WO2021253410A1 - 一种Pif1-like解旋酶及其应用 - Google Patents

一种Pif1-like解旋酶及其应用 Download PDF

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WO2021253410A1
WO2021253410A1 PCT/CN2020/097126 CN2020097126W WO2021253410A1 WO 2021253410 A1 WO2021253410 A1 WO 2021253410A1 CN 2020097126 W CN2020097126 W CN 2020097126W WO 2021253410 A1 WO2021253410 A1 WO 2021253410A1
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helicase
pif1
seq
acid
phenylalanine
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PCT/CN2020/097126
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English (en)
French (fr)
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张周刚
王慕旸
郭倩倩
陈呈尧
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北京齐碳科技有限公司
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Priority to JP2022551797A priority Critical patent/JP2023515597A/ja
Priority to KR1020227026798A priority patent/KR20220124741A/ko
Priority to PCT/CN2020/097126 priority patent/WO2021253410A1/zh
Priority to EP20941459.8A priority patent/EP4063382A4/en
Priority to BR112022014144A priority patent/BR112022014144A2/pt
Priority to IL294925A priority patent/IL294925A/en
Priority to CN202080073171.0A priority patent/CN114599666A/zh
Publication of WO2021253410A1 publication Critical patent/WO2021253410A1/zh
Priority to US17/590,447 priority patent/US20220290223A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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

Definitions

  • the present invention relates to the technical fields of gene sequencing, molecular detection and clinical detection, in particular to a modified Pif1-like helicase, a construct containing Pif1-like helicase, and its use in characterizing target polynucleotides or controlling targets Application in the movement of polynucleotides through pores.
  • Nanopore sequencing technology refers to a gene sequencing technology that uses a single nucleic acid molecule as a measurement unit and uses nanopores to read its sequence information in real time and continuously.
  • Electrophoresis drives the polynucleotide through the nanopore, and since the polynucleotide passes through the nanopore, the current passing through the nanopore can be reduced. Each passing nucleotide or series of nucleotides obtains a characteristic current, and the record of the current level corresponds to the polynucleotide sequence.
  • strand sequencing methods (such as the use of a helicase to control the movement of polynucleotides through a pore), a single polynucleotide chain passes through the pore and enables identification of nucleotides.
  • the advantage of this sequencing technology is that it is simple to build a library and does not require amplification; it has a fast reading speed, which can reach a reading speed of tens of thousands of bases per hour for a single molecule; the read length is very long, usually thousands of bases; it is possible It can directly measure RNA and DNA methylation. These are beyond the reach of the existing second-generation sequencing technology.
  • nanopore sequencing technology also has thorny problems that need to be solved.
  • the translocation of polynucleotides through the nanopore is so fast that the current level of a single nucleotide is too short to be distinguished.
  • the molecular motor that controls the movement of the polynucleotide may be free from the polynucleotide. This allows the polynucleotide to be quickly pulled through the pore in an uncontrolled manner in the direction of the applied field.
  • patent WO2013057495A3 discloses a new method for characterizing target polynucleotides. The method includes controlling the passage of target polynucleotides through Hel308 helicase or molecular motors. The movement of the hole.
  • Patent US20150065354A1 discloses a method for characterizing target polynucleotides using XPD helicase, which includes controlling the movement of target polynucleotides through pores by XPD helicase.
  • Patent CN107109380A discloses a modified enzyme, which is a modified Dda helicase that can control the movement of the target polynucleotide through the pore.
  • the present invention provides a new modified Pif1-like helicase.
  • the modified Pif1-like helicase can be maintained for a longer time. It binds to polynucleotides and controls the movement of polynucleotides through the pores.
  • the Pif1-like helicase of the present invention is a useful tool for controlling the movement of polynucleotides in the process of chain sequencing. It can make the polynucleotides follow or counter the electric field caused by the applied voltage in a controlled and stepwise manner. Perform movement to control the speed at which the polynucleotide passes through the nanopore and obtain a recognizable current level.
  • the Pif1-like helicase of the present invention will still not recover from the polynucleoside Acid-free, that is, it is particularly effective for controlling the movement of 500, 1000, 5000, 10000, 20000, 50000, 100000 or more polynucleotides. Based on the advantages of the Pif1-like helicase of the present invention in the field of chain sequencing to control the smooth movement of polynucleotides from the pore and reduce slippage or irregular movement, it can promote more accurate nucleotides and longer read lengths.
  • a Pif1-like helicase including at least one cysteine introduced into the tower domain, pin domain and/or 1A domain (RecA type motor) of the Pif1-like helicase Amino acid residues and/or at least one unnatural amino acid, wherein the helicase retains its ability to control the movement of the polynucleotide.
  • it includes introducing at least one cysteine residue and/or at least one unnatural amino acid in any of the following groups:
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more cysteine residues can be introduced into the tower domain, pin domain and/or 1A domain , Or introduce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more unnatural amino acids, or introduce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more cysteine residues and unnatural amino acids.
  • the introduction of cysteine residues and/or at least one unnatural amino acid makes the binding of the Pif1-like helicase to the polynucleotide more stable and enhances the ability to control its movement.
