WO2023123347A1 - 解旋酶bch1x及其用途 - Google Patents

解旋酶bch1x及其用途 Download PDF

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WO2023123347A1
WO2023123347A1 PCT/CN2021/143662 CN2021143662W WO2023123347A1 WO 2023123347 A1 WO2023123347 A1 WO 2023123347A1 CN 2021143662 W CN2021143662 W CN 2021143662W WO 2023123347 A1 WO2023123347 A1 WO 2023123347A1
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helicase
amino acid
pore
polynucleotide
complex structure
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French (fr)
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徐讯
郭斐
胡巧霞
李登辉
陈俊毅
王乐乐
赵子裕
季州翔
曾涛
王欧
黎宇翔
董宇亮
章文蔚
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深圳华大生命科学研究院
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Priority to PCT/CN2021/143662 priority Critical patent/WO2023123347A1/zh
Priority to CN202180104688.6A priority patent/CN118355130A/zh
Priority to US18/723,588 priority patent/US20250066747A1/en
Priority to EP21969698.6A priority patent/EP4458987A1/en
Publication of WO2023123347A1 publication Critical patent/WO2023123347A1/zh

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C12Y306/04012—DNA helicase (3.6.4.12)

Definitions

  • the invention relates to the field of biology, in particular to a helicase and its application. More specifically, the present invention relates to the helicase BCH1X and its use in the control and characterization of nucleic acids and nanopore sequencing.
  • Nanopore sequencing is a third-generation sequencing technology emerging in recent years. Due to its advantages of long read length, high throughput, low cost, and portability, it has brought disruptive changes to the gene sequencing industry. Nanopore sequencing technology has a wide range of applications in basic theoretical research of life sciences and clinical practice of biomedicine.
  • Nanopore sequencing is a sequencing technology based on electrical signals. It uses single-stranded nucleic acid molecules as sequencing units, and a nanopore (protein or solid state) inserted in the membrane separates two electrolytic chambers filled with electrolyte. When a voltage is applied between the two electrolytic chambers, a steady perforation current is generated. Different molecules entering the nanopore create a barrier to the flow of ions, known as the nanopore signal. When the nucleic acid passes through the nanopore, due to the difference of the base, the size of the current resistance will be different. By detecting the current fluctuation signal of the nanopore and analyzing the current signal through machine learning, the sequence of the nucleic acid that is perforated is determined.
  • Nanopore sequencing technology has the following advantages: it can easily build a library without amplification; the reading speed is fast, and the reading speed of single-stranded molecules can reach tens of thousands of bases per hour; the reading length is longer, usually Up to thousands of bases; direct measurement of methylated DNA or RNA.
  • nanopore sequencing Due to the extremely fast speed of nucleic acid molecules passing through the nanopore channel, it is impossible to accurately obtain polynucleotide sequence information. Therefore, effectively reducing and controlling the movement of nucleic acid molecules through the nanopore is a key technical issue in realizing nanopore sequencing.
  • the movement of nucleic acid molecules through the nanopore can be controlled by the idea of polymerase amplification or helicase unwinding, and the detection accuracy can be improved by increasing the residence time of nucleic acid molecules at the nanopore.
  • the helicase needs to have better salt tolerance and stability in order to maintain better sequencing speed and sequencing uniformity.
  • the helicase in current commercial nanopore sequencers is generally DDA helicase derived from bacteriophage T4, which has poor yield, stability and salt tolerance. There is a need in the art for novel helicases.
  • An object of the present invention is to provide a new helicase for the characterization of nucleic acids, thereby solving the problem of low salt tolerance and low stability of the conventional helicase, and improving the yield of the helicase for recombinant expression, obviously Improve the accuracy of polynucleotide characterization.
  • the present invention provides a new type of helicase BCH1X, which is screened from a deep-sea metagenomic library (from Shenzhen National Gene Bank).
  • the helicase The gyrase BCH1X is extremely stable and salt tolerant.
  • this type of helicase has a high expression level in the E. coli recombinant protein expression system, and the yield is extremely large.
  • this new type of helicase has a special pin structure, which makes it have good single-stranded DNA binding and double-stranded DNA unwinding activities.
  • the helicase can be used for the control and characterization of nucleic acid, and can be applied to single-molecule nanopore sequencing.
  • the helicase BCH1X comprises:
  • said amino acid difference comprises amino acid substitution, deletion and/or insertion or N-terminal and/or C-terminal extension, provided that the amino acid sequence having said amino acid difference retains that of SEQ ID NO: 1 or 2 Helicase activity.
  • the amino acid substitution is a conservative amino acid substitution.
  • the helicase BCH1X comprises the following amino acid fragments:
  • X represents any amino acid residue
  • Y1 A or G
  • Y2 T or S
  • Y3 V or L
  • Y4 F or Y.
  • the amino acid fragment is a key region where the helicase BCH1X exhibits better performance in sequencing. Specifically, the amino acid fragment is a relatively important segment related to the unwinding speed in the protein structure.
  • the helicase BCH1X consists of the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the helicase BCH1X shown in SEQ ID NO: 1 is named BCH105
  • the helicase BCH1X shown in SEQ ID NO: 2 is named BCH178.
  • the helicase or its complex structure of the present invention can move the target polynucleotide through the nanopore in a controllable and step-by-step manner through the magnetic field generated by the applied voltage, thereby controlling the rate at which the polynucleotide passes through the nanopore, and obtaining resolved current levels.
  • the helicase BCH1X or its complex structure will function efficiently under high salt concentration and have extremely high stability.
  • the helicase or its complex structure of the present invention has a very high expression level when expressed recombinantly (for example, expressed in Escherichia coli), and it is easy to obtain high yield.
  • the present invention provides a nucleotide sequence encoding the helicase BCH1X of the first aspect.
  • the nucleotide sequence encoding the helicase BCH1X comprises a nucleotide sequence encoding the following amino acid sequence:
  • nucleotide sequence encoding the helicase BCH1X comprises a nucleotide sequence encoding the following amino acid fragments:
  • X represents any amino acid residue
  • Y1 A or G
  • Y2 T or S
  • Y3 V or L
  • Y4 F or Y.
  • the nucleotide sequence can be codon-optimized for the cell used for expression to obtain a desired expression level in the cell.
  • the present invention provides a recombinant vector comprising the nucleotide sequence of the second aspect of the present invention.
  • the recombinant vector is a recombinant expression vector, eg, a recombinant expression vector suitable for expression in prokaryotic cells or eukaryotic cells.
  • the prokaryotic cells include, but are not limited to: Escherichia coli cells, Bacillus subtilis cells and the like.
  • the eukaryotic cells include, but are not limited to: yeast cells, insect cells, mammalian cells (eg, CHO cells, HEK293 cells) and the like.
  • the recombinant expression vector is suitable for expressing the helicase BCH1X in Escherichia coli cells.
  • a purification tag can be added to the recombinant helicase.
  • the label can be removed by chemical method or enzymatic reaction.
  • the present invention provides a cell comprising the nucleotide sequence of the second aspect or the recombinant vector of the third aspect.
  • the cells may be prokaryotic cells or eukaryotic cells, such as Escherichia coli cells, Bacillus subtilis cells, yeast cells, insect cells, mammalian cells (eg, CHO cells, HEK293 cells) and the like.
  • the present invention provides a complex structure comprising the helicase BCH1X described in the first aspect and a binding portion for binding polynucleotides, wherein the helicase BCH1X is attached to On the binding moiety, the complex structure is capable of controlling the sequence of polynucleotides.
  • the complex structure is a natural structure or a non-natural structure.
  • the complex structure is an artificial non-natural structure.
  • the binding moiety may be a binding moiety that binds to a base of a polynucleotide, and/or a binding moiety that binds to a sugar of a polynucleotide, and/or a binding moiety that binds to a phosphate in a polynucleotide.
