DNA polymerase at the early stage of a laboratory, and can efficiently replicate, transcribe and reverse transcribe 2'-F/2' -OMe modified RNA. Thus, we have this patent that a series of mutants were obtained by transplanting part or all of the mutation sites of SFM4-3/4-6/4-9 into Tth DNA polymerase.
Disclosure of Invention
In order to modify the substrate specificity of wild-type Tth DNA polymerase so that it can recognize non-natural substrates such as NTPs,2'-F-NTPs and partial 2' -OMe-NTPs, the primary object of the present invention is to provide a series of Tth polymerase mutants capable of synthesizing not only natural DNA products but also RNA,2'-F-RNA or partial 2' -OMe-RNA.
The mutant strain comprises at least one mutation site consisting of V520A, N585S, I616E, E617G, D657N, L659M, E683V/K, E744Q and M749R.
Another object of the present invention is to provide a gene and an amino acid sequence of the above Tth polymerase mutant strain.
The aim of the invention is achieved by the following technical scheme.
A Tth polymerase mutant ssTth has an amino acid sequence shown in SEQ ID NO. 1.
A Tth polymerase mutant ssTth M1-1 has an amino acid sequence shown in SEQ ID NO. 2.
A Tth polymerase mutant ssTth M1-2 has an amino acid sequence shown in SEQ ID NO. 3.
A Tth polymerase mutant ssTth M1-3 has an amino acid sequence shown in SEQ ID NO. 4.
A Tth polymerase mutant ssTth M1-4 has an amino acid sequence shown in SEQ ID NO. 5.
A Tth polymerase mutant ssTth M1-5 has an amino acid sequence shown in SEQ ID NO. 6.
A Tth polymerase mutant ssTth M1-6 has an amino acid sequence shown in SEQ ID NO. 7.
A Tth polymerase mutant ssTth M1-7 has an amino acid sequence shown in SEQ ID NO. 8.
A Tth polymerase mutant ssTth M1-8 has an amino acid sequence shown in SEQ ID NO. 9.
A Tth polymerase mutant ssTth M1-9 has an amino acid sequence shown in SEQ ID NO. 10.
A Tth polymerase mutant ssTth M1-10 has an amino acid sequence shown in SEQ ID NO. 11.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth, and the gene sequence of the gene is shown as SEQ ID NO. 12.
The invention provides a gene for coding the Tth polymerase mutant ssTth M1-1, and the gene sequence of the gene is shown as SEQ ID NO. 13.
The invention provides a gene for coding the Tth polymerase mutant ssTth M1-2, and the gene sequence of the gene is shown as SEQ ID NO. 14.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-3, and the gene sequence of the gene is shown as SEQ ID NO. 15.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-4, and the gene sequence of the gene is shown as SEQ ID NO. 16.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-5, and the gene sequence of the gene is shown as SEQ ID NO. 17.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-6, and the gene sequence of the gene is shown as SEQ ID NO. 18.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-7, and the gene sequence of the gene is shown as SEQ ID NO. 19.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-8, and the gene sequence of the gene is shown as SEQ ID NO. 20.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-9, and the gene sequence of the gene is shown as SEQ ID NO. 21.
The invention also provides a gene for encoding the Tth polymerase mutant ssTth M1-10, and the gene sequence of the gene is shown as SEQ ID NO. 22.
Further, the Tth polymerase mutant is each Tth polymerase mutant obtained by mutating a wild-type Tth DNA polymerase with an enzyme molecule.
Further, sso7D small peptide gene and point mutations V520A, N585S, I616E, E617G, D657N, L659M, E683V/K, E744Q, M747R were introduced into the wild type Tth DNA polymerase gene by oligonucleotide primers.
Further, the transcriptional activity of each Tth polymerase mutant strain on NTPs substrates was measured, the transcriptional activity of each Tth polymerase mutant strain on 2'-F-ATP/2' -F-GTP/2'-OMe-CTP/2' -OMe-UTP substrates was measured, the transcriptional activity of each Tth polymerase mutant strain on 2'-OMe-CTP/2' -OMe-UTP/dATP/dGTP substrates was measured, and the transcriptional activity of each Tth polymerase mutant strain on 2'-F-CTP/2' -F-UTP/ATP/GTP substrates was measured.