  • the Pif1-like helicase is selected from Pba-PM2, Aph-Acj61, Aph-PX29, Avi-Aeh1, Sph-CBH8, Eph-Pei26, Aph-AM101, PphPspYZU05, Eph-EcS1, Eph- Cronus2 or Mph-MP1.
  • the wild-type Pif1-like helicase is shown in Table 1.
  • Table 2 identifies the residues that constitute each domain in each Pif1-like helicase
  • the Pif1-like helicase is selected from Mph-MP1, Sph-CBH8, Eph-Pei26 or PphPspYZU05.
  • the Pif1-like helicase includes a variant of SEQ ID NO: 11, in which the tower domain (residues E264-P278 and N296-P389) and/or the pin domain (residues K97-A113) ) And/or 1A domain (residues M1-L96 and P114-K184) introduced at least one cysteine residue and/or at least one unnatural amino acid.
  • the Pif1-like helicase includes a variant of SEQ ID NO: 1, in which the tower domain (residues E264-P278 and N296-A394) and/or the pin domain (residues K89-E105) ) And/or 1A domain (residues M1-L88 and M106-V181) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 2, in which the tower domain (residues E265-P279 and N297-A392) and/or the pin domain (residues K89-D105) ) And/or 1A domain (residues M1-L88 and I106-M180) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 3, in which the tower domain (residues T266-P280 and N298-S403) and/or the pin domain (residues K89-A109) ) And/or 1A domain (residues M1-L88 and K110-V182) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 4, in which the tower domain (residues T266-P280 and N298-S404) and/or the pin domain (residues K89-A109) ) And/or 1A domain (residues M1-L88 and K110-V182) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 5, in which the tower domain (residues E260-P274 and N292-A391) and/or the pin domain (residues K86-E102) ) And/or 1A domain (residues M1-L84 and M103-K177) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 6, in which the tower domain (residues E266-P280 and N298-A396) and/or the pin domain (residues K91-E107 ) And/or 1A domain (residues M1-L90 and M108-M183) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 7, in which the tower domain (residues T276-P290 and N308-P402) and/or the pin domain (residues K100-D116) ) And/or 1A domain (residues M1-L99 and D117-M191) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 8, in which the tower domain (residues D274-P288 and N306-A404) and/or the pin domain (residues K95-E112) ) And/or 1A domain (residues M1-L95 and I113-K187) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 9, in which the tower domain (residues E260-P274 and N292-A391) and/or the pin domain (residues K86-E102 ) And/or 1A domain (residues M1-L85 and M103-K177) introduced at least one cysteine residue and/or at least one unnatural amino acid;
  • the Pif1-like helicase includes a variant of SEQ ID NO: 10, in which the tower domain (residues E265-P279 and H297-A393) and/or the pin domain (residues K88-E104 ) And/or 1A domain (residues M1-L87 and I105-K180) introduced at least one cysteine residue and/or at least one unnatural amino acid.
  • the tower domain (residues E265-P279 and H297-A393) and/or the pin domain (residues K88-E104 ) And/or 1A domain (residues M1-L87 and I105-K180) introduced at least one cysteine residue and/or at least one unnatural amino acid.
  • the Pif1-like helicase includes a variant of SEQ ID NO: 11, which includes (i) E105C and/or A362C; (ii) E104C and/or K360C; (iii) E104C and/or A362C ; (Iv) E104C and/or Q363C; (v) E104C and/or K366C; (vi) E105C and/or M356C; (vii) E105C and/or K360C; (viii) E104C and/or M356C; (ix) E105C And/or Q363C; (x) E105C and/or K366C; (xi) F108C and/or M356C; (xii) F108C and/or K360C; (xiii) F108C and/or A362C; (xiv) F108C and/or Q363C; (xv) F108C and/or K366C; (xvi) K134C
  • the Pif1-like helicase includes a variant of any one of SEQ ID NO: 1-10, which is defined by any one of (i) to (liv) in SEQ ID NO: 11
  • the position corresponding to the position includes a cysteine residue.
  • the amino acid sequence of the Pif1-like helicase is the amino acid sequence shown in SEQ ID NO: 1 to 11 or has at least 30%, at least 40%, or at least 50% of the amino acid sequence shown in SEQ ID NO: 1 to 11. %, 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 the ability to control the movement of polynucleotides.
  • the amino acid corresponding to the 105th and/or 362th position of SEQ ID NO: 11 introduces a cysteine residue, for example, the 97th position of SEQ ID NO: 2 Position and/or position 363, position 96 and/or 371 of SEQ ID NO: 3, position 94 and/or 361 of SEQ ID NO: 5, position 99 and/or position of SEQ ID NO: 6 / Or the 366th position, the 104th and/or the 375th position of SEQ ID NO: 8, etc.
  • the introduced cysteine and cysteine are connected to each other,
  • the non-natural amino acids of serotonin and the natural amino acids on the helicase are connected to each other.
  • cysteines and/or unnatural amino acids can be linked to each other.
  • 3, 4, 5, 6, 7, 8 or more cysteines and/or unnatural amino acids can be linked to each other.
  • One or more cysteines can be linked to one or more cysteines.
  • One or more cysteines can be linked to one or more unnatural amino acids such as Faz.