  • a binding moiety that binds to a base of a polynucleotide
  • a binding moiety that binds to a sugar of a polynucleotide and/or a binding moiety that binds to a phosphate in a polynucleotide.
  • the complex structure of the present invention is an effective tool for controlling the movement of polynucleotides in the sequencing process.
  • the complex structure comprising the helicase described in the present invention is stable in combination with the polynucleotide, and will not be separated during the sequencing process.
  • This complex structure can provide longer read lengths of polynucleotides while controlling the translocation of polynucleotides through the nanopore.
  • bound polynucleotides in the binding fraction are compatible with strand sequencing and polynucleotide characterization processes.
  • the binding part is more active at high salt concentration (such as 0.3-1M KCl), which is due to its good salt tolerance, and the improvement of the binding part of the complex structure can improve the synthesis ability, Stability and half-life.
  • the helicase and binding moiety are bound via each other's terminal amino acids.
  • the amino terminus of the binding moiety is bound to the carboxy terminus of the helicase or the carboxy terminus of the binding moiety is bound to the amino terminus of the helicase.
  • the binding moiety is inserted into the sequence of the helicase. Such a structure can well combine the helicase and the binding part through two points.
  • a tag was added to the complex structure.
  • the label needs to be removed, it can be removed by chemical method or enzymatic reaction.
  • the present invention provides the use of the helicase BCH1X in the control and characterization of nucleic acids, or in single-molecule nanopore sequencing.
  • the helicase BCH1X or its complex structure of the present invention can control the moving rate of the target polynucleotide through the nanopore.
  • the present invention provides a method of controlling and characterizing a polynucleotide of interest, said method comprising the steps of:
  • the helicase is the helicase BCH1X described in the first aspect of the present invention
  • the complex structure comprises the helicase BCH1X and a binding part for binding polynucleotides
  • the one or more features may be selected from, but not limited to: changes in the magnitude of the current signal, changes in the duration of the current signal, changes in the magnitude and duration of the voltage signal, and the like.
  • the method of controlling and characterizing a polynucleotide of interest is a method of single-molecule nanopore sequencing.
  • the present invention provides a kit for controlling and characterizing a target polynucleotide, said kit comprising a helicase BCH1X or complex structure thereof and a pore.
  • the pores are nanopores.
  • the kit comprises a plurality of helicases or a plurality of complex structures, and a plurality of wells.
  • the pore is a transmembrane pore
  • the transmembrane pore is a biological pore, a solid-state pore, or a pore hybridized between a biological and a solid state.
  • the biological pores are selected from the group consisting of ⁇ -hemolysin protein ( ⁇ -HL), Mycobacterium smegmatis porin A (MspA), curli-specific transport channel protein (CsgG), type III secretion system protein ( InvG) and so on.
  • the kit further comprises a chip comprising a lipid bilayer.
  • the pores span the lipid bilayer.
  • the kit of the present invention comprises one or more lipid bilayers, each lipid bilayer comprising one or more of said pores.
  • kits of the present invention also include reagents or devices for performing the characterization of the target polynucleotide.
  • the reagents include buffers, enzymes or buffers required for PCR amplification.
  • the kit is a kit for single molecule nanopore sequencing.
  • the present invention also provides a sensor for characterizing a target polynucleotide, comprising a complex formed between a pore and a helicase BCH1X or a complex structure thereof, the target polynucleotide interacts with the pore, and thereby A sensor is formed for characterizing the polynucleotide of interest.
  • the pore and the helicase BCH1X or complex structure thereof are contacted in the presence of the polynucleotide of interest and an electrical potential is applied across the pore.
  • the potential is selected from a voltage potential or a chemical potential.
  • said pore is covalently linked to said helicase or said complex structure.
  • the present invention provides a device for characterizing a target polynucleotide, the device comprising a helicase BCH1X or a complex structure thereof, and a pore.
  • the device comprises sensor means supporting the plurality of pores and capable of transmitting signals of interaction of the pores with the polynucleotide, and at least one memory for storing the target polynucleotide, and performing the characterization process. solution.
  • the device comprises a plurality of helicases or a plurality of complex structures, and a plurality of pores.
  • the pore is a transmembrane pore
  • the transmembrane pore is a biological pore, a solid-state pore, or a pore hybridized between biology and solid state.
  • the biological pores are selected from ⁇ -hemolysin protein ( ⁇ -HL), Mycobacterium smegmatis porin A (MspA), curli-specific transport channel protein (CsgG), type III secretion system protein (InvG) et al.
  • the present invention provides a method for preparing the helicase BCH1X or its complex structure described in the first aspect, the method comprising: constructing a recombinant protein that expresses the helicase BCH1X or its complex structure
  • the expression vector is transformed into an appropriate host cell for recombinant expression.
  • the host cells may be prokaryotic or eukaryotic cells, such as E. coli cells, Bacillus subtilis cells, yeast cells, insect cells, mammalian cells (eg, CHO cells, HEK293 cells), etc.
  • prokaryotic or eukaryotic cells such as E. coli cells, Bacillus subtilis cells, yeast cells, insect cells, mammalian cells (eg, CHO cells, HEK293 cells), etc.
  • nucleotides include but are not limited to: adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytosine nucleus glycoside monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate ( dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP).
  • the nucleotides are selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
  • the "conservative amino acid substitution” in the present invention refers to the substitution of one amino acid by another amino acid in the same category, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or a middle amino acid is replaced by another amino acid. A neutral amino acid is replaced by another neutral amino acid.
  • amino acids can be grouped by the properties of common side chains:
  • hydrophobicity norleucine, Met, Ala, Val, Leu, Ile;
  • a conservative amino acid substitution may refer to the substitution of one amino acid in the above group by another amino acid in the same group.
  • the conservative amino acid substitution will not substantially change the activity of the amino acid sequence described in the present invention.
  • Figure 1 shows the results of molecular sieve Superdex 200 purification of the helicase BCH105.
  • Lane M represents molecular weight markers, and lanes 1-9 represent fractions 1-9 purified by molecular sieves. Fractions 6-9 are selected and combined as protein samples to be tested for subsequent experiments.
  • Figure 2 shows the binding results of BCH105 to ssDNA.
  • Figure 3 shows the verification results of the unwinding activity of BCH105. Fluorescence quenching method was used to detect the unwinding double-stranded DNA activity of BCH105 protein. Annealing of a single-stranded DNA (labeled with the BHQ-1 quencher at the 3' end) and another single-stranded DNA (labeled with the FAM fluorescent group at the 5' end) to form a 5' protruding double-stranded DNA as a substrate, prokaryotic expression and Purify and obtain BCH105 protein.
  • Figure 4 shows a schematic diagram of adapters used to construct a sequencing library, a: sense strand (top strand); b: antisense strand (bottom strand).
  • Figure 5 shows a schematic diagram of the sequencing library containing BCH105, a: sense strand (top strand); b: antisense strand (bottom strand); c: double-stranded target fragment; d: BCH105; e: chol-ssDNA.
  • Fig. 6 shows the translocation diagram of the change of the perforation current of the nucleic acid fragment to be tested.
  • Fig. 7 shows the experimental results of comparing the helicase BCH105 and the helicase DDA known in the art under the same conditions.
  • A shows the statistical results of DNA library passing speed under the control of BCH105
  • B shows the statistical results of DNA library passing speed under the control of DDA.
  • the inventor screened two helicase BCH1X sequences from a deep-sea metagenomic library (from Shenzhen National Gene Bank): SEQ ID NO: 1, which was named BCH105; SEQ ID NO: 2, which was named BCH178. Both helicases contain stretches of amino acids:
  • X represents any amino acid residue
  • Y1 A or G
  • Y2 T or S
  • Y3 V or L
  • Y4 F or Y.
  • X represents any amino acid residue
  • Y1 A or G
  • Y2 T or S
  • Y3 V or L
  • Y4 F or Y.
