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WO2020061997A1 - 一种解旋酶及其应用 - Google Patents

一种解旋酶及其应用 Download PDF

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Publication number
WO2020061997A1
WO2020061997A1 PCT/CN2018/108227 CN2018108227W WO2020061997A1 WO 2020061997 A1 WO2020061997 A1 WO 2020061997A1 CN 2018108227 W CN2018108227 W CN 2018108227W WO 2020061997 A1 WO2020061997 A1 WO 2020061997A1
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WIPO (PCT)
Prior art keywords
helicase
target polynucleotide
amino acid
acid sequence
complex structure
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Ceased
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PCT/CN2018/108227
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English (en)
French (fr)
Inventor
陈呈尧
王慕旸
周雅
付童
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Qitan Technology Ltd Beijing
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Qitan Technology Ltd Beijing
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Priority to CN201880095718.XA priority Critical patent/CN112805393A/zh
Priority to PCT/CN2018/108227 priority patent/WO2020061997A1/zh
Publication of WO2020061997A1 publication Critical patent/WO2020061997A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • the invention relates to the technical field of characterization of nucleic acid characteristics, in particular to a method for characterizing a target polynucleotide, and a F8813 helicase, a complex structure containing a helicase, and the method for characterizing the target polynucleotide or controlling the same. Application of the movement of a target polynucleotide through a pore.
  • Nanopore sequencing technology is a single-stranded nucleic acid molecule as a sequencing unit, using a nanopore that can provide ion current channels, so that single-stranded nucleic acid molecules pass through the nanopore under the driving of electrophoresis.
  • nucleic acid passes through the nanopore, it will reduce Nanopore current, gene sequencing technology that reads sequence information of different signals generated in real time.
  • Nanopore sequencing technology has the following advantages: easy to build libraries without the need for amplification; fast reading speed, single-stranded molecules can read tens of thousands of bases per hour; longer read length, usually can Up to thousands of bases; measurements of methylated DNA or RNA can be made directly.
  • the recorded current signal corresponds to the sequence of the polynucleotide, it is usually 3-4 nucleotides that control a certain These levels of current levels, so still need to improve accuracy.
  • accuracy can be improved by changing the structure of the polynucleotide and the duration at the nanopore to control the translocation of the polynucleotide.
  • the method of changing the duration at the nanopore to control the translocation of the polynucleotide mainly solves the problem that the translocation rate of the polynucleotide through the nanopore is too fast and the single nucleotide is too short.
  • an emerging method for characterizing polynucleotides includes transmembrane pores, contact and interaction of helicases with polynucleotides, and thus helicases control the movement of target polynucleotides through nanopores to increase multinuclei. Retention time of the uronic acid at the nanopore.
  • patent WO2013057495A3 discloses a new method for characterizing a target polynucleotide.
  • the method uses a pore and Hel308 helicase or a molecular motor capable of binding the internal nucleotide of the target polynucleotide.
  • the helicase or molecular motor according to the invention can effectively control the movement of the target polynucleotide through the pore.
  • Patent US20150065354A1 discloses a method for characterizing a target polynucleotide using XPD helicase, which uses a pore and XPD helicase.
  • the XPD helicase of the invention can control the movement of the target polynucleotide through the pore.
  • Patent US20170268055A1 discloses a composition and method for sequencing a polynucleotide, which uses a step-by-step translocation step of translocating a target polynucleotide through a pore to characterize the target polynucleotide, including characterizing the sequence of the polynucleotide Methods and compositions.
  • Patent CN106103741A discloses a method for linking one or more polynucleotide binding proteins to a target polynucleotide, and the invention also relates to a new method for characterizing a target polynucleotide.
  • a conductive substrate solution is required as the necessary environment, and a charge carrier (such as a salt) is used as voltage compensation to capture or transfer the target polynucleotide, and measure as the polynucleotide passes
  • a charge carrier such as a salt
  • a high salt concentration is advantageous for enhancing the signal strength obtained, so measuring the signal of a polynucleotide requires that the salt concentration be above a certain level.
  • the high salt concentration provides a high signal for the noise ratio and allows current indications to determine the presence of nucleotides in the context of normal current fluctuations.
  • the present invention provides a new helicase for the characterization of nucleic acids, solves the problem of low salt tolerance of conventional helicases, and significantly improves the accuracy of characterization of polynucleotide characteristics.
  • the present inventor provided a method for characterizing a nucleotide sequence under a high salt concentration, and thus designed and prepared a new helicase and a complex structure containing the helicase, the helicase or complex
  • the structure has a surprisingly high salt tolerance, and is particularly suitable for characterizing nucleotide sequences at high salt concentrations.
  • the ideal salt concentration is greater than 100 mM, and the salt concentration greater than 500 mM is also a good state. Under high salt conditions, it can provide a high signal for the noise ratio, and allow the current to flow under the background of normal current fluctuations. Indicates the presence of a determined nucleotide.
  • the helicase or complex structure provided by the present invention for characterizing a nucleotide sequence can work under a high salt concentration, and more importantly, can improve the accuracy of detecting a nucleic acid sequence.
  • the invention solves the problem of controlling the salt concentration in the traditional sequencing field.
  • a first aspect of the present invention relates to a method for characterizing a target polynucleotide, including:
  • the complex structure includes a helicase and a binding portion for binding a polynucleotide, and the helicase or the complex structure has a helicase activity under a high salt concentration.
  • the rate at which the target polynucleotide passes through the nanopore is controlled by the helicase or complex structure, thereby obtaining an identifiable current level for determining the target polynucleotide. the sequence of.
  • steps (a), (b) are repeated one or more times.
  • said characterizing includes applying an improved Viterbi algorithm.
  • the high salt concentration is at least 100 mM, at least 250 mM, at least 300 mM, at least 500 mM, at least 1000 mM, at least 1500 mM, at least 1800 mM, at least 2000 mM, at least 2500 mM, at least 3000 mM, or at least 3500 mM, wherein the salt is selected From KCl buffer, MgCl 2 buffer or NaCl buffer.
  • the salt concentration is 250 mM-2000 mM.
  • the amino acid sequence of the helicase is the amino acid sequence shown in SEQ ID NO: 1 or has at least 60%, at least 70%, at least 80%, at least 85%, and the amino acid sequence shown in SEQ ID NO: 1. At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of homology and have a solution Rotase activity.
  • the complex structure comprises a helicase and a binding portion for binding a polynucleotide
  • the helicase is a helicase of the Hel308 family, or the amino acid sequence of the helicase Is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, or the amino acid sequence shown in SEQ ID NO: 1, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of homology and helicase activity.
  • the binding portion is selected from a eukaryotic single-stranded binding protein, a bacterial single-stranded binding protein, an archaic single-stranded binding protein, a viral single-stranded binding protein or a double-stranded binding protein.
  • the one or more characteristics are selected from the source, length, identity, sequence, secondary structure, or whether the target polynucleotide is modified.
  • the feature is a sequence.
  • the one or more features are performed by electrical measurement and / or optical measurement.
  • an electrical signal and / or an optical signal are generated by electrical measurement and / or optical measurement, and each nucleotide corresponds to a signal level, and then the electrical signal and / or optical signal is converted into a sequence characteristic of the nucleotide.
  • the electrical measurement according to the present invention is selected from the group consisting of current measurement, impedance measurement, field effect transistor (FET) measurement, tunnel measurement or wind tunnel measurement.
  • FET field effect transistor
  • the electrical signal according to the present invention is selected from the measurement values of current, voltage, tunneling, resistance, potential, conductivity or lateral electrical measurement.
  • the electrical signal is a current passing through the hole.