  • One or more unnatural amino acids such as Faz can be linked to one or more unnatural amino acids such as Faz.
  • One or more cysteines can be linked to one or more natural amino acids on the helicase.
  • One or more unnatural amino acids such as Faz can be linked to one or more natural amino acids on the helicase.
  • connection can be in any manner, including direct connection or indirect connection.
  • connection can be a short-term contact or a permanent connection. More preferably, the connection may be a non-covalent connection or a covalent connection.
  • the covalent connection can be connected by a chemical crosslinking agent, linear molecule or catalyst.
  • the chemical crosslinking agent includes, but is not limited to, maleimide, active ester, succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO), difluorocycloalkyne Hydrocarbons and linear alkynes), etc.; the length of the chemical crosslinking agent can vary from one carbon (phosgene connector) to multiple angstroms.
  • the linear molecules include but are not limited to polyethylene glycol (PEGs), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and Unsaturated hydrocarbon, polyamide.
  • the catalyst includes, but is not limited to, TMAD, etc., which can make between cysteine residues, between unnatural amino acids, between cysteine residues and unnatural amino acids, between unnatural amino acids and natural amino acids, Or a catalyst that creates a covalent bond between a cysteine residue and a natural amino acid.
  • the Pif1-like helicase is further modified to remove one or more cysteine residues.
  • the Pif1-like helicase further includes at least one or more natural cysteine substituted. More preferably, alanine, serine or valine is substituted for cysteine.
  • the Pif1-like helicase includes a variant of SEQ ID NO: 5, and the one or more substituted natural cysteine residues are one of C109, C114, C136 or C414 Or more.
  • the Pif1-like helicase includes a variant of any one of SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 9, 10, and 11, and the one or more The substituted natural cysteine residues correspond to one or more of C109, C114, C136 or C414 in SEQ ID NO: 5.
  • Table 3 shows the amino acid positions in SEQ ID NOs: 1, 2, 3, 4, 6, 7, 8, 9, 10, and 11 corresponding to C109, C114, C136, and C414 in SEQ ID NO: 5.
  • the Pif1-like helicase is further modified to reduce the negative charge on its surface.
  • the Pif1-like helicase further includes a substitution that increases the net positive charge.
  • the substitutions that increase the net positive charge include substitution or modification of negatively charged amino acids, polar or non-polar amino acids, or negatively charged amino acids, polar or non-polar amino acids on the surface. A positively charged amino acid is introduced in the adjacent position.
  • the substitutions that increase the net positive charge include substitution of positively charged amino acids for negatively charged amino acids, uncharged amino acids, aromatic amino acids, polar or non-polar amino acids.
  • the substitutions that increase the net positive charge include substitution of uncharged amino acids for negatively charged amino acids, aromatic amino acids, polar or non-polar amino acids.
  • Suitable positively charged amino acids include, but are not limited to, histidine (H), lysine (K) and/or arginine (R).
  • Uncharged amino acids have no net charge.
  • Suitable uncharged amino acids include, but are not limited to, cysteine (C), serine (S), threonine (T), methionine (M), asparagine (N) or glutamine (Q) .
  • Non-polar amino acids have non-polar side chains.
  • Non-polar amino acids include, but are not limited to, glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L) or valine (V).
  • Aromatic amino acids have aromatic side chains. Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W) or tyrosine (Y).
  • the positively charged amino acids, uncharged amino acids, polar, non-polar amino acids or aromatic amino acids may be natural or unnatural amino acids, which may be artificially synthesized or modified natural amino acids .
  • substitutions include, but are not limited to, arginine (R) for glutamic acid (E), lysine (K) for glutamic acid (E), and asparagine (N) for glutamic acid ( E), substituting lysine (K) for aspartic acid (D), and substituting arginine (R) for aspartic acid (D).
  • the Pif1-like helicase includes a variant of SEQ ID NO: 11 and the one or more negatively charged amino acids are D5, E9, E24, E87, I65, S58, D209 or D216.
  • the one or more negatively charged amino acids are D5, E9, E24, E87, I65, S58, D209 or D216.
  • Any number of these amino acids can be neutralized, such as 1, 2, 3, 4, 5, 6, 7 or 8 of these amino acids. Can neutralize any combination.
  • the Pif1-like helicase includes a variant of any one of SEQ ID NO: 1 to 10, and the one or more negatively charged amino acids correspond to D5, E9 in SEQ ID NO: 11. , E24, E87, I65, S58, D209 or D216 one or more.
  • the amino acids in SEQ ID NOs: 1 to 10 corresponding to D5, E9, E24, E87, I65, S58, D209, and D216 in SEQ ID NO: 11 can be determined using the alignment in FIG. 11.
  • Preferred substitutions also include a variant of SEQ ID NO: 1, and the one or more negatively charged amino acids also include S171.
  • Preferred substitutions also include variants of SEQ ID NO: 9, and the one or more negatively charged amino acids also include S173.