  • Y1 A or G
  • Y2 T or S
  • Y3 V or L
  • Y4 F or Y.
  • the full-length BCH105 DNA sequence was ligated into the PET.28a(+) plasmid, and the double restriction sites used were Nde1 and Xho1, so the N-terminus of the expressed BCH105 protein had a 6*His tag and a thrombin restriction site .
  • Buffer A 20mM Tris-HCl pH 7.5, 250mM NaCl, 20mM imidazole;
  • Buffer B 20mM Tris-HCl pH 7.5, 250mM NaCl, 300mM imidazole;
  • Buffer C 20mM Tris-HCl pH 7.5, 50mM NaCl;
  • Buffer D 20mM Tris-HCl pH 7.5, 1000mM NaCl;
  • Buffer E 20mM Tris-HCl pH 7.5, 100mM NaCl;
  • thrombin thrombin
  • Amino acid sequence of helicase Dda (SEQ ID NO: 3): MTFDDLTEGQ KNAFNIVMKA IKEKKHHVTI NGPAGTGKTT LTKFIIEALI STGGTGIILA APTHAAKKIL SKLSGKEAST IHSILKINPV TYEENVLFEQ KEVPDLAKCR VLICDEVSMY DRKLFKILLS TIPPWCT IIG IGDNKQIRPV EPGENTAYIS PFFTHKDFYQ CELTEVKRSN APIIDVATDV RNGKWNYDKV VDGHGVRGFT GDTALRDFMV NYFSIVKSLD DLFENRVMAF TNKSVDKLNS IIRKKIFETD KDFIVGEIIV MQEPLFKTYK IDGKPVSEII FN NGQLVRII EAEYTSTFVK ARGVPGEYLI RHWDLTVETY GDDEYYREKI KIISSDEELY KFNLFLAKTA ETYKNWNKGG KAP
  • reaction system Mix the substrate ssDNA and BCH105 protein thoroughly in reaction buffer A.
  • the final concentration of the DNA substrate (ssDNA) was 20nM, and the final concentrations of the BCH105 protein used were 0nM, 20nM, 50nM, 100nM, and 500nM, respectively, wherein the reaction buffer A was: 50mM HEPES, 100mM KCl, pH 8.0.
  • the substrate ssDNA used was 3'cy3-labeled single-stranded DNA, and its sequence was TTTTTTTTTTTTCTGAATCACGTACTATATGACACAGTAAAT-cy3
  • reaction buffer solution B In the reaction buffer solution B, fully mix the double-stranded DNA substrate with 5'overhang (overhang), BCH105 protein, and unlabeled single-stranded capture DNA.
  • the final concentration of DNA substrate was 20 nM
  • the final concentration of protein was 100 nM
  • the final concentration of captured DNA was 400 nM.
  • the reaction buffer solution B is: 470mM KCl, 25mM HEPES, 2mM ATP, 10mM MgCl 2 , pH 8.0.
  • the 5' protruding double-stranded DNA is formed by annealing a single-stranded DNA labeled with a BHQ-1 quencher at the 3' end and a single-stranded DNA labeled with a FAM fluorescent group at the 5' end.
  • the sequences are:
  • the positive control group (positive) replaced the 5' protruding double-stranded DNA with the single-stranded DNA labeled with the FAM fluorescent group at the 5' end, and the others were the same as the experimental group.
  • Nuclease-Free Water was used to replace the protein in the negative control group (negative), and the others were the same as the experimental group.
  • reaction solution 40 ⁇ L was added to the microplate plate, and the FAM fluorescence was measured in real time using a microplate reader (excitation wavelength: 492 nm, emission wavelength: 518 nm), at a temperature of 30° C., and a total time of 30 min. Three replicate wells were performed for each group of samples.
  • the experimental results are shown in Figure 3: the fluorescence value of the positive control group (positive) remained unchanged during the measurement process, around 9500; the fluorescence value of the negative control group (negative) remained unchanged during the measurement process, around 2600 Left and right; the fluorescence value of the BCH105 experimental group increased gradually with the increase of the reaction time, and the fluorescence value of the BCH105 experimental group increased from 3200 (0min) to 5600 (30min).
  • the experimental results in Figure 3 show that BCH105 has the activity of unwinding double-stranded DNA, and the unwinding direction is 5'-3'.
  • a patch clamp amplifier or other electrical signal amplifier is used to acquire the current signal.
  • the Teflon membrane with micron-sized holes (50-200 ⁇ m in diameter) in the middle divides the electrolytic cell into two chambers, the cis chamber and the trans chamber. Place a pair of Ag/AgCl electrodes in the cis chamber and the trans chamber respectively.
  • Add nanoporin Mycobacterium smegmatis porin A (MspA, SEQ ID NO: 5), prepared according to Example 5) after forming a layer of bimolecular phospholipid film at the micropores of the two chambers; Electrical measurements were obtained after porin insertion into phospholipid membranes. After applying 180mV, the opening current of a single channel was obtained.
  • the opening current is about 220pA.
  • the DNA single strand of the sequencing library enters the nanopore, under the unwinding action of BCH105, the DNA passes through the nanopore, and part of the current is blocked and the current becomes smaller. Due to the different sizes of different nucleotides, the size of the hindered current is also different, so the fluctuating current signal can be seen.
  • Target sequencing nucleic acid sequence SEQ ID NO: 4
  • the hole passing speed is the time length for observing the change of the current signal.
  • FIG. 7A shows the statistical results of the passing speed of the DNA library under the control of BCH105, with an average value of 242nt/s
  • Figure 7B shows the passing speed of the DNA library under the control of DDA.
  • the statistical result of the hole velocity, the average value is 161nt/s. This shows that under the control of BCH105, the passing speed of the DNA library is faster, indicating that the helicase of the present invention has higher sequencing speed, better salt tolerance and better stability.
  • Embodiment 5 Preparation of MspA protein
  • the gene sequence encoding the MspA protein was inserted into the cloning region of the vector pET24a, so the N-terminus of the expressed MspA protein had 6 His, which could be used as a purification tag.
  • the tag is kanamycin, and the constructed expression vector is named pET24a-MspA.
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • the recombinant expression vector pET24a-MspA was transformed into the Escherichia coli expression strain E.coli BL21(DE3), and the bacterial liquid was evenly spread on the LB solid culture plate with 50 ⁇ g/mL kanamycin, and cultivated overnight at 37°C. Pick a single colony and culture it in 5ml LB medium (containing 50 ⁇ g/mL kanamycin) at 37°C and 200rpm overnight. Inoculate the bacterial liquid obtained above into 50ml LB (containing 50 ⁇ g/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200rpm, for 4h.
  • OD600 value reaches about 0.6-0.8
  • add IPTG to a final concentration of 0.5mM , 18°C, 200rpm, cultivate overnight, about 16-18h.
  • the grown bacteria were collected by centrifugation at 8000 rpm, and the bacteria were frozen and stored at -20°C until use.
  • Buffer A1 (equilibrium buffer): 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol, pH 7.9;
  • Buffer B1 (elution buffer): 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol+500mM imidazole, pH 7.9.
