WO2023168758A1 - Dna聚合酶、核酸适配体、热启动dna聚合酶及方法和应用 - Google Patents

Dna聚合酶、核酸适配体、热启动dna聚合酶及方法和应用 Download PDF

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WO2023168758A1
WO2023168758A1 PCT/CN2022/082834 CN2022082834W WO2023168758A1 WO 2023168758 A1 WO2023168758 A1 WO 2023168758A1 CN 2022082834 W CN2022082834 W CN 2022082834W WO 2023168758 A1 WO2023168758 A1 WO 2023168758A1
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dna polymerase
quadruplex
nucleic acid
sequence
plasmid
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French (fr)
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郑克威
蔡婷婷
王成林
梁慧婷
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深圳市麒御生物科技有限公司
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Priority to US17/835,949 priority Critical patent/US20230287363A1/en
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Definitions

  • the invention relates to the field of biotechnology, and in particular to a modified DNA polymerase, a nucleic acid aptamer, a hot-start DNA polymerase, a test kit, a method for biosynthesizing a DNA polymerase, a method for preparing a hot-start DNA polymerase, and DNA polymerization.
  • the combination of enzymes and nucleic acid aptamers is used to detect nucleic acids or synthesize nucleic acids.
  • Nucleic acid amplification technology is widely used to study human diseases and detect pathogens.
  • isothermal amplification technologies that can be used in nucleic acid diagnostic technologies, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), strand displacement amplification (SDA) and rolling circle Amplification (RCA).
  • LAMP loop-mediated isothermal amplification
  • RPA recombinase polymerase amplification
  • SDA strand displacement amplification
  • RCA rolling circle Amplification
  • a key challenge in nucleic acid detection technology is nonspecific amplification caused by primer mismatches and non-template amplification.
  • Non-specific amplification strongly hinders or reduces the amplification of the target template, reducing the sensitivity of the detection.
  • An effective way to avoid non-specific amplification is to block the activity of thermophilic DNA polymerase at low temperatures.
  • blocking the activity of DNA polymerase with antibodies or specific aptamers is the most common method to prevent non-specific amplification.
  • Covalent modification of DNA polymerases such as Taq DNA polymerase is another way to block its low-temperature phase activity.
  • introducing specific mutation sites in the amino acids of DNA polymerase can also change its properties and reduce the activity of the polymerase.
  • one of the purposes of the present invention is to provide a modified DNA polymerase that can provide a broad-spectrum technical strategy for preparing hot-start DNA polymerase.
  • a modified DNA polymerase including a DNA polymerase fragment and a G-quadruplex binding peptide fused to the N-terminus of the DNA polymerase fragment.
  • the second object of the present invention is to provide a nucleic acid aptamer for regulating the above-mentioned DNA polymerase activity.
  • the second object of the present invention is achieved by adopting the following technical solution: a nucleic acid aptamer for regulating the above-mentioned DNA polymerase activity, the nucleotide sequence of the nucleic acid aptamer includes a G-quadruplex core sequence, The secondary structure of the G-quadruplex core sequence is G-quadruplex;
  • the G-quadruplex is used to bind to the G-quadruplex binding peptide of the DNA polymerase at a first preset temperature to inhibit the activity of the DNA polymerase, and is used at a second preset temperature
  • the G-quadruplex binding peptide is separated from the DNA polymerase to restore the activity of the DNA polymerase; wherein the second preset temperature is higher than the first preset temperature.
  • the third object of the present invention is to provide a hot-start DNA polymerase.
  • the third object of the present invention is achieved by adopting the following technical solution: a hot-start DNA polymerase, including the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer, the G-quadruplex of the nucleic acid aptamer is in the first pre-stage Set a temperature to bind to the G-quadruplex binding peptide of the DNA polymerase to inhibit the activity of the DNA polymerase, and dissociate from the G-quadruplex binding peptide of the DNA polymerase at a second preset temperature , to restore the activity of the DNA polymerase; wherein the second preset temperature is higher than the first preset temperature.
  • the fourth object of the present invention is to provide a kit.
  • the fourth object of the present invention is achieved by adopting the following technical solution: a kit including the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer.
  • the fifth object of the present invention is to provide a method for biosynthesizing the above-mentioned DNA polymerase.
  • the fifth object of the present invention is achieved by adopting the following technical solution: a method for biosynthesizing the above-mentioned DNA polymerase, including the following steps:
  • the sixth object of the present invention is to provide a method for preparing hot-start DNA polymerase.
  • the sixth object of the present invention is achieved by adopting the following technical solution: a method for preparing hot-start DNA polymerase, the DNA polymerase/aptamer complex includes the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer, The nucleic acid aptamer is bound to the DNA polymerase through the G-quadruplex binding to the G-quadruplex binding peptide, and the method includes the following steps:
  • Dissolve the nucleic acid aptamer in the first buffer denature it at 90°C to 100°C for 2 to 8 minutes, and then cool to 20°C to 30°C to obtain the processed nucleic acid aptamer;
  • the first buffer solution includes 10mM pH 7.4 Tris-HCl, 75mM KCl, 0.5mM EDTA and 0.2mg/ml bovine serum albumin
  • the second buffer solution includes 20mM pH 8.8 Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2-8mM MgSO 4 and 0.1% Tween-20.
  • the seventh object of the present invention is to provide the application of the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer for detecting nucleic acid or synthesizing nucleic acid.
  • the seventh object of the present invention is achieved by adopting the following technical solution: the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer are used in combination to detect nucleic acids or synthesize nucleic acids.
  • the present invention constructs a modified DNA polymerase and a nucleic acid aptamer that specifically binds to the DNA polymerase.
  • the activity of the DNA polymerase is inhibited at low temperature.
  • the bound nucleic acid aptamer is strictly inhibited, but completely recovered when heated to the reaction temperature. When applied to isothermal amplification, non-specific amplification will not occur.
  • Figure 1A is a schematic structural diagram of Bst DNA polymerase, Bst-LF and DNA polymerase provided in Embodiment 1 of the present invention, in which the DNA polymerase is fused with a G-quadruplex binding peptide, represented by G4P-Bst and RHAU23-Bst;
  • Figure 1B is an electrophoresis pattern of purified Bst-LF, RHAU23-Bst and G4P-Bst provided in Example 1 of the present invention
  • Figure 1C is a schematic diagram of the principle of G-quadruplex nucleic acid aptamer regulating DNA polymerase activity at different temperatures provided in Embodiment 1 of the present invention
  • Figure 1D is a map of the pCold-I-G4P-Bst plasmid provided in Example 1 of the present invention.
  • Figure 1E is a map of the pCold-I-RHAU23-Bst plasmid provided in Example 1 of the present invention.
  • Figures 2A to 2C show the DNA polymerase G4P-Bst and the nucleic acid aptamers T4B1 core G4, PDGFRB core G4, T4B1 Fs-G4, PDGFRB Fs-G4, T4B1 Fd-G4 and PDGFRB Fd- provided in Embodiment 2 of the present invention.
  • Figure 2D is a diagram showing the binding ability results of DNA polymerase RHAU23-Bst and nucleic acid aptamers T4B1 Fd-G4 and PDGFRB Fd-G4 provided in Example 2 of the present invention;
  • Figure 2E is a graph showing the results of the binding ability of DNA polymerase RHAU23-Bst and nucleic acid aptamers HG53, GH5, and HG3 provided in Example 2 of the present invention
  • Figure 3A is a schematic diagram of the primer extension experiment provided in Embodiment 3 of the present invention.
  • Figures 3B to 3D are diagrams showing the results of primer extension experiments using DNA polymerase G4P-Bst provided in Embodiment 3 of the present invention.
  • FIGS 3E to 3H are diagrams showing the results of primer extension experiments using DNA polymerase RHAU23-Bst provided in Embodiment 3 of the present invention.
  • Figure 4A is a graph showing the results of detecting the binding ability of DNA polymerase G4P-Bst to non-G4 single-stranded DNA and non-G4 double-stranded DNA through the EMSA method provided in Embodiment 4 of the present invention;
  • Figure 4B is a graph showing the results of detecting the binding ability of Bst-LF to T4B1 Fd-G4 and T4B1 Fs-G4 through the EMSA method provided in Embodiment 4 of the present invention;
  • Figure 4C is a graph showing the results of detecting the activity of G4P-Bst in the presence of non-G4 single-stranded DNA and non-G4 double-stranded DNA through primer extension experiments provided in Embodiment 4 of the present invention;
  • Figure 4D is a graph showing the results of detecting the activity of G4P-Bst in the presence of T4B1 Fd-G4 and T4B1 Fs-G4 through primer extension experiments provided in Embodiment 4 of the present invention;
  • Figure 5 is a graph showing the effects of the G-quadruplex type, "hairpin" structure length and loop length provided in Example 5 of the present invention on the ability of the nucleic acid aptamer (HG53) to inhibit the activity of DNA polymerase RHAU23-Bst;
  • Figure 6A is a diagram showing the results of a primer extension experiment performed on Bst-LF at different temperatures in the absence of a nucleic acid aptamer provided in Example 6 of the present invention
  • Figure 6B is a graph showing the results of primer extension experiments performed by Bst 2.0 hot-start DNA polymerase at different temperatures in the absence of nucleic acid aptamers provided in Embodiment 6 of the present invention
  • Figure 6C is a graph showing the results of primer extension experiments performed on RHAU23-Bst at different temperatures in the absence of nucleic acid aptamers provided in Example 6 of the present invention.
  • Figure 6D is a diagram showing the results of primer extension experiments performed on RHAU23-Bst at different temperatures in the presence of the nucleic acid aptamer HG53-GVBQ1 provided in Example 6 of the present invention
  • Figure 6E is a diagram showing the results of primer extension experiments performed on RHAU23-Bst at different temperatures in the presence of the nucleic acid aptamer HG53-H7 provided in Example 6 of the present invention
  • Figure 6F is a graph showing the normalized activity results of RHAU23-Bst provided in Example 6 of the present invention in the presence of nucleic acid aptamer HG53-H7;
  • Figure 7 is a diagram of the results of detecting HPV 16 gene DNA using fluorescent LAMP provided in Embodiment 7 of the present invention.
  • Figure 8A is a graph showing the results of fluorescent RT-LAMP detecting SARS-CoV-2 RNA
  • Figure 8B is a graph showing the results of pH-mediated colorimetric RT-LAMP detecting SARS-CoV-2 RNA
  • Figure 9A is a map of the pCold-I-RHAU23-Taq plasmid provided in Example 9 of the present invention.
  • Figure 9B is a graph showing the results of primer extension experiments performed on RHAU23-Taq at different temperatures in the absence of nucleic acid aptamers provided in Example 9 of the present invention.
  • Figure 9C is a diagram showing the results of primer extension experiments performed on RHAU23-Taq at different temperatures in the presence of the nucleic acid aptamer HG53-H7 provided in Example 9 of the present invention.
  • Figure 9D is the ratio of the activity of RHAU23-Taq in the presence of the nucleic acid aptamer HG53-H7 compared to the absence of the nucleic acid aptamer provided in Embodiment 9 of the present invention.
  • Figure 10A is a map of the pCold-I-RHAU23-RT plasmid provided in Example 10 of the present invention.
  • Figure 10B is a diagram showing the results of primer extension experiments performed on RHAU23-RT at different temperatures in the presence of the nucleic acid aptamer HG53-H7 provided in Example 10 of the present invention
  • Figure 10C is a graph showing the results of a primer extension experiment performed by existing MMLV reverse transcriptase at different temperatures in the absence of a nucleic acid aptamer provided in Embodiment 10 of the present invention
  • Figure 10D is the ratio of the reverse transcriptase activity of RHAU23-RT in the presence of the nucleic acid aptamer HG53-H7 compared to the existing MMLV reverse transcriptase activity in the absence of the nucleic acid aptamer provided in Example 10 of the present invention.
  • the invention provides a modified DNA polymerase, which includes a DNA polymerase fragment and a G-quadruplex binding peptide fused to the N-terminus of the DNA polymerase fragment.