  • the unnatural amino acid is selected from 4-azido-L-phenylalanine (Faz), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenoalkyl)-L-alanine, O-2-propyn-1-yl -L-tyrosine, 4(dihydroxyboronyl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino- 3- ⁇ 4-[(Propan-2-ylsulfanyl)carbonyl]phenyl ⁇ propionic acid, (2S)-2-amino-3- ⁇ 4-[(2-amino-3-sulfanylpropionyl )Amino]phenyl ⁇ propionic acid,
  • the Pif1-like helicase further includes:
  • At least one amino acid that interacts with one or more nucleotides in single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) is substituted; and/or,
  • the Pif1-like helicase has the ability to control the movement of polynucleotides.
  • At least one of the sugars and/or bases that interact with one or more nucleotides in single-stranded or double-stranded DNA is replaced with an amino acid containing a larger side chain (R group) Of amino acids.
  • the Pif1-like helicase includes replacing at least one sugar and/or base with one or more nucleotides of single-stranded or double-stranded DNA with an amino acid containing a larger side chain (R group).
  • Base interacting amino acids Any number of amino acids can be substituted, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more Amino acids.
  • Each amino acid can interact with a base, a sugar, or a base and a sugar.
  • Protein modeling can be used to identify amino acids that interact with the sugars and/or bases of one or more nucleotides in single-stranded or double-stranded DNA.
  • the Pif1-like helicase comprises a variant of SEQ ID NO: 11, wherein the at least one sugar and/or base is linked to one or more nucleotides in single-stranded or double-stranded DNA
  • the interacting amino acid is at least one of P73, H93, N99, F109, I280, A161, F130, D132, D162, D163, E277, K415, Q291, H396, Y244 or P100.
  • These numbers correspond to the relevant positions in SEQ ID NO: 11, and compared to SEQ ID NO: 11, it may be necessary to change when one or more amino acids have been inserted or deleted in the variant. As mentioned above, those skilled in the art can determine the corresponding positions in the variant.
  • the Pif1-like helicase is a variant of any one of SEQ ID NO: 1 to 10, wherein the at least one sugar is associated with one or more nucleotides in single-stranded or double-stranded DNA.
  • the base interacting amino acid is at least one of P73, H93, N99, F109, I280, A161, F130, D132, D162, D163, E277, K415, Q291, H396, Y244 or SEQ ID NO: 11 The amino acid corresponding to P100.
  • Table 4 shows the SEQ ID NOs corresponding to P73, H93, N99, F109, I280, A161, F130, D132, D162, D163, E277, K415, Q291, H396, Y244 and P100 in SEQ ID NO: 11: Amino acids from 1 to 10.
  • the larger side chain (R group) preferably (a) contains an increased number of carbon atoms (b) has an increased length (c) has an increased molecular volume and/or (d) has an increased van der Waals volume.
  • Larger side chains (R groups) are preferably (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a) ), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d).
  • Standard methods in the art can be used to measure each of (a) to (d).
  • the larger side chain (R group) increases the (i) electrostatic interaction between the at least one amino acid and one or more nucleotides in the single-stranded or double-stranded DNA; ii) Hydrogen bonding and/or (iii) cation-pi ((cation- ⁇ )) interaction.
  • positively charged amino acids such as arginine (R), histidine (H), and lysine (K) have R groups that increase electrostatic interaction.
  • amino acids such as asparagine (N), serine (S), glutamine (Q), threonine (T), and histidine (H) have R groups that increase hydrogen bonding. group.
  • aromatic amino acids such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) have increased cation-pi (cation- ⁇ ) Interacting R groups.
  • the amino acid containing the larger side chain (R) may be an unnatural amino acid.
  • the unnatural amino acid can be any of the ones discussed below.
  • the amino acids of the larger side chain (R group) are not alanine (A), cysteine (C), glycine (G), selenocysteine (U), methionine (M), aspartic acid (D) or glutamic acid (E).
  • the Pif1-like helicase contains one or more of the following substitutions:
  • Histidine (H) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q) or asparagine (N); or (iii) phenylpropanine Amino acid (F), tyrosine (Y) or tryptophan (W) substitution. Histidine (H) is more preferably substituted with (a) N, Q or W or (b) Y, F, Q or K.
  • Asparagine (N) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q) or histidine (H); or (iii) phenylpropanine Amino acid (F), tyrosine (Y) or tryptophan (W) substitution. Asparagine (N) is more preferably substituted by R, H, W or Y.
  • Proline (P) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N), threonine (T) Or histidine (H); (iii) tyrosine (Y), phenylalanine (F) or tryptophan (W); or (iv) leucine (L), valine (V) Or isoleucine (I) substitution.
  • Proline (P) is more preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N), threonine (T) or Histidine (H); (iii) Phenylalanine (F) or tryptophan (W) or (iv) Leucine (L), Valine (V) or Isoleucine (I) substitution .
  • Proline (P) is more preferably substituted by (a) F, (b) L, V, I, T or F or (c) W, F, Y, H, I, L or V.
  • Valine (V) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H) ; (Iii) Phenylalanine (F), tyrosine (Y) or tryptophan (W); or (iv) isoleucine (I) or leucine (L) substitution.
  • Valine (V) is more preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H); (iii) Tyrosine (Y) or tryptophan (W); or (iv) isoleucine (I) or leucine (L) substitution.
  • Valine (V) is more preferably substituted by I or H or I, L, N, W or H.