  • the method further comprises the step of applying a potential difference across the pores in contact with the helicase or complex structure and the target polynucleotide.
  • the method of the present invention may include: a) contacting the target polynucleotide with a helicase or a complex structure containing a helicase that remains active at a high pore and high salt concentration such that the helicase or complex
  • the body structure controls the movement of the target polynucleotide through the pore; and b) applying a potential difference across the pore in contact with the helicase or the complex structure and the target polynucleotide to determine the target polynucleotide
  • the current when the nucleotide in the acid interacts with the pore and c) converting the current signal level corresponding to each nucleotide to obtain the sequence of the target polynucleotide.
  • the target polynucleotide is single-stranded, double-stranded, or at least partly double-stranded.
  • the target polynucleotide may be DNA or RNA.
  • the target polynucleotide is double-stranded.
  • the double-stranded portion constitutes a Y-adaptive structure, and the Y-adaptive structure comprises a leader sequence which is preferentially screwed into the hole.
  • the target polynucleotide according to the present invention is a macromolecule containing one or more nucleotides.
  • the target polynucleotide according to the present invention may be naturally occurring or artificially synthesized.
  • one or more nucleotides in the target polynucleotide may be modified, such as methylation, oxidation, damage, abasic, protein labeling, tagging or polynucleotide sequence.
  • a spacer is connected in the middle.
  • the artificially synthesized nucleic acid is selected from peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers having nucleoside side chains.
  • the pores are transmembrane pores, and the transmembrane pores are biological pores, solid pores, or pores that hybridize with solids.
  • the transmembrane pore according to the present invention is a structure that allows hydrated ions to flow from one side of the membrane to another layer of the membrane under the driving of an applied potential.
  • the transmembrane pore provides a channel for movement of the target polynucleotide.
  • the film is a double-layer film.
  • the membrane is a lipid bilayer membrane.
  • the Y-joint structure of the present invention includes a leader sequence that is preferentially screwed into the pore.
  • the 3 'end of the leader sequence is connected with thiol, biotin or cholesterol, and is used to bind to a layer of a lipid bilayer membrane. It is thought that the target polynucleotide points in the right direction and has a pulling effect.
  • the 3 'end of the leader sequence is connected to cholesterol, and is used to bind to a layer of a lipid bilayer membrane.
  • the biological pore is selected from the group consisting of hemolysin, leukocidin, M. smegmatis membrane porin A (MspA), M. smegmatis membrane porin B, M. smegmatis membrane porin C, Mycobacterium smegmatis membrane porin D, lysin, MZA, outer membrane protein F (OmpF), outer membrane protein G (OmpG), outer membrane phospholipase A or Neisseria autotransport lipoprotein (NalP).
  • MspA M. smegmatis membrane porin A
  • M. smegmatis membrane porin B M. smegmatis membrane porin C
  • Mycobacterium smegmatis membrane porin D lysin
  • MZA outer membrane protein F
  • OmpG outer membrane protein G
  • NalP Neisseria autotransport lipoprotein
  • the second aspect of the present invention relates to a helicase (F8813 helicase), the amino acid sequence of the helicase is the amino acid sequence shown in SEQ ID NO: 1 or has the amino acid sequence shown in SEQ ID NO: 1 At least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, or at least 99.9% homology and helicase activity.
  • the sequence having homology with the amino acid sequence shown in SEQ ID NO: 1 and having helicase activity includes: the difference from the amino acid sequence shown in SEQ ID NO: 1 is not more than 20, 15, 10, A sequence of 5, 4, 3, 2 or 1 amino acid and having helicase activity; or a variant of SEQ ID NO: 1, wherein the variant differs from SEQ ID NO: 1 including substitutions, deletions and A sequence that inserts one or more amino acid residues or at least one N- / C-terminal extension and has a helicase activity.
  • the substitution is a conservative amino acid substitution.
  • the helicase is bound to an internal nucleotide of a single-stranded polynucleotide or a double-stranded polynucleotide.
  • the helicase maintains the helicase at a salt concentration of at least 100 mM, at least 250 mM, at least 300 mM, at least 500 mM, at least 1000 mM, at least 1500 mM, at least 1800 mM, at least 2000 mM, at least 2500 mM, at least 3000 mM, or at least 3500 mM.
  • Activity wherein the salt is selected from KCl buffer, MgCl 2 buffer or NaCl buffer.
  • the helicase or complex structure of the present invention can move the target polynucleotide through the nanopore in a controlled and stepwise manner through a magnetic field generated by an applied voltage, thereby controlling the rate of the polynucleotide passing through the nanopore to obtain Recognizable current level.
  • the F8813 helicase or complex structure will effectively function at high salt concentrations.
  • a third aspect of the present invention relates to a nucleotide sequence encoding the amino acid sequence of the helicase according to the second aspect.
  • the nucleotide sequence is at least 60%, at least 70%, at least 80%, or at least 60%, at least 70%, at least 80%, or at least the nucleotide sequence shown in SEQ ID NO: 2. 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% homology the sequence of.
  • a fourth aspect of the present invention relates to a complex structure including a helicase and a binding moiety for binding a polynucleotide.
  • the helicase is attached to the binding part, and the complex structure can control the movement of the polynucleotide.
  • the helicase is a helicase of the Hel308 family, or the amino acid sequence of the helicase is the amino acid sequence shown in SEQ ID NO: 1 or has the amino acid sequence shown in SEQ ID NO: 1 At least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, or at least 99.9% homology and helicase activity.
  • the complex structure is a natural structure or a non-natural structure.
  • the complex structure is an artificially produced 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.
  • the amino acid sequence of the helicase is at least 60%, at least 70%, at least 80%, or at least 85% of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1. , At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of homology and have Helicase activity.
  • the complex structure according to the present invention is an effective tool for controlling the movement of polynucleotides during sequencing.
  • the structure of the helicase-containing complex structure and the polynucleotide of the present invention is stable in binding, and will not detach during the sequencing process.
  • the complex structure can provide a larger read length of the polynucleotide.
  • the binding polynucleotide in the binding moiety matches the process of strand sequencing and polynucleotide characterization.
  • the binding moiety is more active at high salt concentrations (such as 100 mM to 2M). This is due to its salt tolerance.
  • the improvement of the binding moiety of the complex structure can improve the synthesis ability, stability and half life.
  • the complex structure can be inherited.
  • the helicase and the binding moiety pass through each other's terminal amino acids.
  • the amino terminus of the binding moiety is bound to the carboxyl terminus of the helicase or the carboxy terminus of the binding moiety is bound to the amino terminus of the helicase.
  • said binding portion is inserted into the sequence of a helicase.
  • the helicase is the F8813 helicase according to the present invention.
  • the binding portion is inserted into a loop region of a F8813 helicase.
  • F8813 helicase is stably attached to the binding moiety through one or more (preferably 2 or 3) linkers.
  • the linkers can restrict the movement of the binding part.
  • the F8813 helicase is modified and / or the binding moiety is modified to enhance linker ligation.
  • tags are added to the complex structure. When it is necessary to remove the label, it can be removed by chemical method or enzymatic reaction.
  • the binding moiety that binds to the polynucleotide is selected from one or more of eukaryotic single-stranded binding proteins (SSBs), bacterial SSBs, archaeological SSBs, viral SSBs, and double-stranded binding proteins.
  • SSBs eukaryotic single-stranded binding proteins
  • bacterial SSBs bacterial SSBs
  • archaeological SSBs bacterial SSBs
  • viral SSBs double-stranded binding proteins
  • a fifth aspect of the present invention relates to a helicase according to the second aspect or the nucleotide sequence of the third aspect or the complex structure according to the fourth aspect to characterize a target polynucleotide or control a target multinucleus.