Compared with wild Tth DNA polymerase, the invention has the following advantages and beneficial effects: in the series of Tth polymerase mutant strains provided by the invention, mutant strains ssTth M1-7 to ssTth M1-10 can be used for efficiently transcribing NTPs,2'-F-NTPs and partial 2' -OMe-NTPs, and compared with a wild type, the activity of the mutant strain is obviously improved.
Drawings
FIG. 1 is a schematic representation of the mutation sites contained in wild-type and mutant strains of Tth DNA polymerase and the fusion protein Sso7 d. Tth (exo-) is a wild-type Tth DNA polymerase that does not contain a 5'-3' exonuclease active domain (exo); ssTth is Tth (exo-) fused with an Sso7d small peptide; ssTth M1-1 is Tth (exo-) fused with an Sso7d small peptide and a mutation site V520A; ssTth M1-2 is Tth (exo-) fused with an Sso7d small peptide and a mutation site N585S; ssTth M1-3 is Tth (exo-) fused with an Sso7d small peptide, and mutation sites I616E and E617G; ssTth M1-4 is Tth (exo-) fused with Sso7D small peptide, and mutation sites D657N and L659M; ssTth M1-5 is Tth (exo-) fused with an Sso7d small peptide and mutation site E683V; ssTth M1-6 is Tth (exo-) fused with an Sso7d small peptide, and mutation sites E744N and M749R; ssTth M1-7 is Tth (exo-) fused with an Sso7D small peptide, and mutation sites V520A, N585S, I616E, E617G, D657N, E683K, E744N and M749R; ssTth M1-8 is Tth (exo-) fused with an Sso7D small peptide, and mutation sites I616E, E617G, D657N, L659M, E683K, E744N and M749R; ssTth M1-9 is Tth (exo-) fused with an Sso7D small peptide, and mutation sites V520A, I616E, E617G, D657N, E683V, E744N and M749R; ssTth M1-10 is Tth (exo-) fused with an Sso7D small peptide, and mutation sites V520A, N585S, I616E, E617G, D657N, E683V, E744N and M749R.
FIG. 2 is a schematic representation of an alignment of Tth (exo-) DNA polymerase and SFM4-3/4-6/4-9 polymerase sequences. The amino acid at the black dot position is the amino acid position referred to in the present invention.
FIG. 3 is a schematic diagram of substrate molecular structures of dNTPs, NTPs,2'-F-NTPs and 2' -OMe-NTPs, and structures of five bases A, G, C, T and U.
FIG. 4 shows the homology modeling of Tth structure and the distribution of mutation sites in the structure, the left is Stofel fragment of Taq DNA polymerase, and the right is Tth (exo-) DNA polymerase.
FIG. 5 shows the results of gel electrophoresis analysis of Tth DNA polymerase mutant strain transcribed from NTPs substrates using T40 as a template. In FIG. 5, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of RNA transcripts using T40 as a template.
FIG. 6 shows the results of gel electrophoresis analysis of Tth DNA polymerase mutant strain transcribed from NTPs substrates using T60 as a template. In FIG. 6, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of RNA transcripts using T60 as a template.
FIG. 7 shows the results of gel electrophoresis analysis of Tth DNA polymerase mutant transcribed from 2' -F-NTPs substrates using T40 as a template. In FIG. 7, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, gel electrophoresis analysis of T40-templated 2' -F-RNA transcripts.
FIG. 8 shows the results of gel electrophoresis analysis of Tth DNA polymerase mutant strains transcribed from 2'-F-ATP,2' -F-GTP,2'-OMe-CTP and 2' -OMe-UTP substrates using T40 as a template. In FIG. 8, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of the T40-templated 2' -F/OMe-RNA transcripts.