  • Phenylalanine (F) is preferably (i) arginine (R) or lysine (K); (ii) histidine (H); or (iii) tyrosine (Y) or color Amino acid (W) substitution. Phenylalanine (F) is more preferably substituted with (a) W, (b) W, Y or H, (c) W, R or K or (d) K, H, W or R.
  • Glutamine (Q) is preferably (i) arginine (R) or lysine (K) or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W )replace.
  • Alanine (A) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H) ; (Iii) Phenylalanine (F), tyrosine (Y) or tryptophan (W) or (iv) isoleucine (I) or leucine (L) substitution.
  • Serine (S) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H); ( iii) Phenylalanine (F), tyrosine (Y) or tryptophan (W); or (iv) isoleucine (I) or leucine (L) substitution.
  • Serine (S) is preferably substituted by K, R, W or F.
  • Lysine (K) is preferably substituted by (i) arginine (R) or (iii) tyrosine (Y) or tryptophan (W).
  • Arginine (R) is preferably substituted by (iii) Tyrosine (Y) or Tryptophan (W).
  • Methionine (M) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H ) Or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W) substitution.
  • L) Leucine (L) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q) or asparagine (N) or (iii) phenylalanine Acid (F), tyrosine (Y) or tryptophan (W) substitution.
  • Aspartic acid (D) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H ); or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W) substitution. Aspartic acid (D) is more preferably substituted by H, Y or K.
  • Glutamic acid (E) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H) Or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W) substitution.
  • Isoleucine (I) is preferably (i) arginine (R) or lysine (K); (ii) glutamine (Q), asparagine (N) or histidine (H ); (iii) Phenylalanine (F), tyrosine (Y) or tryptophan (W) or (iv) leucine (L) substitution.
  • Tyrosine (Y) is preferably substituted by (i) arginine (R) or lysine (K); or (ii) tryptophan (W). Tyrosine (Y) is more preferably substituted by W or R.
  • the Pif1-like helicase more preferably comprises a variant of SEQ ID NO: 11, which comprises:
  • the helicase of the present invention is preferably a helicase in which at least one amino acid that interacts with one or more phosphate groups of one or more nucleotides in ssDNA or dsDNA is substituted. Any number of amino acids can be substituted, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more Amino acids.
  • the nucleotides in ssDNA each contain three phosphate groups. Each amino acid that is substituted can interact with any number of phosphate groups at a time, for example, one, two, or three phosphate groups at a time. Amino acids that interact with one or more phosphate groups can be identified using protein modeling.
  • the substitution preferably increases (i) electrostatic interaction between the at least one amino acid and one or more phosphate groups in the ssDNA or dsDNA; (ii) hydrogen bonding and/or (iii) cation-pi (cation- ⁇ ) )interaction.
  • the preferred substitutions of (i), (ii) and (iii) are discussed using the labels (i), (ii) and (iii).
  • the substitution preferably increases the net positive charge of the site.
  • Methods known in the art can be used to measure the net charge at any site.
  • the isoelectric point can be used to define the net charge of an amino acid.
  • the net charge is usually measured at about 7.5.
  • the substitution is preferably to replace the negatively charged amino acid with a positively charged, uncharged, non-polar or aromatic amino acid.
  • a negatively charged amino acid is an amino acid with a net negative charge.
  • Negatively charged amino acids include, but are not limited to, aspartic acid (D) and glutamic acid (E).
  • a positively charged amino acid is an amino acid with a net positive charge.
  • the positively charged amino acids can be naturally occurring or non-naturally occurring.
  • the positively charged amino acids can be synthetic or modified.
  • modified amino acids with a net positive charge can be specifically designed for use in the present invention.
  • Many different types of modifications to amino acids are well known in the art.
  • Preferred naturally occurring positively charged amino acids include, but are not limited to, histidine (H), lysine (K), and arginine (R).
  • Uncharged amino acids, non-polar amino acids, or aromatic amino acids may be naturally occurring or non-naturally occurring. It can be synthetic or modified. Uncharged amino acids have no net charge. Suitable uncharged amino acids include, but are not limited to, cysteine (C), serine (S), threonine (T), methionine (M), asparagine (N) and glutamine (Q ). Non-polar amino acids have non-polar side chains. Suitable non-polar amino acids include, but are not limited to, glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L) and valine (V) . Aromatic amino acids have aromatic side chains. Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W), and tyrosine (Y).
  • the Pif1-like helicase preferably comprises a variant of SEQ ID NO: 11, wherein at least one amino acid that interacts with one or more phosphate groups of one or more nucleotides in ssDNA or dsDNA is H75, T91, At least one of S94, K97, N246, N247, N284, K288, N297, T394 or K397.
  • SEQ ID NO: 11 At least one of S94, K97, N246, N247, N284, K288, N297, T394 or K397.
  • the Pif1-like helicase preferably comprises a variant of SEQ ID NO: 1 to 10, wherein the at least one interacts with one or more phosphate groups of one or more nucleotides in ssDNA or dsDNA
  • the amino acid of is at least one amino acid corresponding to H75, T91, S94, K97, N246, N247, N284, K288, N297, T394 or K397 in SEQ ID NO: 11.