  • the G-quadruplex binding peptide is RHAU23 peptide, and the amino acid sequence of RHAU23 peptide is shown in Seq ID No. 1; or,
  • the G-quadruplex binding peptide is G4P, and the amino acid sequence of G4P is shown in Seq ID No. 2.
  • the DNA polymerase fragment can withstand temperatures above 40°C and is active when the temperature is higher than 40°C.
  • the DNA polymerase fragment is derived from any one of Bst DNA polymerase, Taq DNA polymerase and MMLV reverse transcriptase.
  • the DNA polymerase fragment comes from Bst DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in Seq ID No. 3.
  • the DNA polymerase fragment comes from Taq DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in Seq ID No. 4.
  • the DNA polymerase fragment comes from MMLV reverse transcriptase, and the amino acid sequence of the DNA polymerase fragment is shown in Seq ID No. 5.
  • the invention also provides a nucleic acid aptamer for regulating the activity of the above-mentioned DNA polymerase.
  • the nucleotide sequence of the nucleic acid aptamer includes a G-quadruplex core sequence, and the secondary structure of the G-quadruplex core sequence is G-quadruplex;
  • the G-quadruplex is used to bind to the G-quadruplex binding peptide of the DNA polymerase at the first preset temperature to inhibit the activity of the DNA polymerase, and is used to detach from the DNA polymerase at the second preset temperature.
  • the G-quadruplex binds the peptide to restore the activity of the DNA polymerase; wherein the second preset temperature is higher than the first preset temperature.
  • the G-quadruplex is a regular three-layer G-quadruplex, a convex G-quadruplex, a G-quadruplex with a G-vacancy, and a regular two-layer G-quadruplex. Any type of chain.
  • the nucleotide sequence of the nucleic acid aptamer also includes the flanking DNA sequence at the 5' end and/or 3' end of the G-quadruplex core sequence.
  • flanking DNA sequence is a flanking single-stranded DNA sequence or a flanking double-stranded DNA sequence or a DNA sequence used to form a "hairpin" structure.
  • the side DNA sequence is preferably a DNA sequence used to form a "hairpin” structure, wherein the length of the formed "hairpin” structure is greater than or equal to 7 bp.
  • the first preset temperature is 0°C to 30°C
  • the second preset temperature is 45°C to 70°C.
  • the second preset temperature is 55°C to 65°C.
  • the G-quadruplex core sequence is the CSTB core sequence, and the nucleotide sequence of the CSTB core sequence is shown in Seq ID No. 6; or,
  • the G-quadruplex core sequence is the KIT-C core sequence, and the nucleotide sequence of the KIT-C core sequence is shown in Seq ID No. 7; or,
  • the G-quadruplex core sequence is the T4B1 core sequence, and the nucleotide sequence of the T4B1 core sequence is shown in Seq ID No. 8; or,
  • the G-quadruplex core sequence is the PDGFRB core sequence, and the nucleotide sequence of the PDGFRB core sequence is shown in Seq ID No. 9; or,
  • the G-quadruplex core sequence is the T1B1 core sequence, and the nucleotide sequence of the T1B1 core sequence is shown in Seq ID No. 10; or,
  • the G-quadruplex core sequence is the GVBQ1 core sequence, and the nucleotide sequence of the GVBQ1 core sequence is shown in Seq ID No. 11; or,
  • the G-quadruplex core sequence is the GVBQ2 core sequence, and the nucleotide sequence of the GVBQ2 core sequence is shown in Seq ID No. 12; or,
  • the core sequence of the G-quadruplex is the G12 core sequence, and the nucleotide sequence of the G12 core sequence is shown in Seq ID No. 13.
  • the invention also provides a hot-start DNA polymerase, which includes the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer.
  • the G-quadruplex of the nucleic acid aptamer binds to the G-quadruplex of the DNA polymerase at a first preset temperature.
  • the G-quadruplex binding peptide is separated from the DNA polymerase at the second preset temperature to restore the activity of the DNA polymerase; wherein the second preset temperature is higher than First preset temperature.
  • the first preset temperature is 0°C to 30°C
  • the second preset temperature is 45°C to 70°C.
  • the present invention also provides a kit, including the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer.
  • the kit is used to detect human papillomavirus DNA or SARS virus RNA.
  • the present invention also provides a method for biosynthesizing the above-mentioned DNA polymerase, which is characterized in that it includes the following steps:
  • the DNA polymerase fragment comes from residues 291-878 of the DNA polymerase of Bacillus stearothermophilus, the plasmid vector is pCold-I, and the first plasmid is pCold-I-Bst-LF plasmid;
  • the G-quadruplex binding peptide is G4P, and the second plasmid is pCold-I-G4P-Bst plasmid; or, the G-quadruplex binding peptide is RHAU23, and the second plasmid is pCold-I-RHAU23-Bst plasmid.
  • the DNA polymerase fragment comes from Taq DNA polymerase, the plasmid vector is pCold-I, and the first plasmid is pCold-I-Taq-LF plasmid;
  • the G-quadruplex binding peptide is G4P, and the second plasmid is pCold-I-G4P-Taq plasmid; or, the G-quadruplex binding peptide is RHAU23, and the second plasmid is pCold-I-RHAU23-Taq plasmid.
  • the DNA polymerase fragment comes from MMLV reverse transcriptase
  • the plasmid vector is pCold-I
  • the first plasmid is pCold-I-RT-LF plasmid
  • the G-quadruplex binding peptide is G4P, and the second plasmid is pCold-I-G4P-RT plasmid; or the G-quadruplex binding peptide is RHAU23, and the second plasmid is pCold-I-RHAU23-RT plasmid.
  • the invention also provides a method for preparing a hot-start DNA polymerase.
  • the hot-start DNA polymerase is the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer.
  • the nucleic acid aptamer is combined with the G-quadruplex binding peptide through the G-quadruplex.
  • the method includes the following steps:
  • Dissolve the nucleic acid aptamer in the first buffer denature it at 90°C to 100°C for 2 to 8 minutes, and then cool to 20°C to 30°C to obtain the processed nucleic acid aptamer;
  • the first buffer solution includes 10mM pH 7.4 Tris-HCl, 75mM KCl, 0.5mM EDTA and 0.2mg/ml bovine serum albumin
  • the second buffer solution includes 20mM pH 8.8 Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2-8mM MgSO and 0.1% Tween-20.
  • the preset molar concentration ratio of DNA polymerase and nucleic acid aptamer ranges from 1:8 to 1:1.
  • the present invention also provides an application of the above-mentioned DNA polymerase and the above-mentioned nucleic acid aptamer for detecting nucleic acid or synthesizing nucleic acid.
  • the nucleic acid when used to detect nucleic acid, is human papillomavirus DNA or SARS virus RNA.
  • the present invention constructs a DNA polymerase fused with a G-quadruplex binding peptide and a nucleic acid aptamer that specifically binds to the polymerase.
  • the activity of the DNA polymerase is strictly inhibited by the combined nucleic acid aptamer at the low temperature stage, but at It is completely recovered when heated to the reaction temperature. When applied to isothermal amplification, non-specific amplification will not occur.
  • the modified DNA polymerase constructed by the present invention and the This nucleic acid aptamer specifically binds to DNA polymerase and successfully solves the non-specific amplification problem of LAMP in detecting HPV DNA and SARS-CoV-2 RNA; the G-quadruplex binding peptide is compatible with the G-quadruplex adapter
  • the combination of polymers provides a new way to develop controllable DNA polymerases.
  • the Bst DNA polymerase large fragment (Bst-LF) was inserted into the pCold-I vector via endonucleases Nde I and Xba I to construct the pCold-I-Bst-LF plasmid.
  • the coding sequence of G4P was inserted into the N-terminus of Bst-LF to construct pCold-I-G4P-Bst plasmid
  • the coding sequence of RHAU23 was inserted into the N-terminus of Bst-LF to construct
  • the pCold-I-RHAU23-Bst plasmid was obtained.
  • Plasmids pCold-I-Bst-LF, pCold-I-G4P-Bst and pCold-I-RHAU23-Bst were transformed into E. coli strain BL21. Cells were cultured in LB medium containing 50 ⁇ g/mL ampicillin at 37°C to OD 0.8-1.0. Then, 0.4mM IPTG was added and cultured at 16°C for another 16h to induce protein expression.
  • FIG. 1A shows a schematic structural diagram of Bst DNA polymerase, Bst-LF and modified DNA polymerase, in which the modified DNA polymerase is fused with a G-quadruplex binding peptide, represented by G4P-Bst and RHAU23-Bst.
  • Figure 1B shows the electropherograms of purified Bst-LF, RHAU23-Bst and G4P-Bst.
  • Bst-LF comes from residues 291-878 of DNA polymerase I of Bacillus stearothermophilus (ARA98840.1), which is the lack of 5'-3' exonuclease in Bacillus stearothermophilus DNA polymerase.
  • ARA98840.1 Bacillus stearothermophilus
  • the amino acid sequence of Bst-LF is shown in Seq ID No. 3.
  • Bst-LF is active in a wide temperature range and has strong strand displacement activity, and is widely used in loop-mediated isothermal amplification (LAMP).
  • RHAU23 is a 23-amino acid peptide derived from human DHX36 protein, which specifically interacts with G-quadruplex.
  • the amino acid sequence of RHAU23 is shown in Seq ID No. 1.
  • G4P is a small protein (64 amino acids) composed of two tandem RHAU23s. G4P has stronger G-quadruplex binding affinity than RHAU23.
  • the amino acid sequence of G4P is shown in Seq ID No. 2.
  • the map of pCold-I-G4P-Bst plasmid is shown in Figure 1D, and the map of pCold-I-RHAU23-Bst plasmid is shown in Figure 1E.
  • the modified DNA polymerase provided in this embodiment can bind to a nucleic acid aptamer having a G-quadruplex by fusing a G-quadruplex binding peptide at the N-terminus.
  • the DNA polymerase fused with the G-quadruplex binding peptide binds to the G-quadruplex, preventing the DNA polymerase from interacting with the substrate DNA.
  • the G-quadruplex binds The DNA is unfolded and separated from the DNA polymerase so that the DNA polymerase can re-act on the substrate.
  • the binding affinity between modified DNA polymerase and nucleic acid aptamer was detected by gel shift assay (EMSA).
  • ssDNA Complementary single-stranded DNA
  • dsDNA double-stranded DNA
  • nM 5'-FAM labeled aptamer DNA (Table 1) in the first buffer, denature at 95°C for 5 minutes, and then cool to 25°C at a rate of 0.1°C/s; where, the first buffer
  • the solution includes 10mM Tris-HCl (pH 7.4), 75mM KCl, 0.5mM EDTA and 0.2mg/ml bovine serum albumin.
  • each nucleic acid aptamer DNA and the DNA polymerase fused to the G-quadruplex binding peptide at the specified concentration were added to the second buffer, and combined at 4°C for 1 hour to form a DNA-protein binding complex; wherein,
  • the second buffer included 20mM Tris-HCl (pH 8.8) , 10mM (NH4)2SO4 , 50mM KCl, 8mM MgSO4 and 0.1% Tween-20.
  • DNA- and protein-bound complexes were electrophoresed on 8% or 12% nondenaturing polyacrylamide gels containing 75mM KCl in 1 ⁇ TBE buffer containing 75mM KCl for 2 h at 4°C. DNA- and protein-bound complexes were photographed by a ChemiDoc MP imaging system (Bio-Rad) and quantitatively analyzed by Image Quant 5.2 software.
  • Figure 2A to Figure 2C show the modified DNA polymerase G4P-Bst and the nucleic acid aptamers T4B1 core G4, PDGFRB core G4, T4B1 Fs-G4, PDGFRB Fs-G4, T4B1 Fd-G4 and PDGFRB Fd-G4 binding ability results diagram, where core G4 represents the core G-quadruplex, Fs-G4 represents the G-quadruplex containing single-stranded DNA on the flanks, and Fd-G4 represents the G-quadruplex containing double-stranded DNA on the flanks.