  • a sixth aspect of the present invention relates to a kit for characterizing a target polynucleotide, the kit comprising the helicase according to the second aspect or the nucleotide sequence of the third aspect or the fourth aspect.
  • the kit includes a plurality of helicases or a plurality of complex structures, and a plurality of wells.
  • the pores are transmembrane pores, and the transmembrane pores are biological pores, solid pores, or pores that hybridize with solids.
  • the biological pore is selected from the group consisting of hemolysin, leukocidin, M. smegmatis membrane porin A (MspA), M. smegmatis membrane porin B, and M. smegmatis membrane porin C. , Mycobacterium smegmatis membrane porin D, cytolysin, MZA, outer membrane protein F (OmpF), outer membrane protein G (OmpG), outer membrane phospholipase A or Neisseria autotransport lipoprotein (NalP) .
  • the kit further comprises a chip comprising a lipid bilayer.
  • the pores span the lipid bilayer.
  • the kit according to the invention comprises one or more lipid bilayers, each lipid bilayer comprising one or more of the wells.
  • the kit according to the present invention also includes a reagent or device for performing characterization of the target polynucleotide.
  • the reagents include buffers and tools required for PCR amplification.
  • the invention also provides a sensor for characterizing a target polynucleotide, which comprises forming a complex between a pore and a helicase or a complex structure, the target polynucleotide interacts with the pore, and the resulting polynucleotide is used to characterize the target multinucleus.
  • Glycine sensor for characterizing a target polynucleotide, which comprises forming a complex between a pore and a helicase or a complex structure, the target polynucleotide interacts with the pore, and the resulting polynucleotide is used to characterize the target multinucleus.
  • the pore is contacted with a helicase or complex structure in the presence of the target polynucleotide, and a potential is applied across the pore.
  • the potential is selected from a voltage potential or a chemical potential.
  • the pore is covalently linked to the helicase or the complex structure.
  • a seventh aspect of the present invention relates to a device for characterizing a target polynucleotide, the device comprising the helicase according to the second aspect or the nucleotide sequence according to the third aspect or the fourth aspect The complex structure, and pores.
  • the device includes a sensor device supporting the plurality of holes and transmitting a signal of interaction between the holes and the polynucleotide, and at least one memory for storing the target polynucleotide, and required for performing the characterization process.
  • the device includes multiple helicases or multiple complex structures, and multiple pores.
  • the pores are transmembrane pores, and the transmembrane pores are biological pores, solid pores, or pores that hybridize with solids.
  • the biological pore is selected from the group consisting of hemolysin, leukocidin, M. smegmatis membrane porin A (MspA), M. smegmatis membrane porin B, and M. smegmatis membrane porin C. , Mycobacterium smegmatis membrane porin D, cytolysin, MZA, outer membrane protein F (OmpF), outer membrane protein G (OmpG), outer membrane phospholipase A or Neisseria autotransport lipoprotein (NalP) .
  • nucleotide in the present invention includes but is not limited to: adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytosine Nucleoside 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 nucleotide is selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
  • Constant amino acid substitutions include, but are not limited to, substitutions between alanine and serine, glycine, threonine, valine, proline, or glutamic acid; and / or, Asparagus Substitutions between glycine and glycine, asparagine or glutamic acid; and / or, substitutions between serine and glycine, asparagine or threonine; and / or, leucine and isoleucine or Substitution between valine; and / or, substitution between valine and leucine, isoleucine; and / or, substitution between tyrosine and phenylalanine; and / or, Replacement between lysine and arginine.
  • the above-mentioned substitutions do not substantially change the activity of the amino acid sequence according to the present invention.
  • the "comprising" in the present invention is an open description, and includes the specified ingredients or steps described, and other specified ingredients or steps that will not substantially affect.
  • the “homology” described in the present invention refers to the aspect of using protein sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to the actual work needs, so that the used sequences are consistent with the sequences obtained in the prior art.
  • Ratio including (but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, 99.1%, 99.2%, 99
  • Figure 1 SDS-PAGE gel electrophoresis purification results of F8813 helicase, where M is Marker (Kd), one lane is the electrophoresis results of F8813 helicase.
  • FIG. 2 Enzyme activity is detected using a fluorescence assay, where the fluorescent substrate strand (B, 100nM final concentration) shown in a) contains a 3 'single-stranded DNA portion and a 5'-end hybridized double-stranded portion.
  • the 5 'end hybrid double-stranded part has a carboxyfluorescein (C) at the 5' end, and the complementary short strand (D) of the hybrid double strand has a BHQ-1 base at the 3 'end ( E).
  • carboxyfluorescein (C) is mixed with BHQ-1 base (E)
  • carboxyfluorescein (C) is annealed by BHQ-1 (E)
  • the substrate is essentially non-fluorescent.
  • the 1 ⁇ M capture strand was complementary to the short strand of the fluorescent substrate.
  • ATP 0.5 mM
  • MgCl 2 10 mM
  • helicase 100 nM
  • the helicase completely dissociates the double strand
  • the helicase falls off, and the main chain emits fluorescence.
  • excess capture strands (F) take precedence over complementary DNA annealing to prevent re-annealing of the original substrate and loss of fluorescence.
  • Figure 3 The results of the changes in fluorescence values of positive control, negative control, and F8813 helicase were measured over time in 250 mM NaCl buffer, where the abscissa is time (min), the ordinate is the fluorescence value.
  • Figure 4 Results of the change in fluorescence value of F8813 helicase over time under conditions of 250 mM, 500 mM, 1 M, and 2 M NaCl buffer solutions, where the abscissa is time (min) and the ordinate Fluorescence value / positive control (%).
  • FIG. 5 Schematic diagram of the states of helicase controlling DNA through the nanopore.
  • f is a nanopore
  • d is a phospholipid bilayer (dividing the space into a cis region and a trans region)
  • c is a cholesterol tag
  • b is a leader sequence with a cholesterol tag (c)
  • a is a Single-stranded DNA matrix of leader (b).
  • Cholesterol label is combined with phospholipid bilayer to make the surface substrate of bilayer more abundant.
  • Helicase (e) binds to a single strand of DNA in process 2.
  • Helicases move along the DNA in the presence of divalent metal ions and the NTP matrix.
  • a single voltage is applied, and the single strand of DNA is guided by the leader sequence to be captured by the nanopore.
  • the single strand of DNA is pulled through the pore until the helicase is in contact with the nanopore, preventing the uncontrolled translocation of the DNA.
  • portions of the double-stranded DNA such as those with a leader sequence, are removed.
  • the helicase moves in the 3'-5 'direction, opposite to the direction of the electric field.
  • the helicase pulls the DNA out of the nanopore and returns to the cis region. The last part coming out of the nanopore is the 5 'end.
  • the helicase removes DNA from the nanopore in the process 5
  • the helicase will fall off the DNA strand and return to the cis region.
  • FIG. 6 Helicases are able to control the translocation of DNA from the nanopore and generate a gradually changing current as the DNA moves.
  • the binding of helicase F8813 to DNA under the conditions of 180mV, 400mM KCl, 10mM Hepes pH 8.0, 0.10nM DNA, 500nM F8813, 2.86mM ATP, 5mM MgCl 2 is indicated by the small arrow at the top of the figure.
  • the abscissa is time (s)
  • the ordinate current (pA).
  • Figure 7 A current map of F8813 helicase (Panel A) and F8813-SSBSsop7D helicase (Panel B) controlling the translocation of a polynucleotide through a nanopore.