FIG. 9 shows the results of gel electrophoresis analysis of Tth DNA polymerase mutant transcribed from 2'-OMe-CTP,2' -OMe-UTP, dATP and dGTP substrates using T40 as a template. In FIG. 9, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, gel electrophoresis analysis of the T40-templated partial 2' -OMe-RNA transcripts.
FIG. 10 shows the results of gel electrophoresis analysis of Tth DNA polymerase mutant transcribed 2'-F-CTP,2' -F-UTP and ATP and GTP substrates using T40 as a template. In FIG. 10, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of the T40-templated 2' -F-RNA/RNA transcripts.
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.
Example 1
By usingThe super-fidelity DNA polymerase is amplified by PCR, and then overlapped extension PCR is carried out to obtain mutant ssTth M1-10 genes containing a plurality of mutation points. Primers used for site-directed mutagenesis are shown in the following table.
The PCR amplification system and reaction conditions used for site-directed mutagenesis are as follows:
DNA fragment 1:
DNA fragment 2:
DNA fragment 3:
DNA fragment 4:
DNA fragment 5:
DNA fragment 6:
DNA fragment 7:
PCR reaction conditions:
after the reaction, DNA fragments were recovered by using a Tiangen ultrathin recovery kit, and an overlap extension PCR reaction was performed.
The amplification system and reaction conditions used for overlap extension PCR were as follows:
overlap extension PCR reaction conditions:
after the reaction, the reaction product was treated with restriction enzymes EcoRI and NdeI. The reaction system is as follows:
and incubating the prepared enzyme digestion reaction system for 4.5 hours at 37 ℃ to finish enzyme digestion of the DNA reaction product. The DNA fragment exposed to EcoRI and NdeI cleavage sites was recovered using the Tiangen ultrathin recovery kit.
The plasmid pET30a-Tth was treated with the restriction enzymes EcoRI and NdeI. The reaction system is as follows:
and incubating the prepared enzyme digestion reaction system for 4.5 hours at 37 ℃ to finish enzyme digestion of the plasmid vector.
The DNA fragment was separated by agarose gel electrophoresis, and the 5268bp DNA vector was recovered by the artificial gel recovery kit.
Connecting the digested DNA fragment and plasmid vector through T4 DNA ligase, wherein the reaction system is that
And (3) incubating the prepared enzyme digestion reaction system at 16 ℃ overnight to complete the enzyme ligation reaction of the plasmid vector and the DNA product. The enzyme-linked product was directly transformed into E.coli DH 5. Alpha. Positive transformants were verified by colony PCR, plasmids were extracted, and sequenced.
According to the invention, recombinant plasmids pET30a-Tth M1-10 with correct sequencing are extracted from recombinant cloning bacteria DH5 alpha/pET 30a-Tth M1-10, and are transformed into an escherichia coli expression strain BL21 (DE 3) pLysS to obtain recombinant expression strain BL21 (DE 3) pLysS/pET30a-Tth M1-10.
Example 2
Positive transformants of BL21 (DE 3) pLysS/pET30a-Tth M1-10 were selected and cultured overnight at 37℃under 220r/min in 40mL of 2 XYT medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL chloramphenicol, and the culture medium was transferred to 4L of 2 XYT medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL chloramphenicol and cultured at 37℃under 220r/min to OD 600 Adding IPTG with final concentration of 0.5mmol/L to about 0.6-0.8, culturing at 37deg.C for 4-5h, centrifuging at 4deg.C for 10min and collecting thallus, suspending thallus into 250ml with buffer (50mM Tris,150mM NaCl), homogenizing under high pressure, crushing thallus, heating at 70deg.C in water bath for 15min, and centrifuging at 4deg.C for 45min and 10000 r/min. The supernatant was filtered through a 0.45 μm aqueous filter, and the filtered supernatant was purified by nickel column affinity chromatography. The general procedure for purification is: 2 times of buffer is used for pre-balancing Ni column (5 ml, protein load is 500 mg), and then the target protein is eluted by using an elution buffer with the concentration of 150mM imidazole, and the obtained solution is the single recombinant protein Tth M1-10 after separation and purification. Finally, concentrating and purifying to less than 200 mu l by using 50kDa Amicon, adding 100% glycerol with equal volume, and preserving at-20 ℃ for later use.