  • Figure 2D shows the binding ability results of modified DNA polymerase RHAU23-Bst and nucleic acid aptamers T4B1 Fd-G4 and PDGFRB Fd-G4.
  • Figure 2E shows the binding ability results of modified DNA polymerase RHAU23-Bst and nucleic acid aptamers.
  • the binding ability results of HG53, GH5, and HG3.
  • the nucleic acid aptamers HG53, GH5, and HG3 are all single-stranded DNA that can be folded into G-quadruplex and "hairpin" structures, and the above-mentioned Fd-G4 aptamer
  • the body is made up of two complementary single-stranded DNA pairs.
  • both the bulged G-quadruplex of T4B1 (bulged G-quadruplex is also called bulged G-quadruplex) and the bulged G-quadruplex of PDGFRB have The flanking DNA sequence, G4P-Bst, showed strong binding affinity to both the bulged G-quadruplex of T4B1 and the bulged G-quadruplex of PDGFRB.
  • the Kd value range is 1-5nM.
  • RHAU23-Bst also has strong binding affinity to nucleic acid aptamers containing G-quadruplex and flanking double-stranded DNA, with Kd at the nM level.
  • the binding affinity of RHAU23-Bst to nucleic acid aptamers HG53, GH5, and HG3 is similar to the binding affinity of RHAU23-Bst to nucleic acid aptamers containing G-quadruplex and flanking double-stranded DNA, with Kd values ranging from 3.4 to 7.4. nM.
  • Primer extension experiments were performed to detect the regulation of DNA polymerase activity by nucleic acid aptamers.
  • each nucleic acid aptamer DNA listed in Table 2 Dissolve each nucleic acid aptamer DNA listed in Table 2 to 10 ⁇ M in the first buffer, heat at 95°C for 5 minutes, and then cool to 25°C at a rate of 0.1°C/s; wherein the first buffer includes 10mM Tris-HCl (pH 7.4), 75mM KCl, 0.5mM EDTA and 0.2mg/ml bovine serum albumin.
  • DNA polymerase 100nM fused to the G-quadruplex binding peptide and the nucleic acid aptamer into the second buffer, and place it at 4°C for 30 minutes to form a DNA and protein-binding complex; where, G is fused -The molar concentration ratios of DNA polymerase and nucleic acid aptamer for quadruplex-binding peptides are 1:0, 1:1, 1:2, 1:4 and 1:8 respectively, and the second buffer includes 20mM Tris-HCl (pH 8.8), 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 8mM MgSO 4 and 0.1% Tween-20.
  • Figure 3A is a schematic diagram of a primer extension experiment.
  • Figures 3B to 3D are results of a primer extension experiment using modified DNA polymerase G4P-Bst.
  • Figure 3E to 3H are primers using modified DNA polymerase RHAU23-Bst. Results of the extension experiment.
  • the activity of the modified DNA polymerase was basically restored at 65°C, as shown in Figure 3C for details.
  • a G-quadruplex containing flanking double-stranded DNA as a nucleic acid aptamer, that is, using the Fd-G4 aptamer, as long as the ratio of DNA polymerase/aptamer is less than 1, G4P-Bst can be maintained at 25°C The activity was almost completely inhibited.
  • the Fd-G4 aptamer strongly inhibited the activity of G4P-Bst and still showed varying degrees of inhibition at 65°C. See Figure 3D for details.
  • the aptamer containing flanking DNA is obviously more effective. Therefore, the DNA in the flanking region of the G-quadruplex is also necessary for the aptamer to function. .
  • RHAU23-Bst and nucleic acid aptamers HG53, GH5, and HG3 were strongly inhibited at 25°C, and the inhibition lasted for at least 40 minutes. At 65°C, RHAU23-Bst fully recovered its activity. This indicates that using single-stranded DNA that can fold into G-quadruplex and "hairpin" structures as nucleic acid aptamers is sufficient to modulate the activity of DNA polymerase fused to G-quadruplex binding peptides.
  • the regulation of the activity of the modified DNA polymerase by the nucleic acid aptamer is affected by the flanking DNA sequence in the nucleic acid aptamer and the G-quadruplex in the hot-start DNA polymerase based on G-quadruplex regulation. Effects of binding peptides.
  • the activities of the modified DNA polymerases G4P-Bst and RHAU23-Bst were inhibited at 25°C, which is a temperature close to room temperature. The inhibition of polymerase activity at this temperature can effectively prevent non-specific amplification.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • the EMSA method is used to detect the binding ability of DNA polymerase G4P-Bst to non-G4 single-stranded DNA and non-G4 double-stranded DNA, and the EMSA method is used to detect Bst-LF and T4B1 Fd-G4 and T4B1 Fs. -G4’s binding ability.
  • Figure 4 shows the experimental results, wherein Figure 4A is the result of detecting the binding ability of DNA polymerase G4P-Bst to non-G4 single-stranded DNA and non-G4 double-stranded DNA by EMSA method, and Figure 4B is the result of detecting Bst by EMSA method.
  • Figure 4C is the result of the primer extension experiment to detect the activity of G4P-Bst in the presence of non-G4 single-stranded DNA and non-G4 double-stranded DNA.
  • Figure 4C 4D is a graph showing the activity results of Bst-LF in the presence of T4B1 Fd-G4 and T4B1 Fs-G4 through primer extension experiments.
  • removing the G-quadruplex from the nucleic acid aptamer or removing the G-quadruplex binding peptide from the DNA polymerase significantly reduces the binding affinity of the DNA polymerase to the nucleic acid aptamer, both of which Resulting in the inability to regulate DNA polymerase activity. Therefore, the G-quadruplex in nucleic acid aptamers and the G-quadruplex-binding peptide in DNA polymerase are two indispensable parts for controlling hot-start DNA polymerase.
  • nucleic acid aptamers with four different G-quadruplex types were selected for the experiment.
  • the eight nucleic acid aptamers were CSTB, Kit-C, PDGFRB, T1B1, T4B1, GVBQ1, GVBQ2 and G12.
  • the G-quadruplex type of the nucleic acid aptamers CSTB and Kit-C is a regular three-layer G-quadruplex
  • the G-quadruplex type of the nucleic acid aptamers PDGFRB, T1B1 and T4B1 is a raised G -Quadruplex
  • the G-quadruplex type of nucleic acid aptamers GVBQ1 and GVBQ2 is a G-quadruplex with G-vacancies
  • the G-quadruplex type of nucleic acid aptamer G12 is a regular two-layer G -Quadruplex.
  • the molar concentration ratio of RHAU23-Bst and nucleic acid aptamer is 1:2.
  • nucleic acid aptamers containing irregular G-quadruplexes T4B1, GVBQ1 and GVBQ2
  • nucleic acid aptamers containing unstable double-layer G-quadruplexes G12
  • HG53 nucleic acid aptamers with "hairpin" structure lengths of 8bp, 7bp, 6bp, 5bp and 4bp were selected.
  • the molar concentration ratio of RHAU23-Bst and nucleic acid aptamers was 1:2.
  • H8 represents a nucleic acid aptamer with a "hairpin” structure length of 8 bp
  • H7 represents a nucleic acid aptamer with a "hairpin” structure length of 7 bp
  • H6 represents a "hairpin” structure length of 6 bp.
  • Nucleic acid aptamer H5 represents a nucleic acid aptamer with a "hairpin” structure length of 5 bp.
  • the length of the "hairpin" region needs to be at least 7 bp for the nucleic acid aptamer to function effectively.
  • HG53 Compared with GH5 and HG3, HG53 has an extra loop connecting the G-quadruplex and the "hairpin” structure. This loop mainly affects the formation of the "hairpin” structure at the end of DNA.
  • HG53 nucleic acid aptamers with loop lengths of 2nt, 4nt, 6nt, 8nt, 10nt and 12nt were used for experiments.
  • RHAU23-Bst The molar concentration ratio to nucleic acid aptamer is 1:2.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • the inhibitory effects of the nucleic acid aptamers HG53-GVBQ1 and HG53-H7 on three Bst DNA polymerases at different temperatures were tested through primer extension experiments.
  • the three Bst DNA polymerases are Bst-LF, Bst 2.0 hot-start DNA polymerase (Bst 2.0 hot-start DNA polymerase is NEB's commercial hot-start DNA polymerase) and modified DNA polymerase RHAU23- Bst; the detection temperature is 25°C-65°C; the final concentration of Bst-LF, RHAU23-Bst reaction is 100nM, Bst 2.0 hot-start DNA polymerase is 0.32U/ ⁇ L; the concentration of nucleic acid aptamer is 200nM; the reaction buffer is The second buffer includes 20mM Tris-HCl (pH 8.8), 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 8mM MgSO 4 and 0.1% T
  • Figure 6 is a diagram of the experimental results of this embodiment. Please refer to Figure 6A to Figure 6C. In the absence of nucleic acid aptamer binding, three Bst DNA polymerases (Bst-LF, Bst 2.0 hot start DNA polymerase and RHAU23 -Bst) showed similar activity when synthesizing DNA at 25°C-65°C. Please refer to Figure 6D and Figure 6E. The activity of the hot-start enzyme after RHAU23-Bst is combined with the nucleic acid aptamers HG53-H7 and HG53-GVBQ1 is greatly inhibited below 30°C.
  • Bst-LF Bst 2.0 hot start DNA polymerase
  • RHAU23 -Bst The activity of the hot-start enzyme after RHAU23-Bst is combined with the nucleic acid aptamers HG53-H7 and HG53-GVBQ1 is greatly inhibited below 30°C.
  • Figure 6F shows the results of comparing the fraction of the amplified full-length product with RHAU23-Bst to determine the normalized activity of RHAU23-Bst in the presence of the nucleic acid aptamer HG53-H7.
  • the nucleic acid aptamer HG53-H7 inhibits the activity of RHAU23-Bst by more than 80%.
  • the temperature is higher than 35°C, the activity of RHAU23-Bst gradually recovers.
  • the temperature is higher than 50°C, the activity of RHAU23-Bst is gradually restored. -The activity of Bst is restored to over 90%.
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • Hot start DNA polymerase is used to detect human papillomavirus (HPV) DNA.
  • LAMP loop-mediated isothermal amplification
  • the concentration of aptamer used was 200nM.
  • Bst-LF and commercial Bst 2.0 hot-start DNA polymerase were used as controls.
  • Figure 7A in the presence of Bst-LF, the amplification curve of the LAMP reaction did not increase in fluorescence within 50 minutes, indicating that Bst-LF failed to detect the DNA sequence of HPV16.
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • Hot start DNA polymerase is used to detect SARS-CoV-2.
  • RT-LAMP Add 5U WarmStart RTx reverse transcriptase (NEB), 100nM nucleic acid aptamer-assembled hot-start DNA polymerase and 0.5 ⁇ gelgreen, modified DNA polymerase of hot-start DNA polymerase into the RT-LAMP reaction system. is RHAU23-Bst, the nucleic acid aptamer is HG53-H7, and the molar ratio of modified DNA polymerase to nucleic acid aptamer is 1:2.
  • RT-LAMP is an existing technology and will not be described in detail here. In this experiment, the primers used are shown in Table 5.
  • FIP/BIP is 1.6 ⁇ M
  • F3/B3 is 0.2 ⁇ M
  • Loop-F/B is 0.4 ⁇ M.
  • Amplification reactions were performed at 65°C on a QuantStudio 7 Flex (Thermo Scientific) real-time fluorescence quantitative PCR system.
  • pH-mediated colorimetric RT-LAMP Add 5U WarmStart RTx reverse transcriptase (NEB), 100nM nucleic acid aptamer-assembled hot-start DNA polymerase and 0.5 ⁇ gelgreen to the pH-mediated colorimetric RT-LAMP reaction system.
  • the modified DNA polymerase of the hot start DNA polymerase is RHAU23-Bst
  • the nucleic acid aptamer is HG53-H7
  • the molar ratio of the modified DNA polymerase to the nucleic acid aptamer is 1:2. Reactions were performed at 65°C for the indicated times and detected by color change in cresol red.