  • the nucleic acid sequence of F8813 helicase (SEQ ID NO: 2) was obtained and optimized by using the correspondence between codons and the frequency of host expression and artificial analysis.
  • the synthetic F8813 helicase nucleic acid sequence was cut by restriction endonucleases and ligated into the expression vector pET15b. It was verified that the recombinant plasmid was correct.
  • the recombinant plasmid was transformed into DE3 competent cells by heat shock and cultured at 37 ° C overnight to obtain monoclonal cells and a large number of E. coli cells containing the recombinant plasmid.
  • the ability of helicase to replace hybrid double-stranded DNA was analyzed by fluorescence.
  • the steps are shown in Figure 2.
  • the fluorescent substrate strand (B, final concentration 100nM) contains a 3 'single-stranded DNA portion and a 5' Terminal hybridized double-stranded portion.
  • the 5 'end hybrid double-stranded part has a carboxyfluorescein (C) at the 5' end, and the complementary short strand (D) of the hybrid double strand has a BHQ-1 base at the 3 'end ( E).
  • Matrix DNA 5'-FAM-SEQ ID NO: 3, SEQ ID NO: 4-BHQ1-3.
  • FAM is carboxyfluorescein and BHQ1 is a fluorescence quencher.
  • Figure 3 shows the change in fluorescence values of positive control, negative control, and F8813 helicase over time in a 250 mM NaCl buffer.
  • the positive control is that only carboxyfluorescein (C ), Does not contain the BHQ-1 base (E); the negative control is the absence of ATP in step b, the abscissa is time (min), and the ordinate is the fluorescence value.
  • NaCl buffer solution (10 mM Hepes pH 8.0, 0.5 mM ATP, 10 mM MgCl2, 100 nM matrix DNA, 1 ⁇ M capture DNA).
  • the NaCl buffer solution was 10 mM Hepes pH 8.0, 0.5 mM ATP, 10 mM MgCl 2 , 100 nM matrix DNA, and 1 ⁇ M capture DNA.
  • Example 4 F8813 helicase controls the movement of DNA strands through nanopores
  • the entire process of DNA passing through the nanopore is recorded in Figure 5.
  • the DNA matrix contains a 30 nt 5 'leader strand captured by the nanopore (SEQ ID NO: 6).
  • the leader strand is a primer with a 3' cholesterol tag (SEQ ID NO: 7), the leader chain is combined with the phospholipid bilayer to enrich the surface DNA of the phospholipid bilayer and improve the capture efficiency.
  • Buffer solution 400 mM KCl, 10 mM Hepes pH 8.0, 2.86 mM ATP, 5 mM MgCl 2 .
  • Helicase F8813 helicase, final concentration from 2 ⁇ L to more than 500 nM.
  • a single nanopore was inserted into a 1,2-2-glycerol-3-phosphate choline lipid bilayer, and a voltage was applied to obtain an electrical measurement value.
  • a pore size of 25 ⁇ m was formed on the PTFE film, and a two-layer membrane separated from two 0.1 mL buffer solutions was formed. All experiments were performed in the specified buffer solution.
  • Single-channel current is measured with an amplifier with a digitizer.
  • the Ag / AgCl electrode is connected to the buffer solution, the cis compartment (the area where nanopores, enzymes and DNA are added) is on the top, and the trans compartment is connected to the probe of the active electrode.
  • DNA polynucleotides and helicase are added to 70 ⁇ L of buffer in the cis region to capture the electrical signals of the helicase and DNA complex as they pass through the nanopore.
  • the final concentration of DNA was 10 nM and the final concentration of enzyme was 0.5 ⁇ M.
  • the helicase ATPase activity was detected by adding a divalent metal ion (5 mM MgCl 2 ) and NTP (2.86 mM ATP) to the cis region, and the experimental constant voltage was +180 mV.
  • Adding a complex of helicase and DNA to the nanopore as shown in FIG. 5 generates a characteristic current as shown in FIG. 6.
  • DNA was captured through the nanopore.
  • DNA that does not bind to the helicase quickly passes through the nanopore, producing a short-term current ( ⁇ 1s).