  • Buffer C1 (equilibrium buffer): 20mM Tris-HCl+50mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Buffer D1 (elution buffer): 20mM Tris-HCl+1000mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Buffer E1 (diluent): 20mM Tris-HCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Amino acid sequence of MspA protein (SEQ ID NO: 5)

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Abstract

提供一种解旋酶BCH1X，其包含SEQ ID NO：1或2所示的氨基酸序列；还提供包含解旋酶BCH1X和用于结合多核苷酸的结合部分的复合体结构及其在控制和表征多核苷酸、单分子纳米孔测序方面的用途。

Description

解旋酶BCH1X及其用途 技术领域
本发明涉及生物领域，具体涉及一种解旋酶及其用途。更具体地，本发明涉及解旋酶BCH1X及其在核酸的控制和表征以及纳米孔测序中的用途。
背景技术
纳米孔测序为近些年新兴起的第三代测序技术，由于其长读长、高通量、低成本和便携性等优势，给基因测序行业带来了颠覆性的改变。纳米孔测序技术在生命科学基础理论研究以及生物医学临床实践中具有广泛的应用。
纳米孔测序(nanopore sequencing)是基于电信号的测序技术。其以单链核酸分子作为测序单元，由一个插在膜上的纳米孔(蛋白或固态)将两个装有电解液的电解室分开。当电压施加给两个电解室之间时，会产生稳定的穿孔电流。进入纳米孔的不同分子会对离子的流动造成阻碍，这被称为nanopore信号。当核酸穿过纳米孔时，由于碱基的不同，会造成电流阻碍的大小不同。通过检测纳米孔的电流波动信号，并通过机器学习分析该电流信号，从而测定得到穿孔的核酸的序列。纳米孔测序技术具有以下优势：在无需扩增的情况下，即可简便的建库；阅读速度快，单链分子的阅读速度能够达到每小时数万个碱基；阅读长度更长，通常可以达到数千个碱基；可以直接进行甲基化的DNA或RNA的测量。
由于核酸分子穿过纳米孔通道时速度极快，无法精确获得多核苷酸序列信息。因此有效地降低并控制核酸分子的穿过纳米孔的运动是实现纳米孔测序的关键技术问题。目前，可以通过聚合酶扩增或解旋酶解旋的思路控制核酸分子的穿过纳米孔运动，通过增加核酸分子在纳米孔处的停留时间而提高检测精度。同时，在纳米孔测序中，需要解旋酶具有较好的盐耐受性及稳定性，才能维持较好的测序速度和测序均一性。
然而，当前商品化的纳米孔测序仪中的解旋酶一般为来源于细菌噬菌体T4的DDA解旋酶，其产量、稳定性及盐耐受性均不佳。本领域需要新型的解旋酶。
发明内容
本发明的一个目的是提供一种新的解旋酶，用于核酸的表征，从而解决常规解旋酶耐盐性和稳定性不高的难题，并提高解旋酶进行重组表达的产量，明显提高多核苷酸特 性表征的精确性。
在第一方面，本发明提供了一类新型的解旋酶BCH1X，其从深海宏基因组文库(来源于深圳国家基因库)筛选得到，与现有技术中的解旋酶相比，所述解旋酶BCH1X具有极高的稳定性和盐耐受性。并且，这类解旋酶在大肠杆菌重组蛋白表达系统中具有很高的表达水平，产量极大。此外，这类新型解旋酶具有特殊的pin结构，使其具有良好的单链DNA结合和双链DNA解旋活性。所述解旋酶可以用于核酸的控制和表征，并应用于单分子纳米孔测序。
在一个实施方案中，所述解旋酶BCH1X包含：
(i)SEQ ID NO：1或2所示的氨基酸序列；或
(ii)与SEQ ID NO：1或2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性并且具有解旋酶活性的氨基酸序列；或
(iii)与SEQ ID NO：1或2所示的氨基酸序列具有不超过20、15、10、5、4、3、2或1个氨基酸差异并且具有解旋酶活性的氨基酸序列。
在一个实施方案中，所述氨基酸差异包括氨基酸取代、缺失和/或插入或N-端和/或C-端延伸，前提是具有所述氨基酸差异的氨基酸序列保留SEQ ID NO：1或2的解旋酶活性。优选地，所述氨基酸取代为保守氨基酸取代。
在一个优选的实施方案中，所述解旋酶BCH1X包含下述氨基酸片段：
GTIH X FLNLKLD XGF  Y1DDG Y2ADNV Y2TKXKLV Y3NK Y4NECL
其中：X表示任意氨基酸残基；Y1＝A或G；Y2＝T或S；Y3＝V或L；Y4＝F或Y。
所述氨基酸片段是解旋酶BCH1X在测序中表现出更好性能的关键区域。具体而言，所述氨基酸片段在蛋白质结构中是比较重要的解旋速度相关区段。
在一个实施方案中，所述解旋酶BCH1X由SEQ ID NO：1或2所示的氨基酸序列组成。
在一个具体的实施方案中，SEQ ID NO：1所示的解旋酶BCH1X命名为BCH105，SEQ ID NO：2所示的解旋酶BCH1X命名为BCH178。其中BCH105(SEQ ID NO：1)和BCH17(SEQ ID NO：2)都包含所述氨基酸片段。
本发明的解旋酶或其复合体结构能够以可控的、逐步的方式通过外加电压产生的磁场将目标多核苷酸移动穿过纳米孔，从而控制多核苷酸过纳米孔的速率，获得可分辨的电流水平。此外，解旋酶BCH1X或其复合体结构将在高盐浓度下有效发挥作用，并具有 极高的稳定性。
本发明的解旋酶或其复合体结构在重组表达时(例如，在大肠杆菌中表达)具有很高的表达水平，容易获得高产量。
在第二方面，本发明提供编码第一方面的解旋酶BCH1X的核苷酸序列。
在一个实施方案中，所述编码解旋酶BCH1X的核苷酸序列包含编码下述氨基酸序列的核苷酸序列：
(i)SEQ ID NO：1或2所示的氨基酸序列；或
(ii)与SEQ ID NO：1或2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性并且具有解旋酶活性的氨基酸序列；或
(iii)与SEQ ID NO：1或2所示的氨基酸序列具有不超过20、15、10、5、4、3、2或1个氨基酸差异并且具有解旋酶活性的氨基酸序列。
在一个优选的实施方案中，所述编码解旋酶BCH1X的核苷酸序列包含编码下述氨基酸片段的核苷酸序列：
GTIH X FLNLKLD XGF  Y1DDG Y2ADNV Y2TKXKLV Y3NK Y4NECL
其中：X表示任意氨基酸残基；Y1＝A或G；Y2＝T或S；Y3＝V或L；Y4＝F或Y。
在一个实施方案中，所述核苷酸序列可以针对用于表达的细胞进行密码子优化，以在所述细胞中获得理想的表达水平。
在第三方面，本发明提供包含本发明第二方面的核苷酸序列的重组载体。
在一个实施方案中，所述重组载体是重组表达载体，例如，适合在原核细胞或真核细胞中表达的重组表达载体。
所述原核细胞包括，但不限于：大肠杆菌细胞、枯草芽孢杆菌细胞等。
所述真核细胞包括，但不限于：酵母细胞、昆虫细胞、哺乳动物细胞(例如，CHO细胞、HEK293细胞)等。
优选地，所述重组表达载体适于在大肠杆菌细胞中表达解旋酶BCH1X。
优选地，为了使解旋酶纯化更便捷，可以在重组解旋酶中加入纯化标签。当需要去除标签时，可以通过化学方法或酶促反应脱去标签。
在第四方面，本发明提供包含第二方面所述的核苷酸序列或第三方面所述的重组载体的细胞。所述细胞可以是原核细胞或真核细胞，例如大肠杆菌细胞、枯草芽孢杆菌细胞、酵母细胞、昆虫细胞、哺乳动物细胞(例如，CHO细胞、HEK293细胞)等。