  • Figure 8A is a graph showing the results of using fluorescent RT-LAMP to detect SARS-CoV-2 pseudoviral RNA with a specified copy number
  • Figure 8B is a graph showing the results of using colorimetric RT-LAMP to detect SARS-CoV-2 pseudoviral RNA, where, The copy number of SARS-CoV-2 pseudoviral RNA is the same as in the fluorescent RT-LAMP assay.
  • the nucleic acid aptamer HG53-H7 combined with RHAU23-Bst or the existing hot-start Bst DNA polymerase can effectively prevent false positive results caused by non-specific amplification.
  • colorimetric RT-LAMP also demonstrates the advantage of hot-start Bst DNA polymerase in preventing false-positive results from non-specific amplification.
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • the combined strategy of G-quadruplex-binding peptides and G-quadruplex-containing nucleic acid aptamers is suitable for Taq DNA polymerase.
  • DNA polymerase RHAU23-Taq was constructed with reference to the construction method of Example 1. Among them, the sequence encoding the large fragment of Bst DNA polymerase in plasmid pCold-I-RHAU23-Bst was replaced with the gene sequence encoding Taq DNA polymerase to construct plasmid pCold-I-RHAU23-Taq, where, pCold-I-RHAU23- The Taq plasmid map is shown in Figure 9A.
  • the activity of RHAU23-Taq was detected with reference to the method in Example 3. Among them, the final concentration of RHAU23-Taq reaction is 100nM, and HG53-H7 is 200nM. Using the sample without nucleic acid aptamer as a control, primer extension was performed at different temperatures for 30 minutes.
  • the reaction buffer is the second buffer, including 20mM Tris-HCl (pH 8.8), 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 and 0.1% Tween-20.
  • the binding strategy of G-quadruplex binding peptide and G-quadruplex-containing nucleic acid aptamer is suitable for Taq DNA polymerase, and Taq DNA polymerase can be transformed into a modified DNA polymerase to enable hot start ability.
  • the conjugation strategy of G-quadruplex binding peptides and G-quadruplex-containing nucleic acid aptamers is suitable for reverse transcriptase.
  • Reverse transcriptase RHAU23-RT was constructed with reference to the construction method of Example 1. Among them, the sequence encoding the Bst large fragment in plasmid pCold-I-RHAU23-Bst was replaced with the gene sequence encoding MMLV reverse transcriptase (RT) to construct plasmid pCold-I-RHAU23-RT, in which pCold-I-RHAU23-RT The plasmid map is shown in Figure 10A.
  • the reverse transcription primer extension experiment was performed according to the method of Example 3. Among them, RNA is used as the template (the nucleotide sequence of the RNA template is such as Seq ID No. 14), and the DNA paired with the RNA is used as the primer (the nucleotide sequence of the DNA primer is such as Seq ID No. 15).
  • the final concentration of RHAU23-RT reaction is 100nM, and HG53-H7 is 200nM.
  • the reaction buffer is the second buffer, including 20mM Tris-HCl (pH 8.8), 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 8mM MgSO 4 and 0.1% Tween-20. Using samples without G-quadruplex binding peptide and without nucleic acid aptamer added as controls, primer extension was performed at different temperatures for 15 minutes.
  • existing MMLV reverse transcriptase has reverse transcriptase activity between 25°C and 65°C.
  • the modified reverse transcriptase is modified using the binding strategy of G-quadruplex binding peptide and G-quadruplex-containing nucleic acid aptamer, the modified reverse transcriptase The reverse transcription activity below 30°C is strongly inhibited. When the temperature rises above 35°C, the modified reverse transcription activity can be mostly restored, especially when the temperature is between 50°C and 60°C, the modified reverse transcription activity Better recovery of activity.
  • the binding strategy of G-quadruplex binding peptide and G-quadruplex-containing nucleic acid aptamer is suitable for reverse transcriptase, and the reverse transcriptase can be transformed into a modified reverse transcriptase to have hot-start properties. .
  • reverse transcriptase G4P-RT by constructing plasmid pCold-I-G4P-RT.
  • the binding strategy of G-quadruplex binding peptides and G-quadruplex-containing nucleic acid aptamers is suitable for a variety of DNA polymerases, and DNA polymerases can be transformed into modified DNA polymerases to make them Has hot start properties.

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Abstract

一种修饰的DNA聚合酶、用于调控DNA聚合酶活性的核酸适配体和热启动DNA聚合酶，修饰的DNA聚合酶包括DNA聚合酶片段和修饰在DNA聚合酶片段N端的G-四链体结合肽；核酸适配体的序列的二级结构包含一个G-四链体；热启动DNA聚合酶包括修饰的DNA聚合酶和核酸适配体，核酸适配体用于在第一预设温度时结合于DNA聚合酶的G-四链体结合肽，以抑制DNA聚合酶的活性，并用于在第二预设温度时脱离于DNA聚合酶的G-四链体结合肽，以恢复DNA聚合酶的活性；其中，第二预设温度高于第一预设温度。将所构建的热启动DNA聚合酶应用于等温扩增时，不会产生非特异性扩增，这在分子诊断中具有很大的应用价值。

Description

DNA聚合酶、核酸适配体、热启动DNA聚合酶及方法和应用 技术领域
本发明涉及生物技术领域，尤其涉及一种修饰的DNA聚合酶、核酸适配体、热启动DNA聚合酶、试剂盒、生物合成DNA聚合酶的方法、制备热启动DNA聚合酶的方法及DNA聚合酶和核酸适配体相配合用于检测核酸或者合成核酸的应用。
背景技术
核酸扩增技术广泛用于研究人类疾病以及检测病原体。除PCR外，还有多种等温扩增技术可以用于核酸诊断技术，如环介导等温扩增(LAMP)、重组酶聚合酶扩增(RPA)、链置换扩增(SDA)和滚环扩增(RCA)。据报道，这些技术还用于检测正在传播的严重急性呼吸综合征冠状病毒-2(SARS-CoV-2)。
核酸检测技术的一个关键挑战是由引物错配和非模板扩增引起的非特异性扩增。非特异性扩增强烈阻碍或降低目标模板的扩增，降低了检测的灵敏度。避免非特异性扩增的有效方法是阻断嗜热DNA聚合酶在低温下的活性。在PCR反应中，用抗体或特异性适配体阻断DNA聚合酶的活性是防止非特异性扩增的最常用方法。对DNA聚合酶(如Taq DNA聚合酶)进行共价修饰是阻断其低温阶段活性的另一种方法。此外，在DNA聚合酶的氨基酸中引入特定突变位点也会改变其性质并降低聚合酶的活性。