  • a DNA fragment that binds to a helicase moving along the DNA strand under ATPase activity), as the DNA moves through the nanopore, generates a long characteristic block current and the current gradually changes level (as shown in Figure 6).
  • Different DNA structures in the nanopore produce unique current block levels.
  • DNA final concentration added to the nanopore is 1nM
  • F8813 helicase final concentration added to the nanopore is 100nM, SEQ ID NO: 1
  • F8813-SSBSsop7D The final concentration added to the nanopore was 100 nM, SEQ ID NO: 17 and buffer (500 mM NaCl, 50 mM Tris-Hcl, pH 8.0, 1 mM DTT).
  • MgCl2 final concentration 5 mM
  • ATP final concentration 2.86 mM
  • buffer 10 mMHEPES, 400 mM KCl pH 8 and 0.5 mg / mL BSA
  • F8813 helicase ( Figure 7A) and F8813-SSBSsop7D helicase ( Figure 7B) control the translocation current of the polynucleotide through the nanopore, respectively.
  • F8813-ssbssop7d controls more DNA movement than F8813-ssbssop7d.
  • F8813-ssbssop7d showed that the improved helicase controls the speed of DNA movement and is quite stable.

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Abstract

提供了一种表征目标多核苷酸的方法，及一种F8813解旋酶、一种包含解旋酶的复合体结构及其在表征目标多核苷酸或控制目标多核苷酸穿过孔的运动中的应用。所述的F8813解旋酶或复合体结构具有极强的耐盐性，其用于表征目标多核苷酸的方法可以提高检测核酸序列的精确度。

Description

一种解旋酶及其应用 技术领域
本发明涉及核酸特性的表征技术领域，具体涉及一种表征目标多核苷酸的方法，及一种F8813解旋酶、一种包含解旋酶的复合体结构及其在表征目标多核苷酸或控制目标多核苷酸穿过孔的运动中的应用。
背景技术
纳米孔测序技术是一种以单链核酸分子作为测序单元，利用一个能够提供离子电流通道的纳米孔，使得单链核酸分子在电泳驱动下通过该纳米孔，当核酸通过纳米孔时，会减少纳米孔的电流，对产生的不同信号实时读取序列信息的基因测序技术。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。
但是，每经过一个或一系列核苷酸时，会产生一种特定的电流，此时记录的电流信号与多核苷酸的序列虽是对应关系，却通常是3-4个核苷酸控制某些级别的电流水平，所以仍然需要提高精确度。目前，可以通过改变多核苷酸结构、在纳米孔处的持续时间从而控制多核苷酸的易位来提高精确度。改变在纳米孔处的持续时间从而控制多核苷酸易位的方法，主要是解决多核苷酸穿过纳米孔的易位速率太快、单个核苷酸太短的问题。目前，新兴的一种表征多核苷酸的方法，包括跨膜孔、解旋酶与多核苷酸的接触及相互作用，从而解旋酶控制目标多核苷酸穿过纳米孔的运动，以增加多核苷酸在纳米孔处的停留时间。
例如：专利WO2013057495A3公开了一种新的表征目标多核苷酸的方法，所述的方法使用孔和Hel308解旋酶或能结合目标多核苷酸内部核苷酸的分子马达。该发明所述的解旋酶或分子马达可以有效控制目标多核苷酸穿过所述孔的运动。
专利US20150065354A1公开了一种使用XPD解旋酶表征目标多核苷酸的方法，所述方法利用孔和XPD解旋酶。该发明所述的XPD解旋酶可以控制目标多核苷酸穿过所述孔的运动。
专利US20170268055A1公开了一种用于多核苷酸测序的组合物和方法，该方法利用用目标多核苷酸通过孔的易位分步易位步骤来表征目标多核苷酸，包括表征多核苷酸的序列的方法和组合物。
专利CN106103741A公开了将一个或多个多核苷酸结合蛋白连接到靶多核苷酸的方法，该发明还涉及表征靶多核苷酸的新方法。
但是上述发明均未公开本发明人设计的一种新的解旋酶。
在利用纳米孔、解旋酶测序的背景下，需要可导电的底物溶液作为必要环境，电荷载体(如盐)作为电压补偿来捕获或转移目标多核苷酸，并测量随着多核苷酸通过孔产生的序列依赖电流的变化。高盐的浓度对于增强获得的信号强度是有利的，因此测量多核苷酸的信号要求盐的浓度在一定的水平之上。高盐浓度为噪音比率提供了一个高的信号，并且允许在正常电流波动背景下，电流指示确定核苷酸的存在。
因此，本发明提供了一种新的解旋酶，用于核酸的表征，解决了常规解旋酶耐盐性不高的难题，明显提高多核苷酸特性表征的精确性。
发明内容
本发明人提供了一种在高盐浓度下进行核苷酸序列的表征的方法，因此设计制备了一种新的解旋酶和包含解旋酶的复合体结构，该解旋酶或复合体结构的耐盐性高的惊人，尤其适用在高盐浓度下进行核苷酸序列的表征。在核酸测序领域，盐的理想浓度为大于100mM，同时大于500mM的盐浓度也是比较好的状态，高盐条件下可以为噪音比率提供了一个高的信号，并且允许在正常电流波动背景下，电流指示确定核苷酸的存在。即本发明提供的用于表征核苷酸序列的解旋酶或复合体结构，可以在高盐浓度下工作，更重要的是，可以提高检测核酸序列的精确度。本发明解决了传统测序领域需要控制盐浓度的难题。
本发明的第一方面，涉及一种表征目标多核苷酸的方法，包括：
(a)将目标多核苷酸与孔，和解旋酶或复合体结构接触，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)用标准方法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；
所述的复合体结构包含解旋酶和用于结合多核苷酸的结合部分，所述的解旋酶或复合体结构在高盐浓度下具有解旋酶活性。
当在纳米孔施加一种力(如电压)，目标多核苷酸通过纳米孔的速率被解旋酶或复合体结构所控制，从而获得一种可识别的电流水平，用于确定目标多核苷酸的序列。
优选的，重复步骤(a)、(b)一次或多次。
优选的，所述的表征包括应用改进型维特比算法。
优选的，所述的高盐浓度为至少100mM、至少250mM、至少300mM、至少500mM、至少1000mM、至少1500mM、至少1800mM、至少2000mM、至少2500mM、至少3000mM或至少3500mM，其中，所述的盐选自KCl缓冲液、MgCl 2缓冲液或NaCl缓冲液。
在本发明的一个具体实施方式中，所述的盐浓度为250mM-2000mM。
优选的，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
优选的，所述的复合体结构包含解旋酶，和用于结合多核苷酸的结合部分，所述的解旋酶为Hel308家族的解旋酶，或者，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
进一步优选的，所述的结合部分选自真核单链结合蛋白、细菌单链结合蛋白、古生单链结合蛋白、病毒单链结合蛋白或双链结合蛋白。
优选的，所述的一个或多个特征选自目标多核苷酸的来源、长度、同一性、序列、二级结构或目标多核苷酸是否被修饰。
在本发明的一个具体实施方式中，所述的特征为序列。
优选的，所述的一个或多个特征通过电测量和/或光学测量进行。
进一步优选的，通过电测量和/或光测量产生电信号和/或光信号，而每种核苷酸对应一种信号水平，继而将电信号和/或光信号转化为核苷酸的序列特征。
本发明所述的电测量选自电流测量、阻抗测量、场效应晶体管(FET)测量、隧道测量或风洞测量。
本发明所述的电信号选自电流、电压、隧穿、电阻、电位、电导率或横向电测量的测量值。
在本发明的一个具体实施方式中，所述的电信号为穿过所述孔的电流。
优选的，所述的方法还包括横跨与所述解旋酶或复合体结构和目标多核苷酸接触的孔施加势差的步骤。
