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WO2020061997A1 - Hélicase et son utilisation - Google Patents

Hélicase et son utilisation 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
Prior art date
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Ceased
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PCT/CN2018/108227
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English (en)
Chinese (zh)
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 PCT/CN2018/108227 priority Critical patent/WO2020061997A1/fr
Priority to CN201880095718.XA priority patent/CN112805393A/zh
Publication of WO2020061997A1 publication Critical patent/WO2020061997A1/fr
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

L'invention concerne un procédé de caractérisation d'un polynucléotide cible, et une hélicase F8813, une structure complexe contenant l'hélicase et son utilisation dans la caractérisation d'un polynucléotide cible ou la régulation du mouvement d'un polynucléotide cible par l'intermédiaire d'un pore. La structure hélicase F8813 ou complexe présente une forte tolérance au sel, qui utilisée dans le procédé de caractérisation d'un polynucléotide cible peut améliorer la précision de la détection de séquence d'acide nucléique.
PCT/CN2018/108227 2018-09-28 2018-09-28 Hélicase et son utilisation Ceased WO2020061997A1 (fr)

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WO2022126304A1 (fr) * 2020-12-14 2022-06-23 北京齐碳科技有限公司 Hélicase modifiée et son application
EP4458987A4 (fr) * 2021-12-31 2025-10-22 Bgi Shenzhen Hélicase bch1x et son utilisation

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CN114230644A (zh) * 2021-12-29 2022-03-25 南京巨匠生物科技有限公司 一种gp32蛋白突变体、重组载体及其构建方法和应用
US20250066748A1 (en) * 2021-12-31 2025-02-27 Bgi Shenzhen Helicase bch2x and use thereof
CN117721101B (zh) * 2022-09-16 2024-09-20 北京普译生物科技有限公司 一种经修饰的CaPif1解旋酶及其应用
WO2024138635A1 (fr) * 2022-12-30 2024-07-04 深圳华大生命科学研究院 Hélicase et son procédé de préparation et son utilisation dans un séquençage à haut débit
WO2024138632A1 (fr) * 2022-12-30 2024-07-04 深圳华大生命科学研究院 Hélicase, son procédé de préparation et son utilisation dans le séquençage
EP4644570A1 (fr) * 2022-12-30 2025-11-05 BGI Shenzhen Mutant d'hélicase, son procédé de préparation et son utilisation dans un séquençage à haut débit

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WO2022126304A1 (fr) * 2020-12-14 2022-06-23 北京齐碳科技有限公司 Hélicase modifiée et son application
CN116601297A (zh) * 2020-12-14 2023-08-15 北京齐碳科技有限公司 一种经修饰的解旋酶及其应用
EP4458987A4 (fr) * 2021-12-31 2025-10-22 Bgi Shenzhen Hélicase bch1x et son utilisation

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