与PCR相比，等温扩增技术中也存在非特异性扩增，严重影响LAMP等相关技术的应用。然而，目前解决等温扩增中非特异性扩增问题的方法很少。据报道，酰胺添加剂可以提高LAMP中核酸扩增的特异性并减少了背景扩增。但有机溶剂的使用也有很多局限性，如无法用于冻干试剂的制备。已发现来自于 腾冲嗜热厌氧菌的解旋酶可抑制非靶核酸的扩增，从而有效降低背景信号。然而，解旋酶水解dATP并释放H +，导致溶液pH改变，限制了其在pH介导的比色LAMP中的使用。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种修饰的DNA聚合酶，可提供广谱的制备热启动DNA聚合酶的技术策略。
本发明的目的之一采用如下技术方案实现：一种修饰的DNA聚合酶，包括DNA聚合酶片段和融合于所述DNA聚合酶片段的N端的G-四链体结合肽。
本发明的目的之二在于提供一种用于调控上述的DNA聚合酶活性的核酸适配体。
本发明的目的之二采用如下技术方案实现：一种用于调控上述DNA聚合酶活性的核酸适配体，所述核酸适配体的核苷酸序列包括G-四链体核心序列，所述G-四链体核心序列的二级结构为G-四链体；
所述G-四链体用于在第一预设温度时结合于所述DNA聚合酶的G-四链体结合肽，以抑制所述DNA聚合酶的活性，并用于在第二预设温度时脱离于所述DNA聚合酶的G-四链体结合肽，以恢复所述DNA聚合酶的活性；其中，所述第二预设温度高于所述第一预设温度。
本发明的目的之三在于提供一种热启动DNA聚合酶。
本发明的目的之三采用如下技术方案实现：一种热启动DNA聚合酶，包括上述的DNA聚合酶和上述的核酸适配体，所述核酸适配体的G-四链体在第一预设温度结合于所述DNA聚合酶的G-四链体结合肽，以抑制所述DNA聚合酶的活性，并在第二预设温度脱离于所述DNA聚合酶的G-四链体结合肽，以恢复所述DNA聚合酶的活性；其中，所述第二预设温度高于所述第一预设温度。
本发明的目的之四在于提供一种试剂盒。
本发明的目的之四采用如下技术方案实现：一种试剂盒，包括上述DNA聚合酶和上述核酸适配体。
本发明的目的之五在于提供一种生物合成上述DNA聚合酶的方法。
本发明的目的之五采用如下技术方案实现：一种生物合成上述DNA聚合酶的方法，包括如下步骤：
将DNA聚合酶片段插入质粒载体，构建第一质粒；
在所述第一质粒的基础上，将G-四链体结合肽的编码序列插入所述DNA聚合酶片段的N端，构建第二质粒；
将所述第二质粒转化到大肠杆菌菌株中进行培养并诱导蛋白质表达；
纯化蛋白质，获得所述的DNA聚合酶。
本发明的目的之六在于提供一种制备热启动DNA聚合酶的方法。
本发明的目的之六采用如下技术方案实现：一种制备热启动DNA聚合酶的方法，所述DNA聚合酶/适配体复合物包括上述的DNA聚合酶和上述的核酸适配体，所述核酸适配体通过所述G-四链体结合于所述G-四链体结合肽而结合于所述DNA聚合酶，所述方法包括如下步骤：
将所述核酸适配体溶解于第一缓冲液中，在90℃～100℃变性2分钟～8分钟后，冷却至20℃～30℃，获得处理后的核酸适配体；
将所述DNA聚合酶和处理后的核酸适配体按预设的摩尔浓度比加入第二缓冲液中，在2℃～6℃下孵育30min～60min；
其中，所述第一缓冲液包括10mM pH 7.4Tris-HCl、75mM KCl、0.5mM EDTA和0.2mg/ml牛血清白蛋白，所述第二缓冲液包括20mM pH 8.8 Tris-HCl、10mM(NH 4) 2SO 4、50mM KCl、2-8mM MgSO 4和0.1％吐温-20。
本发明的目的之七在于提供上述DNA聚合酶和上述核酸适配体相配合用于检测核酸或者合成核酸的应用。
本发明的目的之七采用如下技术方案实现：上述DNA聚合酶和上述核酸适配体相配合用于检测核酸或者合成核酸的应用。
相比现有技术，本发明的有益效果在于：(1)本发明构建一种修饰的DNA聚合酶以及与该DNA聚合酶特异性结合的核酸适配体，DNA聚合酶的活性在低温阶段被结合的核酸适配体严格抑制，但在加热到反应温度时完全恢复，在应用于等温扩增时，不会产生非特异性扩增，因此，它在分子诊断中具有很大的应用价值；(2)采用本发明构建的修饰的DNA聚合酶以及与该DNA聚合酶特异性结合的核酸适配体，成功地解决了LAMP检测HPV DNA和SARS-CoV-2 RNA的非特异性扩增问题；(3)G-四链体结合肽与G-四链体适配体的结合为可控DNA聚合酶的开发提供了一条新途径。
附图说明
图1A为本发明实施例一提供的Bst DNA聚合酶、Bst-LF和DNA聚合酶的结构示意图，其中DNA聚合酶融合有G-四链体结合肽，用G4P-Bst和RHAU23-Bst表示；
图1B为本发明实施例一提供的纯化的Bst-LF、RHAU23-Bst和G4P-Bst的电泳图；
图1C为本发明实施例一提供的不同温度下，G-四链体核酸适配体调控DNA聚合酶活性的原理示意图；
图1D为本发明实施例一提供的pCold-I-G4P-Bst质粒图谱；
图1E为本发明实施例一提供的pCold-I-RHAU23-Bst质粒图谱；
图2A至图2C为本发明实施例二提供的DNA聚合酶G4P-Bst与核酸适配 体T4B1 core G4、PDGFRB core G4、T4B1 Fs-G4、PDGFRB Fs-G4、T4B1 Fd-G4和PDGFRB Fd-G4的结合能力结果图；
图2D为本发明实施例二提供的DNA聚合酶RHAU23-Bst与核酸适配体T4B1 Fd-G4和PDGFRB Fd-G4的结合能力结果图；
图2E为本发明实施例二提供的DNA聚合酶RHAU23-Bst与核酸适配体HG53、GH5、HG3的结合能力结果图；
图3A为本发明实施例三提供的引物延伸实验的原理图；
图3B至图3D为本发明实施例三提供的使用DNA聚合酶G4P-Bst进行引物延伸实验的结果图；
图3E至图3H为本发明实施例三提供的使用DNA聚合酶RHAU23-Bst进行引物延伸实验的结果图；
图4A为本发明实施例四提供的通过EMSA方法检测DNA聚合酶G4P-Bst与非G4单链DNA和非G4双链DNA的结合能力的结果图；
图4B为本发明实施例四提供的通过EMSA方法检测Bst-LF与T4B1 Fd-G4和T4B1 Fs-G4的结合能力的结果图；
图4C为本发明实施例四提供的通过引物延伸实验检测非G4单链DNA和非G4双链DNA存在时G4P-Bst的活性的结果图；
图4D为本发明实施例四提供的通过引物延伸实验检测T4B1 Fd-G4和T4B1 Fs-G4存在时G4P-Bst的活性的结果图；
图5为本发明实施例五提供的G-四链体类型、“发夹”结构长度和环长度对核酸适配体(HG53)抑制DNA聚合酶RHAU23-Bst活性能力的影响结果图；
图6A为本发明实施例六提供的在不存在核酸适配体的情况下，Bst-LF在不同温度下进行引物延伸实验的结果图；
图6B为本发明实施例六提供的在不存在核酸适配体的情况下，Bst 2.0热启动DNA聚合酶在不同温度下进行引物延伸实验的结果图；
图6C为本发明实施例六提供的在不存在核酸适配体的情况下，RHAU23-Bst在不同温度下进行引物延伸实验的结果图；
图6D为本发明实施例六提供的在存在核酸适配体HG53-GVBQ1的情况下，RHAU23-Bst在不同温度下进行引物延伸实验的结果图；
图6E为本发明实施例六提供的在存在核酸适配体HG53-H7的情况下，RHAU23-Bst在不同温度下进行引物延伸实验的结果图；
图6F为本发明实施例六提供的RHAU23-Bst在核酸适配体HG53-H7存在的情况下的标准化活性结果图；
图7为本发明实施例七提供的用荧光LAMP检测HPV 16基因DNA的结果图；
图8A是荧光RT-LAMP检测SARS-CoV-2 RNA的结果图；图8B是pH介导的比色RT-LAMP检测SARS-CoV-2 RNA的结果图；
图9A为本发明实施例九提供的pCold-I-RHAU23-Taq质粒图谱；
图9B为本发明实施例九提供的在不存在核酸适配体的情况下，RHAU23-Taq在不同温度下进行引物延伸实验的结果图；
图9C为本发明实施例九提供的在存在核酸适配体HG53-H7的情况下，RHAU23-Taq在不同温度下进行引物延伸实验的结果图；
图9D为本发明实施例九提供的存在核酸适配体HG53-H7时RHAU23-Taq的活性相比不存在核酸适配体时的比值；
图10A为本发明实施例十提供的pCold-I-RHAU23-RT质粒图谱；
图10B为本发明实施例十提供的在存在核酸适配体HG53-H7的情况下， RHAU23-RT在不同温度下进行引物延伸实验的结果图；
图10C为本发明实施例十提供的在不存在核酸适配体的情况下，现有MMLV逆转录酶在不同温度下进行引物延伸实验的结果图；
图10D为本发明实施例十提供的存在核酸适配体HG53-H7时RHAU23-RT逆转录酶活性相比不存在核酸适配体时现有MMLV逆转录酶活性的比值。
具体实施方式
下面，结合附图以及具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
本发明提供一种修饰的DNA聚合酶，包括DNA聚合酶片段和融合于DNA聚合酶片段的N端的G-四链体结合肽。
作为一种实施方式，G-四链体结合肽为RHAU23肽，RHAU23肽的氨基酸序列如Seq ID No.1所示；或者，
G-四链体结合肽为G4P，G4P的氨基酸序列如Seq ID No.2所示。
作为一种实施方式，DNA聚合酶片段耐受40℃以上的温度，且在温度高于40℃以上时具有活性。
作为一种实施方式，DNA聚合酶片段来自于Bst DNA聚合酶、Taq DNA聚合酶和MMLV逆转录酶中的任意一种。
作为一种实施方式，DNA聚合酶片段来自于Bst DNA聚合酶，DNA聚合酶片段的氨基酸序列如Seq ID No.3所示。
作为一种实施方式，DNA聚合酶片段来自于Taq DNA聚合酶，DNA聚合酶片段的氨基酸序列如Seq ID No.4所示。
作为一种实施方式，DNA聚合酶片段来自于MMLV逆转录酶，DNA聚合 酶片段的氨基酸序列如Seq ID No.5所示。
本发明还提供一种用于调控上述DNA聚合酶活性的核酸适配体，核酸适配体的核苷酸序列包括G-四链体核心序列，G-四链体核心序列的二级结构为G-四链体；
G-四链体用于在第一预设温度时结合于DNA聚合酶的G-四链体结合肽，以抑制DNA聚合酶的活性，并用于在第二预设温度时脱离于DNA聚合酶的G-四链体结合肽，以恢复DNA聚合酶的活性；其中，第二预设温度高于第一预设温度。
作为一种实施方式，G-四链体为规则的三层G-四链体、凸起的G-四链体、带有G-空位的G-四链体和规则的两层G-四链体中的任意一种。
作为一种实施方式，核酸适配体的核苷酸序列还包括G-四链体核心序列的5’端和/或3’端的旁侧DNA序列。
作为一种实施方式，旁侧DNA序列为侧翼单链DNA序列或者侧翼双链DNA序列或者用于形成“发夹”结构的DNA序列。
作为一种实施方式，旁侧DNA序列优选为用于形成“发夹”结构的DNA序列，其中，形成的“发夹”结构的长度大于或等于7bp。
作为一种实施方式，第一预设温度为0℃～30℃，第二预设温度为45℃～70℃。
作为一种实施方式，第二预设温度为55℃～65℃。
作为一种实施方式，G-四链体核心序列为CSTB核心序列，CSTB核心序列的核苷酸序列如Seq ID No.6所示；或者，
G-四链体核心序列为KIT-C核心序列，KIT-C核心序列的核苷酸序列如Seq ID No.7所示；或者，
G-四链体核心序列为T4B1核心序列，T4B1核心序列的核苷酸序列如Seq ID No.8所示；或者，
G-四链体核心序列为PDGFRB核心序列，PDGFRB核心序列的核苷酸序列如Seq ID No.9所示；或者，
G-四链体核心序列为T1B1核心序列，T1B1核心序列的核苷酸序列如Seq ID No.10所示；或者，
G-四链体核心序列为GVBQ1核心序列，GVBQ1核心序列的核苷酸序列如Seq ID No.11所示；或者，
G-四链体核心序列为GVBQ2核心序列，GVBQ2核心序列的核苷酸序列如Seq ID No.12所示；或者，
G-四链体核心序列为G12核心序列，G12核心序列的核苷酸序列如Seq ID No.13所示。
本发明还提供一种热启动DNA聚合酶，包括上述DNA聚合酶和上述核酸适配体，核酸适配体的G-四链体在第一预设温度结合于DNA聚合酶的G-四链体结合肽，以抑制DNA聚合酶的活性，并在第二预设温度脱离于DNA聚合酶的G-四链体结合肽，以恢复DNA聚合酶的活性；其中，第二预设温度高于第一预设温度。
作为一种实施方式，第一预设温度为0℃～30℃，第二预设温度为45℃～70℃。
本发明还提供一种试剂盒，包括上述DNA聚合酶和上述核酸适配体。
作为一种实施方式，试剂盒用于检测人乳头瘤病毒DNA或者SARS病毒RNA。