Protein concentration was determined by Qubit.
All Tth DNA polymerase mutants contained the following mutation sites:
example 3
Transcriptional Activity test study of Tth polymerase mutant on NTPs substrates: the RNA transcriptional activity of the Tth polymerase mutant strain according to the present invention was determined by the following test. The transcribed template T60/T40 and primer FAM-T60-R sequences are as follows
The transcription system is as follows:
the mixture of template T40 and complementary primer FAM-T60-R was denatured at 95℃for 10min, slowly cooled to room temperature and incubated on ice for 5min. The remaining reagents are then replenished. The transcription procedure is: (50 ℃,60min,70 ℃,5 min), 4 cycles. After transcription, the transcription product was added to one volume of 2 XTBE-Urea loading buffer (manufacturing: C506046-0005), denatured at 95℃for 10min, and the activity was verified by 20% denatured polyacrylamide gel electrophoresis. After electrophoresis, the gel was imaged under blue light from a 535nm filter and transcript band size was determined by FAM fluorescent band position. In FIG. 5, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of RNA transcripts using T40 as a template. As can be seen from FIG. 5, the mutant strains ssTth and ssTthM1-1 to ssTth M1-6 have no significant improvement in RNA transcription activity, and the mutant strains ssTthM1-7 to ssTthM 1-10 can extend the full length of T40, and have good and significantly higher RNA transcription activity than the wild type.
Example 4
Transcriptional activity test study of Tth polymerase mutant on NTPs substrates under long template T60 conditions: the RNA transcriptional activity of the mutant strain according to the present invention under the conditions of long template T60 was determined by the following test. The transcription system is as follows:
the mixture of template T60 and complementary primer FAM-T60-R was denatured at 95℃for 10min, slowly cooled to room temperature and incubated on ice for 5min. The remaining reagents are then replenished. The transcription procedure is: (50 ℃,60min,70 ℃,5 min), 4 cycles. After transcription, the transcription product was added to one volume of 2 XTBE-Urea loading buffer (manufacturing: C506046-0005), denatured at 95℃for 10min, and the activity was verified by 20% denatured polyacrylamide gel electrophoresis. After electrophoresis, the gel was imaged under blue light from a 535nm filter and transcript band size was determined by FAM fluorescent band position. In FIG. 6, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of RNA transcripts using T60 as a template. As can be seen from FIG. 6, the mutant strains ssTth and ssTth M1-1 to ssTth M1-6 have no significant improvement in RNA transcription activity, and the mutant strains ssTth M1-7 to ssTth M1-10 have good and significantly higher RNA transcription activity than the wild-type, wherein the mutant strains ssTth M1-7, ssTth M1-9 and ssTth M1-10 are capable of transcribing the full-length RNA product.
Example 5
Transcriptional Activity test study of Tth polymerase mutant on 2' -F-NTPs substrates: the 2' -F-RNA transcriptional activity of the mutant strain Tth polymerase according to the present invention was determined by the following test. The transcription system is as follows:
the mixture of template T40 and complementary primer FAM-T60-R was denatured at 95℃for 10min, slowly cooled to room temperature and incubated on ice for 5min. The remaining reagents are then replenished. The transcription procedure is: (50 ℃,20min,70 ℃,5 min). After transcription, the transcription product was added to one volume of 2 XTBE-Urea loading buffer (manufacturing: C506046-0005), denatured at 95℃for 10min, and the activity was verified by 20% denatured polyacrylamide gel electrophoresis. After electrophoresis, the gel was imaged under blue light from a 535nm filter and transcript band size was determined by FAM fluorescent band position. In FIG. 7, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, gel electrophoresis analysis of T40-templated 2' -F-RNA transcripts. As can be seen from FIG. 7, the mutant strains ssTth and ssTth M1-1 to ssTth M1-6 have no significant improvement in RNA transcription activity, and the mutant strains ssTth M1-7 to ssTth M1-10 can extend the full length of T40 and have good and significantly higher 2' -F-RNA transcription activity than the wild type.