在第五方面，本发明提供一种复合体结构，所述复合体结构包含第一方面所述的解旋酶BCH1X和用于结合多核苷酸的结合部分，其中所述解旋酶BCH1X附着在所述结合部分上，所述复合体结构能够控制多核苷酸的序列。
优选地，所述的复合体结构为天然结构的或非天然结构。
在本发明的一个具体实施方式中，所述的复合体结构为人工制造的非天然结构。
优选地，所述结合部分可以为与多核苷酸的碱基结合的结合部分，和/或与多核苷酸的糖结合的结合部分，和/或与多核苷酸中的磷酸结合的结合部分。本领域技术人员能够根据具体需要选择适当的结合部分。
本发明所述的复合体结构是测序过程中控制多核苷酸移动的一种有效的工具。本发明所述的包含解旋酶的复合体结构与多核苷酸的结合稳定，在测序过程中不会脱离。当控制多核苷酸穿过纳米孔易位时，该复合体结构可以提供多核苷酸更大的读长。在缓冲液中，结合部分中结合多核苷酸与链测序和多核苷酸的表征过程是相匹配的。与在标准生理水平相比，结合部分在高盐浓度(例如0.3-1M KCl)下活性更佳，其原因在于其良好的耐盐性，对复合体结构的结合部分改进后可以提高合成能力、稳定性和半衰期。
优选地，解旋酶与结合部分是通过彼此的末端氨基酸结合。例如，结合部分的氨基末端与解旋酶的羧基末端结合或结合部分的羧基末端与解旋酶的氨基末端结合。进一步优选地，所述结合部分插入解旋酶的序列中。这样的结构可以将解旋酶与结合部分通过两点很好的结合在一起。
为使得复合体结构纯化的更容易，在复合体结构中加入标签。当需要去除标签时，可以通过化学方法或酶促反应脱去标签。
在第六方面，本发明提供解旋酶BCH1X在控制和表征核酸中的用途，或在单分子纳米孔测序中的用途。本发明的解旋酶BCH1X或其复合体结构能够控制目标多核苷酸穿过纳米孔的运动速率。
在第七方面，本发明提供一种控制和表征目标多核苷酸的方法，所述方法包括下述步骤：
(a)将目标多核苷酸与孔、和解旋酶或其复合体结构接触，使得所述解旋酶或其复合体结构控制所述目标多核苷酸穿过所述孔的运动；和
(b)获取目标多核苷酸中的核苷酸与所述孔相互作用时的一个或多个特征，以表征所述目标多核苷酸；
其中所述解旋酶为本发明第一方面所述的解旋酶BCH1X，所述复合体结构包含所述 解旋酶BCH1X和用于结合多核苷酸的结合部分，
所述一个或多个特征可以选自，但不限于：电流信号大小变化、电流信号时长变化、电压信号大小变化及电压信号时长变化等。
在一个实施方案中，所述控制和表征目标多核苷酸的方法是单分子纳米孔测序的方法。
在第八方面，本发明提供一种控制和表征目标多核苷酸的试剂盒，所述试剂盒包含解旋酶BCH1X或其复合体结构和孔。优选地，所述孔是纳米孔。
优选地，所述的试剂盒包括多个解旋酶或多个复合体结构，和多个孔。
优选地，所述孔为跨膜孔，所述跨膜孔为生物孔、固态孔或生物与固态杂交的孔。进一步优选地，所述生物孔选自α-溶血素蛋白(α-HL)、耻垢分枝杆菌孔蛋白A(MspA)、curli-特异性转运通道蛋白(CsgG)、III型分泌系统蛋白(InvG)等。
优选地，所述试剂盒还包括包含脂质双层的芯片。所述孔横跨脂质双层。
本发明所述的试剂盒包含一个或多个脂质双层，每个脂质双层包含一个或多个所述的孔。
本发明所述的试剂盒还包括实施表征目标多核苷酸的试剂或装置。优选地，所述试剂包括缓冲剂、PCR扩增所需的酶或缓冲液。
在一个实施方案中，所述试剂盒是用于单分子纳米孔测序的试剂盒。
在第九方面，本发明还提供一种表征目标多核苷酸的传感器，包括在孔和解旋酶BCH1X或其复合体结构之间形成的复合体，目标多核苷酸与孔相互作用，并由此形成用于表征目标多核苷酸的传感器。
优选地，在所述目标多核苷酸存在下使所述孔和解旋酶BCH1X或其复合体结构接触，并跨所述孔施加电势。所述电势选自电压电势或化学电势。
优选地，所述孔与所述解旋酶或所述复合体结构共价连接。
在第十方面，本发明提供一种表征目标多核苷酸的装置，所述装置包括解旋酶BCH1X或其复合体结构，和孔。
优选地，所述装置包括支撑所述多个孔并可传输孔与多核苷酸相互作用的信号的传感器装置，和至少一个用于存储目标多核苷酸的存储器，和实施表征过程中所需的溶液。
优选地，所述装置包括多个解旋酶或多个复合体结构，和多个孔。
优选地，所述孔为跨膜孔，所述的跨膜孔为生物孔、固态孔或生物与固态杂交的孔。进一步优选地，所述的生物孔选自α-溶血素蛋白(α-HL)、耻垢分枝杆菌孔蛋白A(MspA)、 curli-特异性转运通道蛋白(CsgG)、III型分泌系统蛋白(InvG)等。
在第十一方面，本发明提供一种制备第一方面所述的解旋酶BCH1X或其复合体结构的方法，所述方法包括：构建表达所述解旋酶BCH1X或其复合体结构的重组表达载体，转化到适当的宿主细胞中进行重组表达。
在一个实施方案中，所述宿主细胞可以是原核细胞或真核细胞，例如大肠杆菌细胞、枯草芽孢杆菌细胞、酵母细胞、昆虫细胞、哺乳动物细胞(例如，CHO细胞、HEK293细胞)等。
本领域技术人员能够根据实际需要选择合适的表达载体和宿主细胞。
本发明所述的“核苷酸”包括但不限于：腺苷单磷酸(AMP)、鸟苷单磷酸(GMP)、胸苷单磷酸(TMP)、尿苷单磷酸(UMP)、胞嘧啶核苷单磷酸(CMP)、环状腺苷单磷酸(cAMP)、环状鸟苷单磷酸(cGMP)脱氧腺苷单磷酸(dAMP)、脱氧鸟苷单磷酸(dGMP)、脱氧胸苷单磷酸(dTMP)、脱氧尿苷单磷酸(dUMP)和脱氧胞苷单磷酸(dCMP)。优选地，所述核苷酸选自AMP、TMP、GMP、CMP、UMP、dAMP、dTMP、dGMP或dCMP。
本发明所述的“保守氨基酸取代”是指一个氨基酸被同一类别内的另一氨基酸取代，例如一个酸性氨基酸被另一酸性氨基酸取代，一个碱性氨基酸被另一碱性氨基酸取代，或一个中性氨基酸被另一中性氨基酸取代。
例如，氨基酸可以按照常见侧链的性质进行分组：
(1)疏水性:正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性亲水性:Cys，Ser，Thr，Asn，Gln；
(3)酸性:Asp，Glu；
(4)碱性:His，Lys，Arg；
(5)影响链取向的残基:Gly，Pro；
(6)芳族的:Trp，Tyr，Phe；
保守氨基酸取代可以指上述分组中的一个氨基酸被同组的另一个氨基酸取代。所述的保守氨基酸取代基本不会改变本发明所述的氨基酸序列的活性。
示例性的保守氨基酸取代如下表A所示：
表A.示例性的取代和保守取代
Figure PCTCN2021143662-appb-000001
Figure PCTCN2021143662-appb-000002
附图说明
结合下述附图，本发明的实施方案和优点将更明显。
图1显示解旋酶BCH105的分子筛Superdex 200纯化结果。泳道M表示分子量标记，泳道1-9表示分子筛纯化级分1-9，选取级分6-9合并作为待测蛋白样品，用于后续实验。
图2显示BCH105对ssDNA的结合结果。
图3显示BCH105的解旋活性验证结果。荧光猝灭法检测BCH105蛋白解旋双链DNA活性。由一条单链DNA(3’末端BHQ-1猝灭基团标记)与另一条单链DNA(5’末端FAM荧光基团标记)退火形成5’突出的双链DNA为底物，原核表达并纯化获得BCH105蛋白。在含有ATP和Mg 2+的缓冲溶液中充分混匀DNA底物、蛋白和无标记的单链捕获DNA，30℃孵育60min，使用酶标仪实时测定FAM荧光。Mock为阴性对照(Negative control)，以相同浓度的5’末端FAM荧光标记的单链DNA为底物作为阳性对照(Positive control)。反应时间(Time，min)为横坐标，荧光强度(ΔRFI，a.u.)为纵坐标绘制散点图。