本发明还提供一种生物合成上述DNA聚合酶的方法，其特征在于，包括如 下步骤：
将DNA聚合酶片段插入质粒载体，构建第一质粒；
在第一质粒的基础上，将G-四链体结合肽的编码序列插入DNA聚合酶片段的N端，构建第二质粒；
将第二质粒转化到大肠杆菌菌株中进行培养并诱导蛋白质表达；
纯化蛋白质，获得的DNA聚合酶。
作为一种实施方式，DNA聚合酶片段来自于嗜热脂肪芽孢杆菌的DNA聚合酶的291-878残基，质粒载体为pCold-I，第一质粒为pCold-I-Bst-LF质粒；
G-四链体结合肽为G4P，第二质粒为pCold-I-G4P-Bst质粒；或者，G-四链体结合肽为RHAU23，第二质粒为pCold-I-RHAU23-Bst质粒。
作为一种实施方式，DNA聚合酶片段来自于Taq DNA聚合酶，质粒载体为pCold-I，第一质粒为pCold-I-Taq-LF质粒；
G-四链体结合肽为G4P，第二质粒为pCold-I-G4P-Taq质粒；或者，G-四链体结合肽为RHAU23，第二质粒为pCold-I-RHAU23-Taq质粒。
作为一种实施方式，DNA聚合酶片段来自于MMLV逆转录酶，质粒载体为pCold-I，第一质粒为pCold-I-RT-LF质粒；
G-四链体结合肽为G4P，第二质粒为pCold-I-G4P-RT质粒；或者，G-四链体结合肽为RHAU23，第二质粒为pCold-I-RHAU23-RT质粒。
本发明还提供一种制备热启动DNA聚合酶的方法，热启动DNA聚合酶上述DNA聚合酶和上述核酸适配体，核酸适配体通过G-四链体结合于G-四链体结合肽而结合于DNA聚合酶，方法包括如下步骤：
将核酸适配体溶解于第一缓冲液中，在90℃～100℃变性2分钟～8分钟后，冷却至20℃～30℃，获得处理后的核酸适配体；
将DNA聚合酶和处理后的核酸适配体按预设的摩尔浓度比加入第二缓冲液中，在2℃～6℃下孵育30min～60min；
其中，第一缓冲液包括10mM pH 7.4 Tris-HCl、75mM KCl、0.5mM EDTA和0.2mg/ml牛血清白蛋白，第二缓冲液包括20mM pH 8.8 Tris-HCl、10mM(NH 4) 2SO 4、50mM KCl、2-8mM MgSO 4和0.1％吐温-20。
作为一种实施方式，DNA聚合酶和核酸适配体的预设的摩尔浓度比的范围为1:8～1:1。
本发明还提供一种上述DNA聚合酶和上述核酸适配体相配合用于检测核酸或者合成核酸的应用。
作为一种实施方式，在应用于检测核酸时，核酸为人乳头瘤病毒DNA或者SARS病毒RNA。
本发明构建了G-四链体结合肽融合的DNA聚合酶以及与该聚合酶特异性结合的核酸适配体，DNA聚合酶的活性在低温阶段被结合的核酸适配体严格抑制，但在加热到反应温度时完全恢复，在应用于等温扩增时，不会产生非特异性扩增，因此，它在分子诊断中具有很大的应用价值；采用本发明构建的修饰的DNA聚合酶以及与该DNA聚合酶特异性结合的核酸适配体，成功地解决了LAMP检测HPV DNA和SARS-CoV-2 RNA的非特异性扩增问题；G-四链体结合肽与G-四链体适配体的结合为可控DNA聚合酶的开发提供了一条新途径。
实施例一：
修饰的DNA聚合酶的构建。
通过内切酶Nde I和Xba I将Bst DNA聚合酶大片段(Bst-LF)插入pCold-I载体以构建pCold-I-Bst-LF质粒。在pCold-I-Bst-LF的基础上，将G4P的编码序列插入Bst-LF的N端，构建了pCold-I-G4P-Bst质粒，将RHAU23的编码序列 插入Bst-LF的N端，构建了pCold-I-RHAU23-Bst质粒。
将质粒pCold-I-Bst-LF、pCold-I-G4P-Bst和pCold-I-RHAU23-Bst转化到大肠杆菌菌株BL21中。细胞在含有50μg/mL氨苄青霉素的LB培养基中，37℃培养至OD 0.8-1.0。然后，添加0.4mM IPTG并在16℃下再培养16h来诱导蛋白质表达。根据说明书使用His-Tag钴树脂(Thermo-Scientific)纯化蛋白质，蛋白质储存在含有20mM Tris-HCl、150mM NaCl、1mM DTT、0.5mM EDTA和50％甘油的缓冲液中。图1A示出了Bst DNA聚合酶、Bst-LF和修饰的DNA聚合酶的结构示意图，其中修饰的DNA聚合酶融合有G-四链体结合肽，用G4P-Bst和RHAU23-Bst表示。图1B为纯化的Bst-LF、RHAU23-Bst和G4P-Bst的电泳图。
其中，Bst-LF来自于嗜热脂肪芽孢杆菌(ARA98840.1)的DNA聚合酶I的291-878残基，也就是嗜热脂肪芽孢杆菌DNA聚合酶中缺少5'-3'核酸外切酶结构域的部分，Bst-LF的氨基酸序列如Seq ID No.3所示。Bst-LF在较宽的温度范围内具有活性，并且具有较强的链置换活性，被广泛应用于环介导等温扩增(LAMP)。RHAU23是一种来自人类DHX36蛋白的23个氨基酸的肽，它与G-四链体特异性相互作用，RHAU23的氨基酸序列如Seq ID No.1所示。G4P是一种由两个串联的RHAU23组成的小蛋白质(64个氨基酸)，G4P比RHAU23具有更强的G-四链体结合亲和力，G4P的氨基酸序列如Seq ID No.2所示。pCold-I-G4P-Bst质粒图谱见图1D，pCold-I-RHAU23-Bst质粒图谱见图1E。
本实施例提供的修饰的DNA聚合酶，通过在N端融合有G-四链体结合肽，能够与具有G-四链体的核酸适配体相结合。如图1C所示，在低温阶段，融合G-四链体结合肽的DNA聚合酶与G-四链体结合，阻止DNA聚合酶与底物DNA相互作用，在高温阶段，G-四链体DNA去折叠并从DNA聚合酶中分离，从而 DNA聚合酶可以重新作用于底物。
实施例二：
通过凝胶迁移实验(EMSA)检测修饰的DNA聚合酶与核酸适配体结合亲和力。
寡核苷酸购自Sangon生物技术有限公司(中国)。互补单链DNA(ssDNA)溶解在含有10mM Tris-HCl(pH7.4)、75mM KCl和0.5mM EDTA的缓冲液中，在95℃变性5分钟后，以0.1℃/s的速度冷却至25℃，从而配对成双链DNA(dsDNA)。
将20nM 5'-FAM标记的核酸适配体DNA(表1)溶解在第一缓冲液中，在95℃变性5分钟后，以0.1℃/s的速率冷却至25℃；其中，第一缓冲液包括10mM Tris-HCl(pH值7.4)、75mM KCl、0.5mM EDTA和0.2mg/ml牛血清白蛋白。然后将各核酸适配体DNA与指定浓度的融合G-四链体结合肽的DNA聚合酶加入第二缓冲液中，在4℃下结合1小时，形成DNA和蛋白结合的复合物；其中，第二缓冲液包括20mM Tris-HCl(pH 8.8)，10mM(NH 4) 2SO 4、50mM KCl、8mM MgSO 4和0.1％吐温-20。
核酸适配体和DNA聚合酶的结合通过凝胶迁移实验来检测。具体地，在含有75mM KCl的1×TBE缓冲液中，DNA和蛋白结合的复合物在4℃下在含有75mM KCl的8％或12％非变性聚丙烯酰胺凝胶上电泳2小时。通过ChemiDoc MP成像系统(Bio-Rad)对DNA和蛋白结合的复合物进行拍照，并通过Image Quant 5.2软件进行定量分析。
结果如图2所示，其中图2A至图2C为修饰的DNA聚合酶G4P-Bst与核酸适配体T4B1 core G4、PDGFRB core G4、T4B1 Fs-G4、PDGFRB Fs-G4、T4B1 Fd-G4和PDGFRB Fd-G4的结合能力结果图，其中core G4表示核心G-四链体， Fs-G4表示侧翼含单链DNA的G-四链体，Fd-G4表示侧翼含双链DNA的G-四链体；图2D为修饰的DNA聚合酶RHAU23-Bst与核酸适配体T4B1 Fd-G4和PDGFRB Fd-G4的结合能力结果图，图2E为修饰的DNA聚合酶RHAU23-Bst与核酸适配体HG53、GH5、HG3的结合能力结果图，其中核酸适配体HG53、GH5、HG3均为可折叠成G-四链体和“发夹”结构的单链DNA，而上述的Fd-G4适配体是由两个互补的单链DNA配对而成的。
从图中可以看出，无论T4B1的凸起的G-四链体(凸起的G-四链体也称为bulged G-四链体)和PDGFRB的凸起的G-四链体是否具有旁侧DNA序列，G4P-Bst显示出与T4B1的凸起的G-四链体和PDGFRB的凸起的G-四链体均具有强的结合亲和力。其中，Kd值范围为1-5nM。RHAU23-Bst与含有G-四链体和侧翼双链DNA的核酸适配体也具有强的结合亲和力，Kd处于nM水平。并且RHAU23-Bst与核酸适配体HG53、GH5、HG3的结合亲和力与RHAU23-Bst与含有G-四链体和侧翼双链DNA的核酸适配体的结合亲和力相似，Kd值范围为3.4-7.4nM。
表1
Figure PCTCN2022082834-appb-000001
Figure PCTCN2022082834-appb-000002
实施例三：
通过引物延伸实验检测核酸适配体对DNA聚合酶活性的调节。
将表2中列出的各核酸适配体DNA溶解在第一缓冲液中至10μM，在95℃下加热5min，然后以0.1℃/s的速率冷却至25℃；其中，第一缓冲液包括10mM Tris-HCl(pH值7.4)、75mM KCl、0.5mM EDTA和0.2mg/ml牛血清白蛋白。将指定比例的融合G-四链体结合肽的DNA聚合酶(100nM)与核酸适配体加入第二缓冲液中，在4℃下放置30min形成DNA和蛋白结合的复合物；其中，融合G-四链体结合肽的DNA聚合酶与核酸适配体的摩尔浓度比分别为1:0、1:1、1:2、1:4和1:8，第二缓冲液包括20mM Tris-HCl(pH 8.8)，10mM(NH 4) 2SO 4、50mM KCl、8mM MgSO 4和0.1％吐温-20。添加2.5mM dNTP，100nM引物和100nM模板DNA到DNA和蛋白结合的复合物中，并在25℃或65℃下反应5分钟或者40分钟，其中，引物和模板DNA的序列请参阅表3。反应完成后添加四倍体积的终止缓冲液(99％甲酰胺、0.1％SDS和20mM EDTA)终止反应。样品在95℃下煮沸5分钟，然后在1×TBE缓冲液中的12％尿素变性聚丙烯酰胺凝胶上电泳。通过ChemiDoc MP成像系统(Bio-Rad)上对引物和全长产物进行拍照，并使用Image Quant 5.2软件进行数字化定量。
表2
Figure PCTCN2022082834-appb-000003
Figure PCTCN2022082834-appb-000004
表3
Figure PCTCN2022082834-appb-000005
图3A为引物延伸实验的原理图，图3B至图3D为使用修饰的DNA聚合酶G4P-Bst进行引物延伸实验的结果图，图3E至图3H为使用修饰的DNA聚合酶RHAU23-Bst进行引物延伸实验的结果图。
从图中可以看出，当仅以核心G-四链体(core G4)作为核酸适配体时，也就是核酸适配体不具有任何旁侧DNA序列，无论是在25℃还是65℃，G4P-Bst的活性几乎不受影响，详见图3B；当使用含侧翼单链DNA的G-四链体作为核酸适配体时，也就是使用Fs-G4适配体，G4P-Bst的活性在25℃时表现出不同程度的抑制，该抑制与修饰的DNA聚合酶/核酸适配体的比率相关，在65℃时修饰的DNA聚合酶的活性基本恢复，详见图3C。当使用含侧翼双链DNA的G-四链体作为核酸适配体时，也就是使用Fd-G4适配体，只要DNA聚合酶/核酸适配体的比例小于1，G4P-Bst在25℃时的活性几乎完全被抑制，Fd-G4适配体对G4P-Bst活性的抑制强烈，在65℃下仍表现出不同程度的抑制，详见图3D。由于G4P-Bst与上述三种核酸适配体的结合亲和力相似，但含侧翼DNA的适配体明显效果更好因此，G-四链体的侧翼区域的DNA也是适配体发挥作用所必 需的。
G4P-Bst与Fd-G4适配体结合后，其活性被严重抑制，即使升温到65℃也没有完全恢复；相比之下RHAU23-Bst与Fd-G4适配体结合后，其在65℃下的活性几乎完全恢复，详见图3E。核酸适配体对这两个聚合酶的抑制效果不同与G4P和RHAU23对G4结合能力相关。这说明我们可以通过改变G-四链体结合肽来调节适配体对酶活性的抑制能力。
RHAU23-Bst与核酸适配体HG53、GH5、HG3在25℃下被强烈的抑制，且抑制作用至少持续40分钟。而在65℃时，RHAU23-Bst完全恢复活性。这表明以可折叠成G-四链体和“发夹”结构的单链DNA作为核酸适配体足以调节融合G-四链体结合肽的DNA聚合酶的活性。