  • Table 5 shows the amino acids in SEQ ID NO: 1 to 10 corresponding to H75, T91, S94, K97, N246, N247, N284, K288, N297, T394, and K397 in SEQ ID NO: 11.
  • the Pif1-like helicase comprises any one or more of the following:
  • Histidine is (i) arginine (R) or lysine (K); (ii) asparagine (N), serine (S), glutamine (Q) or threonine Acid (T); or (iii) phenylalanine (F), tryptophan (W) or tyrosine (Y) substitution;
  • Threonine is (i) arginine (R), histidine (H) or lysine (K); (ii) asparagine (N), serine (S), glutamine Amide (Q) or histidine (H); or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) substitution;
  • Serine is (i) arginine (R), histidine (H) or lysine (K); (ii) asparagine (N), glutamine (Q), threonine (T ) Or histidine (H); or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) substitution;
  • Asparagine (N) is (i) arginine (R), histidine (H) or lysine (K); (ii) serine (S), glutamine (Q), threonine Acid (T) or histidine (H); or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) substitution; and/or,
  • Lysine (K) is (i) arginine (R) or histidine (H); (ii) asparagine (N), serine (S), glutamine (Q), threonine Acid (T) or histidine (H); or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) substitution.
  • the Pif1-like helicase is a variant of SEQ ID NO: 11, which includes one or more of (a) to (k), wherein:
  • the helicase of the present invention is also such a helicase, wherein a part of the helicase that interacts with the transmembrane pore contains one or more modifications, preferably one or more substitutions, and it is further preferred that the helicase includes At least one amino acid that interacts with the transmembrane pore is substituted.
  • the part of the helicase that interacts with the transmembrane pore is usually the part of the helicase that interacts with the transmembrane pore when the helicase is used to control the movement of polynucleotides through the pore.
  • the portion When helicases are used to control the movement of polynucleotides through a pore, the portion usually contains amino acids that interact or come into contact with the pore. When an electric potential is applied, when the helicase is bound or linked to a polynucleotide that is moving through the pore, the portion usually contains amino acids that interact or contact with the pore.
  • the part that interacts with the transmembrane pore preferably includes a variant of SEQ ID NO: 11, where the position includes one or more amino acids on E196, W202, N199 or G201 being substituted, for example, 2, 3, 4 Or 5.
  • the part that interacts with the transmembrane pore preferably includes a variant of any one of SEQ ID NO: 1 to 10, which includes SEQ ID NO: 11 in (a) E196; (b) W202; (c); N199; or (d) At least one or more substitutions corresponding to the G201 position.
  • Table 6 shows the amino acids in SEQ ID NO: 1 to 10 corresponding to E196, W202, N199, and G201 in SEQ ID NO: 11.
  • the Pif1-like helicase is a variant of SEQ ID NO. 11, which includes substitutions at the following positions:
  • -F109/E196/H75 for example, F109W/E196L/H75N, F109W/E196L/H75Q, F109W/E196L/H75K or F109W/E196L/H75F;
  • -F109/E196/T91 for example, F109W/E196L/T91K, F109W/E196L/T91Q or F109W/E196L/T91N;
  • F109/S94/E196 for example, F109W/S94H/E196L, F109W/S94T/E196L, F109W/S94R/E196L, F109W/S94Q/E196L, F109W/S94N/E196L, or F109W/S94K/E196L;
  • F109/N99/E196 for example, F109W/N99R/E196L, F109W/N99H/E196L, F109W/N99W/E196L or F109W/N99Y/E196L;
  • -F109/P100/E196 for example, F109W/P100L/E196L, F109W/P100V/E196L, F109W/P100I/E196L or
  • -F109/D132/E196 for example, F109W/D132H/E196L, F109W/D132Y/E196L or F109W/D132K/E196L;
  • -F109/A161/E196 for example, F109W/A161I/E196L, F109W/A161L/E196L, F109W/A161N/E196L, F109W/A161W/E196L or F109W/A161H/E196L;
  • F109/D163/E196 for example, F109W/D163W/E196L, F109W/D163F/E196L, F109W/D163Y/E196L, F109W/D163H/E196L, F109W/D163I/E196L, F109W/D163L/E196L or F109W/D163V/E196L ;
  • F109/Y244/E196 for example, F109W/Y244W/E196L, F109W/Y244Y/E196L or F109W/Y244H/E196L;
  • -F109/E196/I280 for example, F109W/E196L/I280K, F109W/E196L/I280H, F109W/E196L/I280W or F109W/E196L/I280R;
  • -F109/E196/Q291 for example, F109W/E196L/Q291K, F109W/E196L/Q291R, F109W/E196L/Q291W or F109W/E196L/Q291F;
  • F109/N297/E196 for example, F109W/N297Q/E196L, F109W/N297K/E196L or F109W/N297H/E196L;
  • -F109/T394/E196 for example, F109W/T394K/E196L, F109W/T394H/E196L or F109W/T394N/E196L;
  • F109/H396/E196 for example, F109W/H396Y/E196L, F109W/H396F/E196L, F109W/H396Q/E196L or F109W/H396K/E196L;
  • -F109/K397/E196 for example, F109W/K397R/E196L, F109W/K397H/E196L or F109W/K397Y/E196L; or,
  • Pif1-like helicase is an enzyme having an amino acid sequence that is changed from that of a wild-type helicase and retains polynucleotide binding activity.