图4显示用于构建测序文库的接头示意图，a：正义链(top strand)；b：反义链(bottom strand)。
图5显示含有BCH105的测序文库的示意图，a：正义链(top strand)；b：反义链(bottom strand)；c：双链目的片段；d：BCH105；e：chol-ssDNA。
图6显示待测核酸片段穿孔电流变化易位图。
图7显示解旋酶BCH105与本领域已知的解旋酶DDA在相同条件下进行比较实验结果。(A)显示在BCH105控制下的DNA文库过孔速度统计结果，(B)显示在DDA控制下的DNA文库过孔速度统计结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，本领域技术人员应该理解，所述实施例仅用于举例说明的目的，不以任何方式限制本发明的保护范围。基于本发明中的实施例，本领域普通技术人员能够确定所述实施例的等价实施方案也在本发明的保护范围之内。
本领域技术人员还应该理解，除非另有说明，实施例中所用的细胞或菌株、质粒、试剂等均可商购得到。
本发明人从深海宏基因组文库(来源于深圳国家基因库)筛选得到两个解旋酶 BCH1X序列：SEQ ID NO：1，将其命名为BCH105；SEQ ID NO：2，将其命名为BCH178。这两个解旋酶都包含氨基酸片段：
GTIH X FLNLKLD XGF  Y1DDG Y2ADNV Y2 TKXKLV Y3NK Y4NECL
其中：X表示任意氨基酸残基；Y1＝A或G；Y2＝T或S；Y3＝V或L；Y4＝F或Y。本发明人发现，该氨基酸片段是使解旋酶在测序中表现更好的关键区域。
BCH105的氨基酸序列(SEQ ID NO：1)
mkhdlssateeqryiftdfikqaqlalkgdsdyevfaiqgfagsgktwlsaliidellelgmkvavtspthkavrvslnmlknngidtnsplmypgtihhflnlkldhgfaddgtacnvttkaklvvnkfnealeyvdvlivdeasmvsgelydhalktlgdrakiilfigdsyqllpvddedssiflkddifhykltkvvrqaedniiiaksqelikamdqktyypsvndyfvnitedtegikllksnvelfelyfsdfkdkmtgaytnkvvnqfneyirytlyqetkfiadkdelvfqetytdskgniivsngeiievataklttdidkfkiwkivskknelgeavrfnvldpssynefndlldkycadakiakgydrskawkkyfklkekyakvrynfsstihklqgstyqnmyfdmrgldyfyrmnrdnvlrlvyvgitrasdqvfilqd
BCH178的氨基酸序列(SEQ ID NO：2)
mvkdinkanekqqeafngiveaaknieekitkvyslvgsagtgktwtltqiikelldsgikvamttpthkalavvtdmlkeakiddpnlitgtihyflnlkldygfgddgsadnvstkpklvlnkyneclqytdlliidessmiseelykltmeilddrckmilfvgdkyqllpveggeniiydhpeimhyqltetvrqkagstiiekaneirdyikygnhpdnvfnlfhetdeiqmlqesdflpsyfanehnktigsftnamvdqynnyvryvvtneldylanddevvfqkpysnasgdlifqngetvviestkkvfddknsvwywrckgkarmfnvldpdslsvykdklkelldyaktqkgyhksnawkayfklmnrfgiikysfastlhklqgsttesmyfdmrdlkrfynrdkegvlrliyvaitrpseklfilgl
实施例1：BCH105的克隆、表达和纯化
BCH105的克隆和表达
将全长BCH105的DNA序列连接入PET.28a(+)质粒中，使用的双酶切位点是Nde1和Xho1，因此表达得到的BCH105蛋白N端具有6*His标签和凝血酶酶切位点。
将克隆好的PET.28a(+)-BCH105质粒转化入大肠杆菌表达菌BL21(DE3)或其衍生菌中。挑取单个菌落，接入5mL含有卡那霉素的LB培养基中，37℃震荡培养过夜。然后转接入1L的LB(含有卡那霉素)中，37℃震荡培养至OD600＝0.6-0.8,降温至16℃，加入终浓度500μM的IPTG诱导表达过夜。
BCH105的纯化
缓冲液A:20mM Tris-HCl pH 7.5,250mM NaCl,20mM咪唑；
缓冲液B:20mM Tris-HCl pH 7.5,250mM NaCl,300mM咪唑；
缓冲液C:20mM Tris-HCl pH 7.5,50mM NaCl；
缓冲液D:20mM Tris-HCl pH 7.5,1000mM NaCl；
缓冲液E:20mM Tris-HCl pH 7.5,100mM NaCl；
收集表达BCH105的大肠杆菌菌体，使用缓冲液A重悬菌体,用细胞破碎仪破碎菌体，然后离心取上清。将上清与事先用缓冲液A平衡好的Ni-NTA填料混合，结合1h。收集填料，用缓冲液A大量清洗填料，直至没有杂蛋白被洗出。然后在填料中加入缓冲液B洗脱BCH105。将洗脱得到的BCH105蛋白过缓冲液C平衡好的脱盐柱，进行缓冲液更换。然后加入适量的凝血酶(thrombin)，然后加入到缓冲液C平衡好的ssDNA纤维素填料中，4℃酶切和结合过夜。收集ssDNA纤维素填料，用缓冲液C洗3-4次，然后用缓冲液D洗脱。将ssDNA纤维素纯化后的蛋白浓缩后上分子筛Superdex 200，所用分子筛缓冲液为缓冲液E。收集目的蛋白峰，浓缩，冻存。
由图1可见，经过纯化，最终可得到较大量的纯度良好的BCH105蛋白，平均每克菌纯化出目的蛋白3mg左右。相比之下，远高于使用相同表达载体在相同大肠杆菌中相同条件下表达的解旋酶DDA的产量(解旋酶DDA是本领域中常用的解旋酶，每克菌纯化仅得到0.23mg DDA蛋白)。选取级分6-9合并，用于后续实验。
解旋酶Dda的氨基酸序列(SEQ ID NO：3)：MTFDDLTEGQ KNAFNIVMKA IKEKKHHVTI NGPAGTGKTT LTKFIIEALI STGGTGIILA APTHAAKKIL SKLSGKEAST IHSILKINPV TYEENVLFEQ KEVPDLAKCR VLICDEVSMY DRKLFKILLS TIPPWCTIIG IGDNKQIRPV EPGENTAYIS PFFTHKDFYQ CELTEVKRSN APIIDVATDV RNGKWNYDKV VDGHGVRGFT GDTALRDFMV NYFSIVKSLD DLFENRVMAF TNKSVDKLNS IIRKKIFETD KDFIVGEIIV MQEPLFKTYK IDGKPVSEII FNNGQLVRII EAEYTSTFVK ARGVPGEYLI RHWDLTVETY GDDEYYREKI KIISSDEELY KFNLFLAKTA ETYKNWNKGG KAPWSDFWDA KSQFSKVKAL PASTFHKAQG MSVDRAFIYT PCIHYADVEL AQQLLYVGVT RGRYDVFYV*(*表示终止)
实施例2：BCH105的DNA结合和解旋酶活性检测
BCH105的DNA结合能力检测：
50μL反应体系：在反应缓冲液A中充分混匀底物ssDNA、BCH105蛋白。DNA底物(ssDNA)的终浓度为20nM,所用BCH105蛋白的终浓度分别为0nM、20nM、50nM、100nM、和500nM，其中反应缓冲液A为：50mM HEPES,100mM KCl，pH为8.0.