也就是说，本发明的方法可以包括：a)将目标多核苷酸与孔和高盐浓度下保持活性的解旋酶或包含解旋酶的复合体结构接触，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)横跨与所述解旋酶或所述复合体结构和目标多核苷酸接触的孔施加势差，测定目标多核苷酸中的核苷酸与所述孔相互作用时的电流；和c)将每种核苷酸所对应的电流信号水平进行转化，获取的目标多核苷酸的序列。
优选的，所述的目标多核苷酸为单链、双链或至少一部分是双链的。
优选的，所述的目标多核苷酸可以是DNA或RNA。
在本发明所述的一个具体实施方式中，所述的目标多核苷酸为至少一部分是双链的。其中所述的双链部分构成Y衔体结构，所述的Y衔体结构包含优先螺入所述孔的前导序列。
本发明所述的目标多核苷酸为含有一个或多个核苷酸的大分子。
本发明所述的目标多核苷酸可以是天然存在的或人工合成的。优选的，所述的目标多核苷酸中的一个或多个核苷酸可以是经过修饰的，例如甲基化、氧化、损伤、脱碱基的、蛋白标记、带有标签或多核苷酸序列中间连接一段间隔物。优选的，所述人工合成的核酸选自肽核酸(PNA)、甘油核酸(GNA)、苏糖核酸(TNA)、锁定核酸(LNA)、或其他具有核苷侧链的合成聚合物。
优选的，所述的孔为跨膜孔，所述的跨膜孔为生物孔、固态孔或生物与固态杂交的孔。
本发明所述的跨膜孔是允许水合离子在施加的电势的驱动下从膜的一侧流向膜的另一层的结构。所述跨膜孔为目标多核苷酸的移动提供了通道。
优选的，所述的膜为双层膜。优选的，所述的膜为脂质双层膜。
本发明所述的Y衔体结构包含优先螺入所述孔的前导序列，所述的前导序列3’端连接硫醇、生物素或胆固醇，用来与脂质双层膜的一层膜结合，以为目标多核苷酸指向正确的方向并具有拉动的作用。
在本发明的一个具体实施方式中，所述的前导序列3’端连接胆固醇，用来与脂质双层膜的一层膜结合。
优选的，所述的生物孔选自溶血素、杀白细胞素、耻垢分枝杆菌膜孔蛋白A(MspA)、耻垢分枝杆菌膜孔蛋白B、耻垢分枝杆菌膜孔蛋白C、耻垢分枝杆菌膜孔蛋白D、胞溶素、MZA、外膜蛋白F(OmpF)、外膜蛋白G(OmpG)、外膜磷脂酶A或奈瑟氏菌自转运脂蛋白(NalP)。
本发明的第二方面，涉及一种解旋酶(F8813解旋酶)，所述解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
优选的，所述的与SEQ ID NO：1所示氨基酸序列具有同源性并具有解旋酶活性的序列包括：与SEQ ID NO：1所示氨基酸序列的差异不超过20、15、10、5、4、3、2或1个氨基酸并具有解旋酶活性的序列；或为SEQ ID NO：1的变体，其中，所述变体与SEQ ID NO：1的差异包括取代、缺失和/或插入一个或多个氨基酸残基的序列或至少一个N-/C-末端延伸并具有解旋酶活性的序列。进一步优选的，所述的取代为保守的氨基酸取代。
优选的，所述的解旋酶结合到单链多核苷酸或双链多核苷酸的内部核苷酸。
优选的，所述的解旋酶在至少100mM、至少250mM、至少300mM、至少500mM、 至少1000mM、至少1500mM、至少1800mM、至少2000mM、至少2500mM、至少3000mM或至少3500mM的盐浓度下保持解旋酶活性，其中，所述的盐选自KCl缓冲液、MgCl 2缓冲液或NaCl缓冲液。
本发明所述的解旋酶或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，获得可辨认的电流水平。此外，F8813解旋酶或复合体结构将在高盐浓度下有效发挥作用。
本发明的第三方面，涉及一种编码上述第二方面所述的解旋酶的氨基酸序列的核苷酸序列。
优选的，所述的核苷酸序列为SEQ ID NO：2所示的核苷酸序列或与SEQ ID NO：2所示核苷酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性的序列。
本发明的第四方面，涉及一种复合体结构，所述的复合体结构包含解旋酶，和用于结合多核苷酸的结合部分。其中，所述的解旋酶附着在所述结合部分上，所述的复合体结构可以控制多核苷酸的运动。
优选的，所述的解旋酶为Hel308家族的解旋酶，或者，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
优选的，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。
优选的，所述的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。
进一步优选的，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ  ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
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。本发明所述的含有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(如100mM至2M)下活性更佳，其原因在于其耐盐性，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。
在任何情况下，复合体结构都可以被遗传。
优选的，解旋酶与结合部分是通过彼此的末端氨基酸。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。进一步优选的，所述的结合部分插入解旋酶的序列中。这样的结构可以将F8813解旋酶与结合部分通过两点很好的结合在一起。
优选的，所述的解旋酶为本发明所述的F8813解旋酶。
在本发明的一个具体实施方式中，所述的结合部分插入F8813解旋酶的螺旋(loop)区域。通过一个或更多(优选为2个或3个)的连接体(linkers)，将F8813解旋酶稳定的附着在结合部分上。优选的，所述的linkers可以限制结合部分的移动。经过修饰F8813解旋酶和/或修饰结合部分来增强linkers的连接。
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。
与多核苷酸结合的结合部分选自真核单链结合蛋白(SSB)、细菌SSBs、古生SSBs、病毒SSBs、双链结合蛋白中的一个或两个以上。具体序列见表1。
表1与多核苷酸结合的结合部分
Figure PCTCN2018108227-appb-000001
Figure PCTCN2018108227-appb-000002
本发明的第五方面，涉及一种上述第二方面所述的解旋酶或上述第三方面的核苷酸序列或第四方面所述的复合体结构在表征目标多核苷酸或控制目标多核苷酸穿过孔的运动中的应用。
本发明的第六方面，涉及一种表征目标多核苷酸的试剂盒，所述的试剂盒包括上述第二方面所述的解旋酶或上述第三方面的核苷酸序列或上述第四方面所述的复合体结构，和孔。
优选的，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。
优选的，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。进一步优选的，所述的生物孔选自溶血素、杀白细胞素、耻垢分枝杆菌膜孔蛋白A(MspA)、耻垢分枝杆菌膜孔蛋白B、耻垢分枝杆菌膜孔蛋白C、耻垢分枝杆菌膜孔蛋白D、胞溶素、MZA、外膜蛋白F(OmpF)、外膜蛋白G(OmpG)、外膜磷脂酶A或奈瑟氏菌自转运脂蛋白(NalP)。
优选的，所述的试剂盒还包括包含脂质双层的芯片。所述的孔横跨脂质双层。
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。优选的，所述的试剂包括缓冲剂、PCR扩增所需的工具。
本发明还提供了一种表征目标多核苷酸的传感器，包括在孔和解旋酶或复合体结构之间形成复合体，目标多核苷酸与孔相互作用，并由此形成的用于表征目标多核苷酸的传感器。
优选的，在所述目标多核苷酸存在下使所述孔和解旋酶或复合体结构接触，并跨所述孔施加电势。所述的电势选自电压电势或化学电势。
优选的，所述孔与所述解旋酶或所述复合体结构共价连接。
本发明的第七方面，涉及一种表征目标多核苷酸的装置，所述的装置包括上述第二方面所述的解旋酶或上述第三方面所述的核苷酸序列或上述第四方面所述的复合体结构，和孔。
优选的，所述的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。
优选的，所述的装置包括多个解旋酶或多个复合体结构，和多个孔。
优选的，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。进一步优选的，所述的生物孔选自溶血素、杀白细胞素、耻垢分枝杆菌膜孔蛋白A(MspA)、耻垢分枝杆菌膜孔蛋白B、耻垢分枝杆菌膜孔蛋白C、耻垢分枝杆菌膜孔蛋白D、胞溶素、MZA、外膜蛋白F(OmpF)、外膜蛋白G(OmpG)、外膜磷脂酶A或奈瑟氏菌自转运脂蛋白(NalP)。
本发明所述的“核苷酸”包括但不局限于：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)。优选的，所述核苷酸选自AMP、TMP、GMP、CMP、UMP、dAMP、dTMP、dGMP或dCMP。