  • the variant of any one of SEQ ID NO: 1 to 11 is an enzyme having the following amino acid sequence: the amino acid sequence is changed from the amino acid sequence of any one of SEQ ID NO: 1 to 11 and retains the polynucleoside Acid binding activity.
  • the polynucleotide binding activity can be determined using methods known in the art. Suitable methods include, but are not limited to, fluorescence anisotropy method, tryptophan fluorescence method, and electrophoretic migration shift assay (EMSA). For example, the ability of the variant to bind to a single-stranded polynucleotide can be determined as described in the Examples.
  • the variant preferably has at least 20% identity with the sequence. More preferably, based on amino acid identity, the variant polypeptide may have at least 70%, at least 75%, at least 80%, or at least 85% of the entire sequence of the amino acid sequence of any one of SEQ ID NO: 1 to 11, At least 90% and more preferably at least 95%, 97% or 99% identity. It can have at least 70%, such as at least 80%, at least 85%, at least 90%, or at least 95% amino acids over a length of 100 or more, such as 150, 200, 300, 400, or 500 or more consecutive amino acids Identity (strict homology).
  • a construct which comprises at least one or more Pif1-like helicases of the present invention.
  • the construct further comprises a polynucleotide binding part.
  • the construct has the ability to control the movement of polynucleotides.
  • the polynucleotide binding portion may be a portion that binds to the base of the polynucleotide, and/or a portion that binds to the sugar of the polynucleotide, and/or a portion that binds to the phosphate of the polynucleotide.
  • the Pif1-like helicase and polynucleotide binding part constituting the construct can be prepared separately and then directly connected.
  • the construct can be directly prepared by genetic fusion, for example, the nucleotides encoding the Pif1-like helicase and the polynucleotide binding part are connected, and then transferred into host cells for expression and purification.
  • the polynucleotide binding portion is a polypeptide capable of binding to a polynucleotide, including but not limited to eukaryotic single-chain binding protein, bacterial single-chain binding protein, archaea single-chain binding protein, and viral single-chain binding protein.
  • eukaryotic single-chain binding protein including but not limited to eukaryotic single-chain binding protein, bacterial single-chain binding protein, archaea single-chain binding protein, and viral single-chain binding protein.
  • the polynucleotide binding portion includes but is not limited to any one shown in Table 7:
  • the third aspect of the present invention provides a nucleic acid that encodes the Pif1-like helicase or the construct of the present invention.
  • an expression vector is provided, and the expression vector comprises the nucleic acid of the present invention.
  • the nucleic acid is operably linked to a regulatory element in an expression vector, wherein the regulatory element is preferably a promoter.
  • the promoter is selected from T7, trc, lac, ara or ⁇ L.
  • the expression vector includes but is not limited to plasmid, virus or phage.
  • a host cell in the fifth aspect of the present invention, contains the nucleic acid or the expression vector of the present invention.
  • the host cell includes but is not limited to Escherichia coli.
  • the host cell is selected from BL21 (DE3), JM109 (DE3), B834 (DE3), TUNER, C41 (DE3), Rosetta2 (DE3), Origami, Origami B, etc. .
  • a method for preparing Pif1-like helicase includes providing a wild-type Pif1-like helicase, and then performing processing on the wild-type Pif1-like helicase. Modification to obtain the Pif1-like helicase of the present invention.
  • a method for preparing Pif1-like helicase includes culturing and inducing expression of the host cell of the present invention, and obtaining Pif1-like helicase after purification.
  • the method includes obtaining a nucleic acid sequence encoding a Pif1-like helicase according to the amino acid sequence of the Pif1-like helicase of the present invention, and converting it after digestion and ligation into an expression vector In Escherichia coli, induce expression and purification to obtain Pif1-like helicase.
  • the eighth aspect of the present invention provides a method for controlling the movement of a polynucleotide, the method comprising contacting the Pif1-like helicase or the construct of the present invention with the polynucleotide.
  • said controlling the movement of the polynucleotide is controlling the movement of the polynucleotide through the pore.
  • the pores are nanopores, and the nanopores are transmembrane pores.
  • the pores can be natural or man-made, including but not limited to protein pores, polynucleotide pores or solid pores.
  • the transmembrane pores are selected from biological pores, solid pores, or pores hybridized between biological and solid state.
  • the pores include, but are not limited to, those derived from Mycobacterium smegmatis porin A, Mycobacterium smegmatis porin B, Mycobacterium smegmatis porin C, and mycobacterium smegmatis porin.
  • Mycobacterial porin D hemolysin, lysin, interleukin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, WZA or Neisseria autotransport lipoprotein, etc.
  • the method may include one or more Pif1-like helicases to jointly control the movement of the polynucleotide.
  • the ninth aspect of the present invention provides a method for characterizing a target polynucleotide, the method comprising:
  • any number of Pif1-like helicases of the present invention can be used in the method.
  • it may be one or more, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.
  • the two or more Pif1-like helicases of the present invention may be the same or different. It can also contain wild-type Pif1-like helicase or other types of helicases.