Example 6
Transcriptional Activity test Studies of Tth polymerase mutant on 2'-F-ATP,2' -F-GTP,2'-OMe-CTP and 2' -OMe-UTP substrates: the transcriptional activity of the 2' -OMe/F-RNA product of the Tth polymerase mutant strain according to the present invention was determined by the following test. The transcription system is as follows:
the mixture of template T40 and complementary primer FAM-T60-R was denatured at 95℃for 10min, slowly cooled to room temperature and incubated on ice for 5min. The remaining reagents are then replenished. The transcription procedure is: (50 ℃,60min,70 ℃,5 min), 10 cycles. After transcription, the transcription product was added to one volume of 2 XTBE-Urea loading buffer (manufacturing: C506046-0005), denatured at 95℃for 10min, and the activity was verified by 20% denatured polyacrylamide gel electrophoresis. After electrophoresis, the gel was imaged under blue light from a 535nm filter and transcript band size was determined by FAM fluorescent band position. In FIG. 8, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of the T40-templated 2' -OMe/F-RNA transcripts. As can be seen from FIG. 8, the mutant strains ssTth and ssTth M1-1 to ssTth M1-6 have no significant improvement in RNA transcription activity, and the mutant strains ssTth M1-7 to ssTth M1-10 have good and significantly higher 2'-OMe/F-RNA transcription activity than the wild-type, wherein the mutant strain ssTth M1-10 is capable of transcribing the full-length 2' -OMe/F-RNA product, and the mutant strain has the highest activity.
Example 7
Transcriptional Activity test study of Tth polymerase mutant on 2'-OMe-CTP/2' -OMe-UTP/dATP/dGTP substrate: the partial 2' -OMe-RNA transcriptional activity of the Tth polymerase mutant strain according to the present invention was determined by the following test. The transcription system is as follows:
the mixture of template T40 and complementary primer FAM-T60-R was denatured at 95℃for 10min, slowly cooled to room temperature and incubated on ice for 5min. The remaining reagents are then replenished. The transcription procedure is: (50 ℃,60min,70 ℃,5 min), 10 cycles. After transcription, the transcription product was added to one volume of 2 XTBE-Urea loading buffer (manufacturing: C506046-0005), denatured at 95℃for 10min, and the activity was verified by 20% denatured polyacrylamide gel electrophoresis. After electrophoresis, the gel was imaged under blue light from a 535nm filter and transcript band size was determined by FAM fluorescent band position. In FIG. 9, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, gel electrophoresis analysis of the T40-templated partial 2' -OMe-RNA transcripts. As can be seen from FIG. 9, the mutant strains ssTth and ssTth M1-1 to ssTth M1-6 have no significant improvement in RNA transcription activity, and the mutant strains ssTth M1-7 to ssTth M1-10 have good and significantly higher 2'-OMe/F-RNA transcription activity than the wild-type, wherein the mutant strains ssTth M1-9 and ssTth M1-10 are capable of transcribing a part of the full-length 2' -OMe-RNA product.