本发明所述的“保守的氨基酸取代”包括但不限于：丙氨酸与丝氨酸、甘氨酸、苏氨酸、缬氨酸、脯氨酸或谷氨酸之间的替换；和/或，天冬氨酸与甘氨酸、天冬酰胺或谷氨酸之间的替换；和/或，丝氨酸与甘氨酸、天冬酰胺或苏氨酸之间的替换；和/或，亮氨酸与 异亮氨酸或缬氨酸之间的替换；和/或，缬氨酸与亮氨酸、异亮氨酸之间的替换；和/或，酪氨酸与苯丙氨酸之间的替换；和/或，赖氨酸与精氨酸之间的替换。上述所述的取代基本不会改变本发明所述的氨基酸序列的活性。
本发明所述的“和/或”包括择一列出的项目以及任何数量的项目组合。
本发明所述的“包括”是开放式的描述，含有所描述的指定成分或步骤，以及不会实质上影响的其他指定成分或步骤。
本发明所述的“同源性”，是指在使用蛋白序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％，40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，99.9％的同源性。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：SDS-PAGE凝胶电泳纯化F8813解旋酶的结果图，其中，M是Marker(Kd)，1道为F8813解旋酶的电泳结果图。
图2：应用荧光测定法检测酶活，其中，如a)所示荧光底物链(B，终浓度100nM)包含一个3'单链DNA部分和一个5'端杂化双链部分。5'端杂化双链部分中的主链在5'末端带有一个羧基荧光素(C)，而杂化双链的互补短链(D)3'末端带有一个BHQ-1碱基(E)。当将羧基荧光素(C)与BHQ-1碱基(E)混合后，羧基荧光素(C)就会被BHQ-1(E)退火，而底物本质上是非荧光的。在分析中，1μM的捕获链与荧光底物的短链互补。如b)所示，将ATP(0.5mM)、MgCl 2(10mM)和解旋酶(100nM)加入到底物上与荧光底物 的3'尾端结合，沿着主链移动，并取代互补链，开始解旋。如c)所示，解旋酶将双链完全解开，解旋酶脱落，主链上发出荧光。如d所示)，过量的捕获链(F)优先于互补DNA退火，以防止初始底物的重新退火和荧光的丧失。
图3：为在250mM的NaCl缓冲液下，分别测定阳性对照(positive control)、阴性对照(negative control)、F8813解旋酶随时间的推移，其荧光值的变化结果，其中，横坐标为时间(min)，纵坐标为荧光值。
图4：在浓度分别为250mM、500mM、1M和2M的NaCl缓冲溶液条件下，测试F8813解旋酶随时间的推移，其荧光值的变化结果，其中，横坐标为时间(min)，纵坐标为荧光值/阳性对照(％)。
图5：解旋酶控制DNA穿过纳米孔的各状态示意图。其中，过程1中f为纳米孔，d为磷脂双分子层(将空间分为cis区与trans区)，c为胆固醇标签，b为带有胆固醇标签(c)的前导序列，a为带有前导序列(b)的DNA单链基质。胆固醇标签与磷脂双分子层结合，使双分子层的表面底物更加丰富。过程2中解旋酶(e)结合在DNA单链上。在二价金属离子和NTP基质存在的条件下，解旋酶沿DNA移动。过程3中施加电压，DNA单链通过前导序列引导被纳米孔捕获。在力的作用下，DNA单链被拉过孔，直到解旋酶与纳米孔接触时阻碍DNA不受控制的易位。在这个过程中，双链DNA的部分(例如带有前导序列的部分)被移除。解旋酶沿着3'-5'方向移动，与电场方向相反。过程4中解旋酶将DNA从纳米孔中拉出，回到cis区，从纳米孔中出来的最后一部分是5'端。过程5中当解旋酶将DNA从纳米孔中移出时，解旋酶会从DNA链上脱落回到cis区。
图6：解旋酶能够控制DNA从纳米孔中的易位，并随着DNA的移动产生逐步变化的电流。在180mV，400mM KCl，10mM Hepes pH 8.0，0.10nM DNA，500nM F8813，2.86mM ATP，5mM MgCl 2的条件下，解旋酶F8813与DNA的结合由图中顶部的小箭头表示。其中，横坐标为时间(s)，纵坐标为电流(pA)。
图7：F8813解旋酶(图A)与F8813-SSBSsop7D解旋酶(图B)控制多核苷酸通过纳米孔的易位的电流图。
图8：在30分钟的实验过程中，F8813和F8813-ssbssop7d的解旋酶控制DNA运动速度的变化(y轴＝速率，x轴＝时间(秒))。
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，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 F8813解旋酶的重组表达
1、构建重组表达质粒
根据SEQ ID NO：1所示的氨基酸序列，利用密码子与宿主表达的频率的对应关系及人工分析，获得并优化F8813解旋酶的核酸序列(SEQ ID NO：2)。合成的F8813解旋酶核酸序列通过限制性核酸内切酶切割并且连入表达载体pET 15b中。经验证，重组质粒正确。
2、转化重组质粒
重组质粒通过热击转化到DE3感受态细胞，在37℃过夜培养，获得单克隆细胞和大量的包含重组质粒的大肠杆菌细胞。
3、F8813解旋酶的表达和纯化
稀释包含重组质粒的过夜培养液，将稀释液加入至含有1:50相应抗体含量的LB培养基中。37℃扩增培养，并连续不断的测量其OD600值。当OD600＝0.6-0.8时，将LB培养基中的培养液冷却至18℃，并添加异丙基硫代半乳糖苷(Isopropyl β-D-Thiogalactoside，IPTG)诱导表达，使得终浓度达到1mM。12-16h后，18℃收集细菌。高压破碎细菌，通过镍柱、肝素柱和分子筛柱的方法进行离心分离和纯化，收集上清液。纯化后通过SDS-PAGE凝胶电泳检测，结果如图1所示。
实施例2 荧光分析解旋酶取代杂化双链DNA的能力
应用荧光分析解旋酶取代杂化双链DNA的能力，步骤如图2所示，如a)所示荧光底 物链(B，终浓度100nM)包含一个3'单链DNA部分和一个5'端杂化双链部分。5'端杂化双链部分中的主链在5'末端带有一个羧基荧光素(C)，而杂化双链的互补短链(D)3'末端带有一个BHQ-1碱基(E)。当将羧基荧光素(C)与BHQ-1碱基(E)混合后，羧基荧光素(C)就会被BHQ-1(E)退火，而底物本质上是非荧光的。分析结果表明，1μM的捕获链与荧光底物的短链互补。如b)所示，将ATP(0.5mM)、MgCl 2(10mM)和解旋酶(100nM)加入到底物上与荧光底物的3'尾端结合，沿着主链移动，并取代互补链，开始解旋。如c)所示，解旋酶将双链完全解开，解旋酶脱落，主链上发出荧光。如d所示)，过量的捕获链(F)优先与互补DNA退火，以防止初始底物的重新退火和荧光的丧失。
基质DNA：5'-FAM-SEQ ID NO：3、SEQ ID NO：4-BHQ1-3。其中，FAM为羧基荧光素，BHQ1为荧光猝灭剂。
捕获DNA：SEQ ID NO：5。
图3为在250mM的NaCl缓冲液下，分别测定阳性对照、阴性对照、F8813解旋酶随时间的推移，其荧光值的变化结果，其中，阳性对照为步骤a中只含有羧基荧光素(C)，不含有BHQ-1碱基(E)；阴性对照为步骤b中不添加ATP，横坐标为时间(min)，纵坐标为荧光值。
其中，NaCl缓冲液(10mM Hepes pH8.0，0.5mM ATP，10mM MgCl2，100nM基质DNA，1μM捕获DNA)。
实施例3 利用荧光分析测酶活以确定F8813解旋酶的耐盐性
常规的解旋酶不能耐受高盐浓度，在高盐浓度下酶失去结构完整性或无法正常工作。本实施例旨在验证F8813解旋酶的耐盐性。在浓度分别为250mM、500mM、1M和2M的NaCl缓冲溶液条件下，依据实施例2的实验步骤，测试F8813解旋酶随时间的推移，其荧光值的变化结果，其中，横坐标为时间(min)，纵坐标为荧光值，The helicase works at 500mM。结果如图4所示。
其中，NaCl缓冲溶液为10mM Hepes pH8.0，0.5mM ATP，10mM MgCl 2，100nM基 质DNA，1μM捕获DNA。
实施例4 F8813解旋酶控制DNA链穿过纳米孔的移动
图5中记录了DNA通过纳米孔的全过程，DNA基质包含被纳米孔(SEQ ID NO：6)捕获的30nt的5'前导链，所述的前导链是一个带有3'胆固醇标签的引物(SEQ ID NO：7)，所述前导链与磷脂双分子层结合，丰富磷脂双分子层表面DNA，提高捕获效率。
5'Phos-TTGGT TTTTG TTTGT TTTTA GAATT TTTTT ACACT ACCAC TGCTA GCATTTTTCA TTTCT CACTA TCCCG TTCTC ATTGG TGCAC CATCT TTTTT TGGTTTTTTT GCAGC AGCAT-3’(SEQ ID NO：6)
5’-AACCAAAAAAAGATGGTGCACCAATGAGAACGGGATAGTGAGAAATTTTTT TTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTT-3'Chol(SEQ ID NO：7)