所用底物ssDNA为3’cy3标记的单链DNA,其序列为TTTTTTTTTTTTCTGAATCACGTACTATATGACACAGTAAAT-cy3
室温反应1h，取样，进行10％非变性PAGE。结果如图2所示。
图2的实验结果显示，随着ssDNA样品中加入的BCH105蛋白的增多，越来越多的cy3标记的ssDNA条带在非变性PAGE上的条带向上迁移，表明形成了ssDNA-BCH105复合物，从而证明BCH105具有良好的ssDNA结合能力。
BCH105的DNA解旋能力检测：
40μL反应体系：在反应缓冲溶液B中充分混匀5’突出(overhang)的双链DNA底物、BCH105蛋白、无标记的单链捕获DNA。DNA底物的终浓度为20nM，蛋白的终浓度为100nM，捕获DNA的终浓度为400nM。其中反应缓冲溶液B为：470mM KCl，25mM HEPES，2mM ATP，10mM MgCl 2，pH为8.0。
5’突出的双链DNA由一条3’末端BHQ-1猝灭基团标记单链DNA与一条5’末端FAM荧光基团标记的单链DNA退火形成，序列分别为：
5’-GCACCGAACTAGCAGCGTCGAAAAGCAGTACTTAGGCATT-BHQ-1-3’，
5’-FAM-TTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGCTGCTAGTTCGGTGC-3’。
无标记的单链捕获DNA的序列为：
5’-AATGCCTAAGTACTGCTTTTCGACGCTGCTAGTTCGGTGC-3’。
阳性对照组(positive)由5’末端FAM荧光基团标记的单链DNA代替5’突出的双链DNA，其他同实验组。
阴性对照组(negative)用不含核酸酶的水(Nuclease-Free Water)代替蛋白，其他同实验组。
40μL反应溶液加入到酶标板中，使用酶标仪实时测定FAM荧光(激发波长为492nm、发射波长为518nm)，温度为30℃，总时长为30min。每组样品进行3个复孔重复。
实验结果如图3所示：阳性对照组(positive)荧光值在测定过程中一直保持不变，约在9500附近；阴性对照组(negative)荧光值在测定过程中一直保持不变，约在2600 左右；BCH105实验组荧光值随反应时间的增加而逐渐增大，BCH105实验组荧光值由3200(0min)增大到5600(30min)。图3的实验结果表明BCH105具有解旋双链DNA的活性，且解旋方向为5’-3’。
实施例3：使用BCH105表征和控制核酸
使用BCH105表征和控制核酸
将两条部分区域互补的DNA链(正义链(top strand)和反义链(bottom strand))退火后形成接头(如图4所示)，与目标测序双链核酸片段(SEQ ID NO：4)利用T4DNA连接酶在室温下连接并纯化，制备测序文库(如图5所示)。然后该测序文库与BCH105在25℃孵育1h(摩尔浓度比1:8)，形成含有BCH105解旋酶的测序文库；与5’端含有胆固醇的单链DNA(chol-ssDNA)在室温下孵育10min。chol-ssDNA序列与接头反义链(bottom strand)其中一部分区域互补，胆固醇结合磷脂膜后能够降低文库上样量，提高捕获率。
该实验中使用膜片钳放大器或其他电信号放大器采集电流信号。
中间有微米级小孔的(直径50-200μm)Teflon膜将电解池分为两个腔室，cis腔室和trans腔室。在cis腔室和trans腔室各放置一对Ag/AgCl电极。在两个腔室的微孔处形成一层双分子磷脂膜后加入纳米孔蛋白(耻垢分枝杆菌孔蛋白A(MspA，SEQ ID NO：5)，按照实施例5制备)；待单个纳米孔蛋白插入磷脂膜后获得了电测量。施加180mV后，获得单个通道的开孔电流。加入适量含有BCH105解旋酶的测序文库，等待几分钟后，测序文库被纳米孔所捕获并在解旋酶的控制下核酸穿过纳米孔。该实验施加180mV来观测并获得电流信号的变化。该实验所用缓冲液为：0.47M KCl，50mM HEPES，5mM ATP，25mM MgCl 2，pH 7.6。实验结果如图6所示。
由图6的结果可见，开孔电流为220pA左右，随着测序文库的DNA单链进入纳米孔，在BCH105的解旋作用下，DNA穿过纳米孔，部分电流被阻碍，电流变小。由于不同核苷酸大小不同，阻碍的电流大小也因此不同，所以可以看到波动的电流信号。
本领域技术人员能够理解，本实施例验证的BCH105的解旋活性和测序实验结果可以间接证明其稳定性，并且，生理盐水0.9％NaCl换算约为0.15M盐浓度，类似于生理条件下的盐浓度，而本实施例在DNA解旋过程中所使用的KCl浓度为0.47M，可证明本发明的解旋酶相对于生理环境具有高盐耐受性(例如，耐受0.3-1M的KCl)。
接头序列正义链(Top strand)：
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3’(Y＝iSP18)
接头序列反义链(Bottom strand)：
5’-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3’
chol-ssDNA：5’-胆固醇-TTGACCGCTCGCCTC-3’
目标测序核酸序列(SEQ ID NO：4)：
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGATATCGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGG CGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
实施例4：解旋酶BCH105与解旋酶DDA的比较
使用本发明的解旋酶BCH105与本领域已知的解旋酶DDA在相同条件下(与实施例3的实验条件相同)进行比较实验。结果显示在图7中，其中过孔速度＝目标测序核酸序列长度/过孔时间，过孔时间为观测到电流信号变化的时间长度。如图7所示，对目标DNA测序文库过孔速度进行统计，图7A显示在BCH105控制下的DNA文库过孔速度统计结果，均值为242nt/s；图7B显示在DDA控制下的DNA文库过孔速度统计结果，均值为161nt/s。这说明在BCH105控制下DNA文库的过孔速度更快，表明本发明的解旋酶的测序速度更高，盐耐受度更好，稳定性更好。
实施例5：制备MspA蛋白
MspA蛋白表达载体的构建
通过In-fusion的方法，采用NdeI和XhoI酶切后，将MspA蛋白编码的基因序列插入到载体pET24a的克隆区，因此表达得到的MspA蛋白N端具有6个His，可以作为纯化标签，其中筛选标签为卡那霉素，将构建好的表达载体命名为pET24a-MspA。
重组大肠杆菌菌株的培养和诱导
LB液体培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L。
将重组表达载体pET24a-MspA转化到大肠杆菌表达菌株E.coli BL21(DE3)中，将菌液均匀涂抹在50μg/mL卡那霉素的LB固体培养平板上，37℃过夜培养。挑取单菌落，于5ml LB培养基(含有50μg/mL卡那霉素)培养，37℃，200rpm，过夜培养。将上述所得菌液，按1:100接种于50ml LB(含有50μg/mL卡那霉素)中培养，37℃，200rpm，4h。将扩大培养的菌液，按1:100接种于2L LB(含有50μg/mL卡那霉素)中培养，37℃，200rpm，待OD600值达0.6-0.8左右，加入IPTG至终浓度为0.5mM，18℃，200rpm，培养过夜，约16-18h。将长好的菌体于8000rpm离心收集，菌体冻存于-20℃待用。
重组型MspA蛋白提取和纯化
纯化缓冲液配制
1、Ni柱亲和层析
缓冲液A1(平衡缓冲液)：20mM Tris-HCl+250mM NaCl+0.5％Tween-20+5％甘油，pH 7.9；
缓冲液B1(洗脱缓冲液)：20mM Tris-HCl+250mM NaCl+0.5％Tween-20+5％甘油+500mM咪唑，pH 7.9。
2、离子交换层析
稀释缓冲液：
缓冲液C1(平衡缓冲液)：20mM Tris-HCl+50mM NaCl+0.5％Tween-20+5％甘油，pH 6.5。
缓冲液D1(洗脱缓冲液)：20mM Tris-HCl+1000mM NaCl+0.5％Tween-20+5％甘油，pH 6.5。
3、蛋白样品稀释液
缓冲液E1(稀释液)：20mM Tris-HCl+0.5％Tween-20+5％甘油，pH 6.5。
按1g重组大肠杆菌菌体加10ml缓冲液A1的比例重悬菌体，超声破碎细胞，直至菌体溶液至澄清。将破碎后的菌体12000rpm，4℃离心30min，取上清，0.22μm滤膜过滤后于4℃储存。