由此可见，核酸适配体对修饰的DNA聚合酶的活性的调节受到核酸适配体中的旁侧DNA序列和基于G-四链体调控的热启动DNA聚合酶中的G-四链体结合肽的影响。并且，修饰的DNA聚合酶G4P-Bst和RHAU23-Bst的活性在25℃时被抑制，25℃是接近室温的温度，在该温度下聚合酶活性的抑制可以有效阻止非特异性扩增。
实施例四：
通过EMSA和引物延伸实验检测G-四链体和G-四链体结合肽对热启动DNA聚合酶的必要性。
参照实施例二的实验方法，通过EMSA方法检测DNA聚合酶G4P-Bst与非G4单链DNA和非G4双链DNA的结合能力，以及通过EMSA方法检测Bst-LF与T4B1 Fd-G4和T4B1 Fs-G4的结合能力。
参照实施例三的实验方法，通过引物延伸实验检测非G4单链DNA和非G4双链DNA存在时G4P-Bst的活性，以及通过引物延伸实验检测T4B1 Fd-G4和 T4B1 Fs-G4存在时G4P-Bst的活性。
图4示出了实验结果，其中，图4A为通过EMSA方法检测DNA聚合酶G4P-Bst与非G4单链DNA和非G4双链DNA的结合能力的结果图，图4B为通过EMSA方法检测Bst-LF与T4B1 Fd-G4和T4B1 Fs-G4的结合能力的结果图，图4C为通过引物延伸实验检测非G4单链DNA和非G4双链DNA存在时G4P-Bst的活性的结果图，图4D为通过引物延伸实验检测T4B1 Fd-G4和T4B1 Fs-G4存在时Bst-LF的活性结果图。
从图中可以看出，从核酸适配体中去除G-四链体或者从DNA聚合酶中去除G-四链体结合肽都显著降低了DNA聚合酶与核酸适配体的结合亲和力，均导致无法调节DNA聚合酶的活性。因此，核酸适配体中的G-四链体和DNA聚合酶中的G-四链体结合肽是控制热启动DNA聚合酶的两个不可或缺的部分。
实施例五：
通过引物延伸实验验证核酸适配体抑制修饰的DNA聚合酶的能力可通过G-四链体类型和“发夹”结构的长度进行调节。
5.1在温度为25℃下，通过引物延伸实验检测含有不同G-四链体类型的HG53核酸适配体存在下修饰的DNA聚合酶RHAU23-Bst的活性。
本实验中，选用四种不同G-四链体类型的八种核酸适配体进行实验，八种核酸适配体分别为CSTB、Kit-C、PDGFRB、T1B1、T4B1、GVBQ1、GVBQ2和G12。其中，核酸适配体CSTB和Kit-C的G-四链体类型为规则的三层G-四链体，核酸适配体PDGFRB、T1B1和T4B1的G-四链体类型为凸起的G-四链体，核酸适配体GVBQ1和GVBQ2的G-四链体类型为带有G-空位的G-四链体，核酸适配体G12的G-四链体类型为规则的两层G-四链体。RHAU23-Bst与核酸适配体的摩尔浓度比为1:2。
结果如图5A所示，从图中可以看出，所有八种核酸适配体均能有效抑制修饰的DNA聚合酶的活性。其中，含有非规则G-四链体的核酸适配体(T4B1、GVBQ1和GVBQ2)和含有非稳定的双层G-四链体的核酸适配体(G12)都可以抑制修饰的DNA聚合酶的活性至少40分钟。
5.2在温度为25℃下，通过引物延伸实验检测含有不同“发夹”结构长度的HG53核酸适配体存在下修饰的DNA聚合酶RHAU23-Bst的活性。
本实验中，分别选用“发夹”结构长度为8bp、7bp、6bp、5bp和4bp的HG53核酸适配体，RHAU23-Bst与核酸适配体的摩尔浓度比为1:2。
结果如图5B所示，其中H8表示“发夹”结构长度为8bp的核酸适配体，H7表示“发夹”结构长度为7bp的核酸适配体，H6表示“发夹”结构长度为6bp的核酸适配体，H5表示“发夹”结构长度为5bp的核酸适配体。从图中可以看出，“发夹”区域的长度至少需要7bp，核酸适配体才能有效发挥作用。
5.3在温度为25℃下，通过引物延伸实验检测含有不同环长的HG53核酸适配体存在下修饰的DNA聚合酶RHAU23-Bst的活性。
与GH5和HG3相比，HG53有一个额外的环连接G-四链体和“发夹”结构。该环主要影响DNA末端“发夹”结构的形成，本实施例进行的实验中，分别取环长度为2nt、4nt、6nt、8nt、10nt和12nt的HG53核酸适配体进行实验，RHAU23-Bst与核酸适配体的摩尔浓度比为1:2。
结果如图5C所示，从图中可以看出，该环的长度需要为6nt或更多。另外，以删除G-四链体或者突变其序列的核酸适配体进行实验，发现删除G-四链体或突变其序列使核酸适配体不再起到抑制修饰的DNA聚合酶的作用，可见G-四链体是适配体的关键成分。
实施例六：
核酸适配体在不同温度下对Bst DNA聚合酶活性抑制能力的检测。
参照实施例三的实验方法，通过引物延伸实验测试在不同温度下核酸适配体HG53-GVBQ1和HG53-H7对三种Bst DNA聚合酶的抑制作用。其中，三种Bst DNA聚合酶分别为Bst-LF、Bst 2.0热启动DNA聚合酶(Bst 2.0热启动DNA聚合酶是NEB公司的商品化的热启动DNA聚合酶)和修饰的DNA聚合酶RHAU23-Bst；检测温度为25℃-65℃；Bst-LF、RHAU23-Bst反应终浓度为100nM，Bst 2.0热启动DNA聚合酶为0.32U/μL；核酸适配体使用浓度为200nM；反应缓冲液为第二缓冲液，包括20mM Tris-HCl(pH 8.8)、10mM(NH 4) 2SO 4、50mM KCl、8mM MgSO 4和0.1％吐温-20。
图6为本实施例的实验结果图，请参阅图6A至图6C，在没有核酸适配体结合的情况下，三种Bst DNA聚合酶(Bst-LF、Bst 2.0热启动DNA聚合酶和RHAU23-Bst)在25℃-65℃合成DNA时表现出相似的活性。请参阅图6D和图6E，RHAU23-Bst与核酸适配体HG53-H7和HG53-GVBQ1结合后的热启动酶的活性在低于30℃以下受到极大抑制。图6F示出了通过比较扩增后的全长产物与RHAU23-Bst的分数来确RHAU23-Bst在核酸适配体HG53-H7存在的情况下的标准化活性结果图，从图中可以看出，在低于30℃时，核酸适配体HG53-H7使RHAU23-Bst的活性抑制了80％以上，在高于35℃时，RHAU23-Bst的活性逐渐恢复，并在高于50℃时，RHAU23-Bst的活性恢复到90％以上。从实验结果可以看出，结合核酸适配体的RHAU23-Bst的活性可以通过升高温度(热启动)来精确启动，在低温下有效抑制非特异性扩增；而商品化的Bst 2.0热启动DNA聚合酶的活性在低温下几乎没有被抑制。因此，本申请提供的修饰的DNA聚合酶有助于其在检测核酸方面的广泛应用。
实施例七：
热启动DNA聚合酶用于检测人乳头瘤病毒(HPV)DNA。
通过环介导等温扩增(LAMP)进行检测，其中，LAMP为现有技术，在此不再详述。本实验中，将HPV16的E6-E7基因的质粒十倍梯度稀释直到1拷贝每μL，根据E6-E7基因的DNA序列设计如表4所示的引物，LAMP反应中引物使用终浓度为FIP/BIP 1.6μΜ，Loop-F/Loop-B 0.4μΜ，F3/B3 0.2μΜ。RHAU23-Bst反应终浓度为100nM。Bst 2.0热启动DNA聚合酶(NEB)为0.32U/μL。适配体使用浓度为200nM。Bst-LF和商品化的Bst 2.0热启动DNA聚合酶为对照。如图7A所示，在Bst-LF存在的情况下，LAMP反应的扩增曲线在50分钟内荧光没有增加，表明Bst-LF未能检测到HPV16的DNA序列。相比之下，请参阅图7B和图7C，当DNA聚合酶RHAU23-Bst与核酸适配体HG53-H7的DNA和蛋白结合的复合物用于LAMP反应时，或者商品化的Bst 2.0热启动DNA聚合酶(图中用warm-start表示)用于LAMP反应时，典型的扩增曲线根据模板DNA的浓度依次出现。由于RHAU23-Bst的活性受温度控制的限制，修饰的DNA聚合酶RHAU23-Bst与核酸适配体HG53-H7的DNA和蛋白结合的复合物可以在室温下放置8小时而不会影响后续LAMP反应的准确性，详见图7B。相反，随着在室温下放置时间的增加，在Bst 2.0热启动DNA聚合酶存在下的样品的扩增信号逐渐降低，并且需要更长的时间才能达到平台期，详见图7C。因此，Bst 2.0热启动DNA聚合酶不能有效防止低温下非特异性扩增引起的引物耗尽，这与实施例六中图6B中的结果一致。
表4
名称 序列(5’-3’)
FIP CCGACCCCTTATATTATGGAATATGGTGTATTAACTGTCAAAAGCCA(Seq ID No.45)
BIP CGGTCGATGTATGTCTTGTTGTTATGCAATGTAGGTGTATCTCCA(Seq ID No.46)
Loop-F CTTTTTGTCCAGATGTCTTTGCT(Seq ID No.47)
Loop-B CAAGAACACGTAGAGAAACCCAG(Seq ID No.48)
F3 AGAGATGGGAATCCATATGCTG(Seq ID No.49)
B3 ATCTATTTCATCCTCCTCCTCTG(Seq ID No.50)
实施例八：
热启动DNA聚合酶用于检测SARS-CoV-2。
RT-LAMP：在RT-LAMP反应体系中加入5U WarmStart RTx逆转录酶(NEB)，100nM核酸适配体组装的热启动DNA聚合酶以及0.5×gelgreen，热启动DNA聚合酶的修饰的DNA聚合酶为RHAU23-Bst，核酸适配体为HG53-H7，修饰的DNA聚合酶与核酸适配体摩尔比为1:2。其中，RT-LAMP为现有技术，在此不再详述。本实验中，所用引物如表5所示，引物浓度：FIP/BIP为1.6μM，F3/B3为0.2μM，Loop-F/B为0.4μM。扩增反应在65℃下在QuantStudio 7 Flex(Thermo Scientific)的实时荧光定量PCR系统上进行。
pH介导的比色RT-LAMP：在pH介导的比色RT-LAMP反应体系中加入5U WarmStart RTx逆转录酶(NEB)，100nM核酸适配体组装的热启动DNA聚合酶以及0.5×gelgreen，热启动DNA聚合酶的修饰的DNA聚合酶为RHAU23-Bst，核酸适配体为HG53-H7，修饰的DNA聚合酶与核酸适配体的摩尔比为1:2。反应在65℃下进行指定时间，并通过甲酚红的颜色变化进行检测。
图8A为使用荧光RT-LAMP检测具有指定拷贝数的SARS-CoV-2假病毒RNA的结果图；图8B为用比色RT-LAMP检测SARS-CoV-2假病毒RNA的结果图，其中，SARS-CoV-2假病毒RNA的拷贝数与荧光RT-LAMP检测中的相同。
如图8A所示，核酸适配体HG53-H7结合RHAU23-Bst或现有的热启动Bst DNA聚合酶(WarmStart 2.0 Bst)可有效防止非特异性扩增产生的假阳性结果。从图8B中可以看出，比色RT-LAMP也显示了热启动Bst DNA聚合酶在防止非特异性扩增产生的假阳性结果方面的优势。
表5
Figure PCTCN2022082834-appb-000006
实施例九：
G-四链体结合肽和含有G-四链体的核酸适配体的组合策略适用于Taq DNA聚合酶。
参照实施例一的构建方法构建DNA聚合酶RHAU23-Taq。其中，将质粒pCold-I-RHAU23-Bst中编码Bst DNA聚合酶大片段的序列替换成编码Taq DNA聚合酶的基因序列，构建质粒pCold-I-RHAU23-Taq，其中，pCold-I-RHAU23-Taq质粒图谱见图9A所示。
参照实施例三的方法对RHAU23-Taq的活性进行检测。其中，RHAU23-Taq反应终浓度为100nM，HG53-H7 200nM。以不加核酸适配体的样品为对照，在不同温度下进行引物延伸30分钟。反应缓冲液为第二缓冲液，包括20mM Tris-HCl(pH 8.8)、10mM(NH 4) 2SO 4、50mM KCl、2mM MgSO 4和0.1％吐温-20。
在不同温度下，RHAU23-Taq结合含G-四链体的核酸适配体后，RHAU23-Taq的相对活性通过比较其全长(图中用FL表示)延伸产物与RHAU23-Taq的全长产物比例来计算。如图9B所示，RHAU23-Taq在25℃～65℃下都显示有活性。请参阅图9C和9D，当200nM HG53-H7存在时，RHAU23-Taq 的活性在35℃下几乎全部抑制；当温度升高35℃以上时，RHAU23-Taq的活性逐渐恢复，特别是当温度升高50℃以上时，RHAU23-Taq的活性几乎完全恢复。因此，G-四链体结合肽和含有G-四链体的核酸适配体的结合策略适用于Taq DNA聚合酶，可以将Taq DNA聚合酶改造为修饰的DNA聚合酶，使其具备热启动能力。