具体实验试剂及步骤如下：
缓冲溶液：400mM KCl，10mM Hepes pH 8.0，2.86mM ATP，5mM MgCl 2。
解旋酶：F8813解旋酶，终浓度从2μL至大于500nM。
将单个纳米孔插入1,2-2-甘油-3-磷酸胆碱脂质双层中，通入电压，获得电测量值。通过Montal-Mueller技术，在PTFE薄膜上形成了25μm的孔径，形成分离了两个0.1mL的缓冲溶液的双层膜。所有的实验在规定的缓冲溶液中进行。单通道的电流是用带数字化装置的放大器测量的。Ag/AgCl电极连接到缓冲溶液中，cis隔区(加入纳米孔、酶和DNA的区域)在上方，而trans间隔区与活性电极的探头相连。
脂质双层插入单个纳米孔之后，向cis区70μL缓冲液中加入DNA多核苷酸和解旋酶，捕获解旋酶与DNA复合体穿过纳米孔时的电信号。其中，DNA的终浓度为10nM，酶的终浓度为0.5μM。通过向cis区域添加二价金属离子(5mM MgCl 2)和NTP(2.86mM ATP)，检测解旋酶ATP酶活性，实验恒定电压为+180mV。
向纳米孔中添加如图5所示的解旋酶与DNA的复合体，产生了如图6所示的特征电流。在180mV的外力作用下，DNA通过纳米孔被捕获。未结合解旋酶的DNA迅速穿过纳米孔，产生短暂的电流(<<1s)。结合解旋酶(在ATP酶活性下沿着DNA链移动)的 DNA片段，随着DNA通过纳米孔移动，产生长特征块电流且电流逐步改变水平(如图6所示)。在纳米孔中不同的DNA结构产生独特的电流块水平。
实施例5 F8813解旋酶与F8813-SSBSsop7D(SEQ ID NO:17)的解旋酶在控制DNA通过纳米孔时的易位的对比
参见图2及序列，DNA(加入纳米孔的终浓度为1nM)室温下预培养五分钟，与F8813解旋酶(添加到纳米孔的终浓度100nM，SEQ ID NO：1)或F8813-SSBSsop7D(添加到纳米孔的终浓度100nM，SEQ ID NO：17和缓冲液(500mM NaCl,50mM Tris-Hcl,pH值8.0，1mM DTT))。15分钟后，向混合液中加入MgCl2(终浓度5mM)、ATP(终浓度2.86mM)和缓冲液(10mMHEPES，400mM KCl pH值8和0.5mg/mL BSA)。
在缓冲液(10mM HEPES,400mM KCl,pH 8.0,5mM MgCl 2)条件下，将共聚物插入的单个纳米孔，从单个纳米孔中获得了电测量。当将共聚物插入单个孔后，用缓冲液(2mL,10mM HEPES,400mM KCl,pH 8.0,5mM MgCl 2，去除多余的纳米孔。随后加入F8813解旋酶(终浓度100nM)或F8813-ssbssop7d解旋酶(终浓度100nM)，DNA(终浓度1nM)，能量(终浓度ATP 2.86mM)，共70μL预混料。解旋酶控制DNA的易位电流在180mV条件下检测。
F8813解旋酶(图7A)与F8813-SSBSsop7D解旋酶(图7B)分别控制多核苷酸通过纳米孔的易位电流。图8显示了在30分钟的实验过程中，F8813和F8813-ssbssop7d的解旋酶控制DNA运动速度的变化(y轴＝速率，x轴＝时间(秒))。
在同一时期内，F8813-ssbssop 7d与F8813相比，F8813-ssbssop 7d控制的DNA运动的量更多。在整个实验过程中，f8813-ssbssop7d显示了改进的解旋酶控制DNA运动速度且相当稳定。
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，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾 的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
Figure PCTCN2018108227-appb-000003
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Claims (22)

  1. 一种表征目标多核苷酸的方法，其特征在于，包括：
    (a)将目标多核苷酸与孔，和解旋酶或复合体结构接触，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；所述的复合体结构包含解旋酶和用于结合多核苷酸的结合部分，所述的解旋酶或复合体结构在高盐浓度下具有解旋酶活性。
  2. 根据权利要求1所述的方法，其特征在于，所述的高盐浓度为至少100mM、至少250mM、至少300mM、至少500mM、至少1000mM、至少1500mM、至少1800mM、至少2000mM、至少2500mM、至少3000mM或至少3500mM，其中，所述的盐选自KCl缓冲液、MgCl 2缓冲液或NaCl缓冲液。
  3. 根据权利要求1或2所述的方法，其特征在于，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
  4. 根据权利要求1或2任一所述的方法，其特征在于，所述的复合体结构包含解旋酶，和用于结合多核苷酸的结合部分；优选的，所述的解旋酶为Hel308家族的解旋酶，或者，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
  5. 根据权利要求4所述的方法，其特征在于，所述的结合部分选自真核单链结合蛋白、细菌单链结合蛋白、古生单链结合蛋白、病毒单链结合蛋白或双链结合蛋白。
  6. 根据权利要求1-5任一所述的方法，其特征在于，所述的一个或多个特征选自目标多核苷酸的来源、长度、同一性、序列、二级结构或目标多核苷酸是否被修饰。
  7. 根据权利要求1-6任一所述的方法，其特征在于，所述的一个或多个特征通过电测 量和/或光学测量进行。
  8. 根据权利要求1-7任一所述的方法，其特征在于，所述的方法还包括横跨与所述解旋酶或复合体结构，和目标多核苷酸接触的孔施加势差的步骤。
  9. 根据权利要求1-8任一所述的方法，其特征在于，所述的目标多核苷酸为单链、双链或至少一部分是双链的。
  10. 根据权利要求1-9任一所述的方法，其特征在于，所述的孔为跨膜孔，所述的跨膜孔为生物孔、固态孔或生物与固态杂交的孔。
  11. 一种解旋酶，其特征在于，所述解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
  12. 根据权利要求11所述的解旋酶，其特征在于，所述的解旋酶结合到单链多核苷酸或双链多核苷酸的内部核苷酸。
  13. 根据权利要求11所述的解旋酶，其特征在于，所述的解旋酶在至少100mM、至少250mM、至少300mM、至少500mM、至少1000mM、至少1500mM、至少1800mM、至少2000mM、至少2500mM、至少3000mM或至少3500mM的盐浓度下保持解旋酶活性，其中，所述的盐选自KCl缓冲液、MgCl 2缓冲液或NaCl缓冲液。
  14. 一种编码权利要求11-13任一所述的解旋酶的氨基酸序列的核苷酸序列。
  15. 根据权利要求14所述的核苷酸序列，其特征在于，所述的核苷酸序列为SEQ ID NO：2所示的核苷酸序列或与SEQ ID NO：2所示核苷酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性的序列。
  16. 一种复合体结构，其特征在于，所述的复合体结构包含解旋酶，和用于结合多核苷酸的结合部分。
  17. 根据权利要求16所述的复合体结构，其特征在于，所述的结合部分选自真核单 链结合蛋白、细菌单链结合蛋白、古生单链结合蛋白、病毒单链结合蛋白或双链结合蛋白。
  18. 根据权利要求16或17所述的复合体结构，其特征在于，所述的解旋酶为Hel308家族解旋酶。
  19. 根据权利要求16或17所述的复合体结构，其特征在于，所述的解旋酶的氨基酸序列为SEQ ID NO：1所示氨基酸序列或与SEQ ID NO：1所示氨基酸序列具有至少60％、至少70％、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％、至少99％或至少99.9％的同源性并具有解旋酶活性。
  20. 一种权利要求11-13任一所述的解旋酶或权利要求14-15任一所述的核苷酸序列或权利要求16-19任一所述的复合体结构在表征目标多核苷酸或控制目标多核苷酸穿过孔的运动中的应用。
  21. 一种表征目标多核苷酸的试剂盒，其特征在于，所述的试剂盒包括权利要求11-13任一所述的解旋酶或权利要求14-15任一所述的核苷酸序列或权利要求16-19任一所述的复合体结构，和孔。
  22. 一种表征目标多核苷酸的装置，其特征在于，所述的装置包括权利要求11-13任一所述的解旋酶或权利要求14-15任一所述的核苷酸序列或权利要求16-19任一所述的复合体结构，和孔。
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