Example 8
Transcriptional Activity test study of Tth polymerase mutant on 2'-F-CTP/2' -F-UTP/ATP/GTP substrate: the 2' -F-RNA/RNA transcriptional activity of the Tth polymerase mutant strain according to the present invention was determined by the following test. The transcription system is as follows:
the mixture of template T40 and complementary primer FAM-T60-R was denatured at 95℃for 10min, slowly cooled to room temperature and incubated on ice for 5min. The remaining reagents are then replenished. The transcription procedure is: (50 ℃,60min,70 ℃,5 min), 4 cycles. After transcription, the transcription product was added to one volume of 2 XTBE-Urea loading buffer (manufacturing: C506046-0005), denatured at 95℃for 10min, and the activity was verified by 20% denatured polyacrylamide gel electrophoresis. After electrophoresis, the gel was imaged under blue light from a 535nm filter and transcript band size was determined by FAM fluorescent band position. In FIG. 10, from left to right, lane P is control and the black arrow is the full length position of the template; lanes WT are wild-type Tth DNA polymerase, lanes 1-11 are ssTth, ssTth M1-1 to ssTth M1-10 mutants, and gel electrophoresis analysis of the T40-templated 2' -F-RNA/RNA transcripts. As can be seen from FIG. 10, the mutants ssTth and ssTth M1-1, ssTth M1-4 to ssTth M1-6, and the RNA transcriptional activity was not significantly improved, and the mutants ssTth M1-2, ssTth M1-3, ssTth M1-7 to ssTth M1-10 had good and significantly higher 2'-F-RNA/RNA transcriptional activity than the wild type, wherein the mutants ssTth M1-2, ssTth M1-7, ssTth M1-9 and ssTth M1-10 were able to transcribe the full-length 2' -F-RNA/RNA product.

Claims (10)

1. A mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity, characterized in that said mutant strain recognizes both natural dNTPs substrates and non-natural substrates of NTPs,2'-F-NTPs or part of 2' -OMe-NTPs.
2. A Tth DNA polymerase mutant having RNA and XNA synthesis activity according to claim 1, wherein the mutant comprises at least one mutation site consisting of V520A, N585S, I616E, E617G, D657N, L659M, E683V/K, E744Q, M749R.
3. The mutant Tth DNA polymerase having RNA and XNA synthesizing activity according to claim 1, wherein the mutant Tth DNA polymerase is each mutant Tth DNA polymerase obtained by mutating a wild-type Tth DNA polymerase with an enzyme molecule;
the method comprises the following steps: introducing an Sso7D small peptide gene and point mutations V520A, N585S, I616E, E617G, D657N, L659M, E683V/K, E744Q, M747R into a wild type Tth DNA polymerase gene by oligonucleotide primers;
the transcriptional activity of each Tth DNA polymerase mutant on NTPs substrate is measured, the transcriptional activity of each Tth DNA polymerase mutant on 2'-F-ATP/2' -F-GTP/2'-OMe-CTP/2' -OMe-UTP substrate is measured, the transcriptional activity of each Tth DNA polymerase mutant on 2'-OMe-CTP/2' -OMe-UTP/dATP/dGTP substrate is measured, and the transcriptional activity of each Tth DNA polymerase mutant on 2'-F-CTP/2' -F-UTP/ATP/GTP substrate is measured.
4. The mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity according to claim 1, wherein the amino acid sequence of the mutant Tth polymerase is shown in SEQ ID No.1 to SEQ ID No. 4.
5. The mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity according to claim 1, wherein the amino acid sequence of the mutant Tth polymerase is shown in SEQ ID No.5 to SEQ ID No. 8.
6. The mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity according to claim 1, wherein the amino acid sequence of the mutant Tth polymerase is shown in SEQ ID No.9 to SEQ ID No. 11.
7. The mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity according to claim 4, wherein the gene sequence of the mutant of Tth DNA polymerase is shown in SEQ ID No.12 to SEQ ID No. 15.
8. The mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity according to claim 5, wherein the gene sequence of the mutant of Tth DNA polymerase is shown in SEQ ID No.16 to SEQ ID No. 19.
9. The mutant strain of Tth DNA polymerase having RNA and XNA synthesis activity according to claim 6, wherein the gene sequence of the mutant Tth polymerase is shown in SEQ ID No.20 to SEQ ID No. 22.
10. Use of a mutant Tth DNA polymerase having RNA and XNA synthesis activity according to any of claims 1 to 9 for transcription of NTPs,2'-F-NTPs and part of 2' -OMe-NTPs.
CN202211297381.5A 2022-08-18 2022-10-21 Tth DNA polymerase mutant strain with RNA and XNA synthesis activity and application thereof Pending CN117586984A (en)

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