可以理解，在其他实施例中，通过构建质粒pCold-I-G4P-Taq来构建修饰的DNA聚合酶G4P-Taq也是可以的。
实施例十：
G-四链体结合肽和含有G-四链体的核酸适配体的结合策略适用于逆转录酶。
参照实施例一的构建方法构建逆转录酶RHAU23-RT。其中，将质粒pCold-I-RHAU23-Bst中编码Bst大片段的序列替换成编码MMLV逆转录酶(RT)的基因序列，构建质粒pCold-I-RHAU23-RT，其中pCold-I-RHAU23-RT质粒图谱见图10A所示。
参照实施例三的方法进行逆转录引物延伸实验。其中，使用RNA为模板(RNA模板的核苷酸序列如Seq ID No.14)，与RNA配对的DNA为引物(DNA引物的核苷酸序列如Seq ID No.15)。RHAU23-RT反应终浓度为100nM，HG53-H7 200nM。反应缓冲液为第二缓冲液，包括20mM Tris-HCl(pH 8.8)、10mM(NH 4) 2SO 4、50mM KCl、8mM MgSO 4和0.1％吐温-20。以未融合G-四链体结合肽以及未添加核酸适配体的样品为对照，在不同温度下进行引物延伸15分钟。
请参阅图10C，现有的MMLV逆转录酶在25℃-65℃之间具有逆转录酶活性。请参阅图10B和图10D，当使用G-四链体结合肽和含有G-四链体的核酸适 配体的结合策略对现有的MMLV逆转录酶进行改造之后，改造后的逆转录酶在30℃以下的逆转录活性被强烈抑制，当温度升高到35℃以上，改造后的逆转录活性能够大部分恢复，特别是在温度为50℃～60℃之间，改造后的逆转录活性恢复的更佳。因此，G-四链体结合肽和含有G-四链体的核酸适配体的结合策略适用于逆转录酶，可以将逆转录酶改造为修饰的逆转录酶，使其具备热启动的性质。
可以理解，在其他实施例中，通过构建质粒pCold-I-G4P-RT来构建逆转录酶G4P-RT也是可以的。
综上所述，G-四链体结合肽和含有G-四链体的核酸适配体的结合策略适用于多种DNA聚合酶，可以将DNA聚合酶改造为修饰的DNA聚合酶，使其具备热启动的性质。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Figure PCTCN2022082834-appb-000007
Figure PCTCN2022082834-appb-000008
Figure PCTCN2022082834-appb-000009
Figure PCTCN2022082834-appb-000010
Figure PCTCN2022082834-appb-000011
Figure PCTCN2022082834-appb-000012
Figure PCTCN2022082834-appb-000013
Figure PCTCN2022082834-appb-000014
Figure PCTCN2022082834-appb-000015
Figure PCTCN2022082834-appb-000016
Figure PCTCN2022082834-appb-000017
Figure PCTCN2022082834-appb-000018
Figure PCTCN2022082834-appb-000019
Figure PCTCN2022082834-appb-000020
Figure PCTCN2022082834-appb-000021
Figure PCTCN2022082834-appb-000022
Figure PCTCN2022082834-appb-000023
Figure PCTCN2022082834-appb-000024
Figure PCTCN2022082834-appb-000025
Figure PCTCN2022082834-appb-000026
Figure PCTCN2022082834-appb-000027
Figure PCTCN2022082834-appb-000028
Figure PCTCN2022082834-appb-000029
Figure PCTCN2022082834-appb-000030
Figure PCTCN2022082834-appb-000031
Figure PCTCN2022082834-appb-000032

Claims (27)

  1. 一种修饰的DNA聚合酶，其特征在于，包括DNA聚合酶片段和融合于所述DNA聚合酶片段的N端的G-四链体结合肽。
  2. 根据权利要求1所述的DNA聚合酶，其特征在于，所述G-四链体结合肽为RHAU23肽，所述RHAU23肽的氨基酸序列如Seq ID No.1所示；或者，
    所述G-四链体结合肽为G4P，所述G4P的氨基酸序列如Seq ID No.2所示。
  3. 根据权利要求1或2所述的DNA聚合酶，其特征在于，所述DNA聚合酶片段耐受40℃以上的温度，且在温度高于40℃以上时具有活性。
  4. 根据权利要求3所述的DNA聚合酶，其特征在于，所述DNA聚合酶片段来自于Bst DNA聚合酶、Taq DNA聚合酶和MMLV逆转录酶中的任意一种。
  5. 根据权利要求4所述的DNA聚合酶，其特征在于，所述DNA聚合酶片段来自于Bst DNA聚合酶，所述DNA聚合酶片段的氨基酸序列如Seq ID No.3所示。
  6. 根据权利要求4所述的DNA聚合酶，其特征在于，所述DNA聚合酶片段来自于Taq DNA聚合酶，所述DNA聚合酶片段的氨基酸序列如Seq ID No.4所示。
  7. 根据权利要求4所述的DNA聚合酶，其特征在于，所述DNA聚合酶片段来自于MMLV逆转录酶，所述DNA聚合酶片段的氨基酸序列如Seq ID No.5所示。
  8. 一种用于调控如权利要求1至7任一项所述的DNA聚合酶活性的核酸适配体，其特征在于，所述核酸适配体的核苷酸序列包括G-四链体核心序列，所述G-四链体核心序列的二级结构为G-四链体；
    所述G-四链体用于在第一预设温度时结合于所述DNA聚合酶的G-四链体结合肽，以抑制所述DNA聚合酶的活性，并用于在第二预设温度时脱离于所述 DNA聚合酶的G-四链体结合肽，以恢复所述DNA聚合酶的活性；其中，所述第二预设温度高于所述第一预设温度。
  9. 根据权利要求8所述的核酸适配体，其特征在于，所述G-四链体为规则的三层G-四链体、凸起的G-四链体、带有G-空位的G-四链体和规则的两层G-四链体中的任意一种。
  10. 根据权利要求8所述的核酸适配体，其特征在于，所述核酸适配体的核苷酸序列还包括所述G-四链体核心序列的5’端和/或3’端的旁侧DNA序列。
  11. 根据权利要求10所述的核酸适配体，其特征在于，所述旁侧DNA序列为侧翼单链DNA序列或者侧翼双链DNA序列或者用于形成“发夹”结构的DNA序列。
  12. 根据权利要求10所述的核酸适配体，其特征在于，所述旁侧DNA序列优选为用于形成“发夹”结构的DNA序列，其中，形成的所述“发夹”结构的长度大于或等于7bp。
  13. 根据权利要求8所述的核酸适配体，其特征在于，所述第一预设温度为0℃～30℃，所述第二预设温度为45℃～70℃。
  14. 根据权利要求13所述的核酸适配体，其特征在于，所述第二预设温度为55℃～65℃。
  15. 根据权利要求8至14任一项所述的核酸适配体，其特征在于，所述G-四链体核心序列为CSTB核心序列，所述CSTB核心序列的核苷酸序列如Seq ID No.6所示；或者，
    所述G-四链体核心序列为KIT-C核心序列，所述KIT-C核心序列的核苷酸序列如Seq ID No.7所示；或者，
    所述G-四链体核心序列为T4B1核心序列，所述T4B1核心序列的核苷酸序 列如Seq ID No.8所示；或者，
    所述G-四链体核心序列为PDGFRB核心序列，所述PDGFRB核心序列的核苷酸序列如Seq ID No.9所示；或者，
    所述G-四链体核心序列为T1B1核心序列，所述T1B1核心序列的核苷酸序列如Seq ID No.10所示；或者，
    所述G-四链体核心序列为GVBQ1核心序列，所述GVBQ1核心序列的核苷酸序列如Seq ID No.11所示；或者，
    所述G-四链体核心序列为GVBQ2核心序列，所述GVBQ2核心序列的核苷酸序列如Seq ID No.12所示；或者，
    所述G-四链体核心序列为G12核心序列，所述G12核心序列的核苷酸序列如Seq ID No.13所示。
  16. 一种热启动DNA聚合酶，其特征在于，包括如权利要求1至7任一项所述的DNA聚合酶和如权利要求8至15任一项所述的核酸适配体，所述核酸适配体的G-四链体在第一预设温度结合于所述DNA聚合酶的G-四链体结合肽，以抑制所述DNA聚合酶的活性，并在第二预设温度脱离于所述DNA聚合酶的G-四链体结合肽，以恢复所述DNA聚合酶的活性；其中，所述第二预设温度高于所述第一预设温度。
  17. 根据权利要求16所述的热启动DNA聚合酶，其特征在于，所述第一预设温度为0℃～30℃，所述第二预设温度为45℃～70℃。
  18. 一种试剂盒，其特征在于，包括如权利要求1至7任一项所述的DNA聚合酶和如权利要求8至15任一项所述的核酸适配体。
  19. 根据权利要求18所述的试剂盒，其特征在于，所述试剂盒用于检测人乳头瘤病毒DNA或者SARS病毒RNA。
  20. 一种生物合成如权利要求1至7任一项所述的DNA聚合酶的方法，其特征在于，包括如下步骤：
    将DNA聚合酶片段插入质粒载体，构建第一质粒；
    在所述第一质粒的基础上，将G-四链体结合肽的编码序列插入所述DNA聚合酶片段的N端，构建第二质粒；
    将所述第二质粒转化到大肠杆菌菌株中进行培养并诱导蛋白质表达；
    纯化蛋白质，获得所述的DNA聚合酶。
  21. 根据权利要求20所述的方法，其特征在于，所述DNA聚合酶片段来自于嗜热脂肪芽孢杆菌的DNA聚合酶的291-878残基，所述质粒载体为pCold-I，所述第一质粒为pCold-I-Bst-LF质粒；
    所述G-四链体结合肽为G4P，所述第二质粒为pCold-I-G4P-Bst质粒；或者，所述G-四链体结合肽为RHAU23，所述第二质粒为pCold-I-RHAU23-Bst质粒。
  22. 根据权利要求20所述的方法，其特征在于，所述DNA聚合酶片段来自于Taq DNA聚合酶，所述质粒载体为pCold-I，所述第一质粒为pCold-I-Taq-LF质粒；
    所述G-四链体结合肽为G4P，所述第二质粒为pCold-I-G4P-Taq质粒；或者，所述G-四链体结合肽为RHAU23，所述第二质粒为pCold-I-RHAU23-Taq质粒。
  23. 根据权利要求20所述的方法，其特征在于，所述DNA聚合酶片段来自于MMLV逆转录酶，所述质粒载体为pCold-I，所述第一质粒为pCold-I-RT-LF质粒；
    所述G-四链体结合肽为G4P，所述第二质粒为pCold-I-G4P-RT质粒；或者，所述G-四链体结合肽为RHAU23，所述第二质粒为pCold-I-RHAU23-RT质粒。
  24. 一种制备热启动DNA聚合酶的方法，所述热启动DNA聚合酶包括如 权利要求1至7任一项所述的DNA聚合酶和如权利要求8至15任一项所述的核酸适配体，所述核酸适配体通过所述G-四链体结合于所述G-四链体结合肽而结合于所述DNA聚合酶，其特征在于，所述方法包括如下步骤：
    将所述核酸适配体溶解于第一缓冲液中，在90℃～100℃变性2分钟～8分钟后，冷却至20℃～30℃，获得处理后的核酸适配体；
    将所述DNA聚合酶和处理后的核酸适配体按预设的摩尔浓度比加入第二缓冲液中，在2℃～6℃下孵育30min～60min；
    其中，所述第一缓冲液包括10mM pH 7.4 Tris-HCl、75mM KCl、0.5mM EDTA和0.2mg/ml牛血清白蛋白，所述第二缓冲液包括20mM pH 8.8 Tris-HCl、10mM(NH 4) 2SO 4、50mM KCl、2-8mM MgSO 4和0.1％吐温-20。
  25. 根据权利要求24所述的方法，其特征在于，所述DNA聚合酶和所述核酸适配体的预设的摩尔浓度比的范围为1:8～1:1。
  26. 如权利要求1至7任一项所述的DNA聚合酶和如权利要求8至15任一项所述的核酸适配体相配合用于检测核酸或者合成核酸的应用。
  27. 根据权利要求26所述的应用，其特征在于，在应用于检测核酸时，所述核酸为人乳头瘤病毒DNA或者SARS-CoV-2病毒RNA。
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