将Ni柱亲合层析柱水洗5个柱体积(5CV)，缓冲液B1清洗5CV，缓冲液A1进行平衡10CV后，进行上样。上样完成后，用缓冲液A1平衡15CV，使用缓冲液B1进行线性洗脱(0-8％缓冲液B1，30CV)去除杂质，使用缓冲液B1进行线性洗脱(8-100％缓冲液B1，5CV)收集目的蛋白。
将Ni柱收集到的蛋白用缓冲液E1稀释2倍,将阴离子交换Q柱水洗5CV，缓冲液C1平衡5CV，蛋白样品上样。用缓冲液C1平衡5CV后，用洗脱缓冲液缓冲液D1线性洗脱(0-9％缓冲液D1，30CV)，用洗脱缓冲液缓冲液D1线性洗脱(9-100％缓冲液D1，10CV)并收集蛋白。收集蛋白进行4℃过夜透析，透析液为缓冲液A1，随后储存于-80℃。
MspA蛋白的氨基酸序列(SEQ ID NO：5)
GLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLELGYQIGFPWSLGVGINFSYTTPNILIDDGDITRPPFGLNSVITPNLFPGVSISADLGNGPGIQEVATFSVDVSGAEGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSV TTYGEPWNMN
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，本领域技术人员可以对本发明的技术方案进行多种改变并且仍然能够获得所需要的技术效果，这些改变均属于本发明的保护范围。

Claims (16)

  1. 一种解旋酶，其包含：
    (i)SEQ ID NO：1或2所示的氨基酸序列；或
    (ii)与SEQ ID NO：1或2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性并且具有解旋酶活性的氨基酸序列；或
    (iii)与SEQ ID NO：1或2所示的氨基酸序列具有不超过20、15、10、5、4、3、2或1个氨基酸差异并且具有解旋酶活性的氨基酸序列。
  2. 根据权利要求1所述的解旋酶，其包含下述氨基酸片段：
    GTIH X FLNLKLD XGF  Y1DDG Y2ADNV Y2 TKXKLV Y3NK Y4 NECL
    其中：X表示任意氨基酸残基；Y1表示A或G；Y2表示T或S；Y3表示V或L；Y4表示F或Y。
  3. 根据权利要求1所述的解旋酶，其中所述氨基酸差异包括氨基酸取代、缺失和/或插入或N-端和/或C-端延伸，优选地，所述氨基酸取代为保守氨基酸取代。
  4. 根据权利要求1所述的解旋酶，其由SEQ ID NO：1或2所示的氨基酸序列组成。
  5. 一种编码权利要求1-4中任一项所述的解旋酶的核苷酸序列。
  6. 一种包含权利要求5所述的核苷酸序列的重组载体，优选为重组表达载体。
  7. 一种包含权利要求5所述的核苷酸序列或权利要求6所述的重组载体的细胞，优选地，所述细胞是原核细胞或真核细胞，更优选地，所述细胞是大肠杆菌细胞、酵母细胞、昆虫细胞或哺乳动物细胞。
  8. 一种复合体结构，其包含权利要求1-4中任一项所述的解旋酶BCH1X和用于结合多核苷酸的结合部分。
  9. 根据权利要求8所述的复合体结构，其中所述结合部分为与多核苷酸的碱基结合的结合部分，和/或与多核苷酸的糖结合的结合部分，和/或与多核苷酸中的磷酸结合的结合部分。
  10. 一种控制和表征目标多核苷酸的方法，所述方法包括下述步骤：
    (a)将目标多核苷酸与孔、和权利要求1-4中任一项所述的解旋酶或权利要求8所述的复合体结构接触，使得所述解旋酶或复合体结构控制所述目标多核苷酸穿过所述孔的运动；和
    (b)获取目标多核苷酸中的核苷酸与所述孔相互作用时的一个或多个特征，以表征所述目标多核苷酸；
    其中所述一个或多个特征选自电流信号大小变化、电流信号时长变化、电压信号大小变化及电压信号时长变化。
  11. 根据权利要求10所述的方法，所述方法是单分子纳米孔测序方法。
  12. 一种控制和表征多核苷酸的试剂盒，所述试剂盒包含权利要求1-4中任一项所述的解旋酶或其复合体结构和孔。
  13. 一种用于单分子纳米孔测序的试剂盒，所述试剂盒包含权利要求1-4中任一项所述的解旋酶或其复合体结构和孔。
  14. 一种表征目标多核苷酸的传感器，所述传感器包括在孔和权利要求1-4中任一项所述的解旋酶或其复合体结构之间形成的复合体。
  15. 一种表征目标多核苷酸的装置，所述装置包括权利要求1-4中任一项所述的解旋 酶或其复合体结构和孔。
  16. 根据权利要求10或11所述的方法、根据权利要求12或13所述的试剂盒、根据权利要求14所述的传感器或根据权利要求15所述的装置，其中所述孔是跨膜孔，优选是生物孔、固态孔或生物与固态杂交的孔，更优选地，所述生物孔选自α-溶血素蛋白(α-HL)、耻垢分枝杆菌孔蛋白A(MspA)、curli-特异性转运通道蛋白(CsgG)或III型分泌系统蛋白(InvG)。
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US18/723,588 US20250066747A1 (en) 2021-12-31 2021-12-31 Helicase bch1x and use thereof
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160319344A1 (en) * 2013-12-19 2016-11-03 Centre National De La Recherche Scientifique (Cnrs) Nanopore sequencing using replicative polymerases and helicases
CN108699540A (zh) * 2016-02-29 2018-10-23 吉尼亚科技公司 用于纳米孔测序的聚合酶-模板复合物
CN110267974A (zh) * 2017-02-10 2019-09-20 牛津纳米孔技术公司 修饰的纳米孔、包含其的组合物及其用途
US20200216887A1 (en) * 2016-01-21 2020-07-09 Genia Technologies, Inc. Nanopore sequencing complexes
CN112805393A (zh) * 2018-09-28 2021-05-14 北京齐碳科技有限公司 一种解旋酶及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160319344A1 (en) * 2013-12-19 2016-11-03 Centre National De La Recherche Scientifique (Cnrs) Nanopore sequencing using replicative polymerases and helicases
US20200216887A1 (en) * 2016-01-21 2020-07-09 Genia Technologies, Inc. Nanopore sequencing complexes
CN108699540A (zh) * 2016-02-29 2018-10-23 吉尼亚科技公司 用于纳米孔测序的聚合酶-模板复合物
CN110267974A (zh) * 2017-02-10 2019-09-20 牛津纳米孔技术公司 修饰的纳米孔、包含其的组合物及其用途
CN112805393A (zh) * 2018-09-28 2021-05-14 北京齐碳科技有限公司 一种解旋酶及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRINKERHOFF, H. ET AL.: "Multiple Rereads of Single Proteins at Single-Amino Acid Resolution Using Nanopores", SCIENCE, vol. 374, 17 December 2021 (2021-12-17), XP093033008, DOI: 10.1126/science.abl4381 *
CAO YING /, LIU HAIZHOU, CAO YING, LI WEI, CHU XIN, WU KE, LIU DI: "Research progress and application of nanopore sequencing technology", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 36, no. 5, 25 May 2020 (2020-05-25), pages 811 - 819, XP055882525, DOI: 10.13345/j.cjb.190368 *

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