  • two or more helicases can be connected or just arranged by being respectively bound to the polynucleotide to perform the function of controlling the movement of the polynucleotide.
  • the rate at which the target polynucleotide passes through the pore is controlled by the Pif1-like helicase or construct, so as to obtain a recognizable and stable current level for Determine the characteristics of the target polynucleotide.
  • steps I) and II) are repeated one or more times.
  • the method further includes the step of applying a potential difference across the hole contacting the helicase or the construct and the target polynucleotide.
  • the pore is a structure that allows hydrated ions to flow from one side of the membrane to the other layer of the membrane under the driving of an applied electric potential.
  • the pores are nanopores, and the nanopores are transmembrane pores.
  • the transmembrane pore provides a channel for the movement of the target polynucleotide.
  • the membrane can be any membrane existing in the prior art, and is preferably an amphiphilic molecular layer, that is, a layer formed of amphiphilic molecules having at least one hydrophilic part and at least one lipophilic or hydrophobic part, such as phospholipids. , Amphoteric molecules can be synthetic or naturally occurring. Further preferably, the membrane is a lipid bilayer membrane.
  • the target polynucleotide can be attached to the membrane using any known method.
  • the membrane is an amphiphilic molecular layer, such as a lipid bilayer
  • the polynucleotide is preferably connected to the membrane via a polypeptide present in the membrane or via a hydrophobic anchor present in the membrane.
  • the hydrophobic anchors are preferably lipids, fatty acids, sterols, carbon nanotubes or amino acids.
  • the pores are selected from the group consisting of biological pores, solid pores, or pores hybridized between biological and solid state.
  • the pores include, but are not limited to, those derived from Mycobacterium smegmatis porin A, Mycobacterium smegmatis porin B, Mycobacterium smegmatis porin C, and mycobacterium smegmatis porin.
  • Mycobacterial porin D hemolysin, lysin, interleukin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, WZA or Neisseria autotransport lipoprotein, etc.
  • the Pif1-like helicase moves in the 5'-3' direction along the DNA, but the orientation of the DNA in the hole (depending on which end of the DNA is captured) means Enzymes can be used to move DNA out of the hole against the direction of the applied field, or move DNA into the hole in the direction of the applied field.
  • the target polynucleotide is single-stranded, double-stranded, or at least part of it is double-stranded.
  • the target polynucleotide can be modified by means of tags, spacers, methylation, oxidation or damage.
  • the target polynucleotide is double-stranded.
  • the double-stranded part constitutes a Y adapter structure, and the Y adapter structure includes a leader sequence that is preferentially screwed into the pore.
  • the length of the target polynucleotide may be 10-100000 or more.
  • the length of the target polynucleotide can be at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000, etc.
  • the helicase is incorporated into the internal nucleotide of the single-stranded polynucleotide.
  • the one or more characteristics are selected from the source, length, identity, sequence, secondary structure of the target polynucleotide, or whether the target polynucleotide is modified.
  • the one or more features are carried out by electrical measurement and/or optical measurement.
  • electrical and/or optical signals are generated by electrical measurement and/or optical measurement, and each nucleotide corresponds to a signal level, and then the electrical signals and/or optical signals are converted into nucleotide characteristics.
  • the electrical measurement includes, but is not limited to, current measurement, impedance measurement, tunnel measurement, wind tunnel measurement or field effect transistor (FET) measurement, etc.
  • FET field effect transistor
  • the electrical signal of the present invention is selected from the measured values of current, voltage, tunneling, resistance, potential, conductivity or lateral electrical measurement.
  • the electrical signal is a current passing through the hole.
  • the characterization further includes the application of an improved Viterbi algorithm.
  • the tenth aspect of the present invention provides a product for characterizing a target polynucleotide, the product comprising the Pif1-like helicase of the present invention, the construct, the nucleic acid, the Expression vector or said host cell, and pore.
  • the product contains multiple Pif1-like helicases and/or multiple constructs.
  • the product contains multiple holes.
  • the pores are nanopores, and the nanopores are transmembrane pores.
  • the transmembrane pores are selected from biological pores, solid pores, or pores hybridized between biological and solid state.
  • the pores include, but are not limited to, those derived from Mycobacterium smegmatis porin A, Mycobacterium smegmatis porin B, Mycobacterium smegmatis porin C, and mycobacterium smegmatis porin.
  • Mycobacterial porin D hemolysin, lysin, interleukin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, WZA or Neisseria autotransport lipoprotein, etc.
  • the product contains multiple Pif1-like helicases or multiple constructs, and multiple pores.
  • the product is selected from kits, devices or sensors.
  • the kit also includes a chip containing a lipid bilayer.
  • the pores span the lipid bilayer.
  • the kit of the present invention contains one or more lipid bilayers, and each lipid bilayer contains one or more of the pores.
  • the kit of the present invention also includes reagents or devices for performing characterization of target polynucleotides.
  • the reagents include buffers and tools required for PCR amplification.
  • the eleventh aspect of the present invention provides the Pif1-like helicase, the construct, the nucleic acid, the expression vector, the host cell or the product of the present invention To characterize the target polynucleotide or to control the movement of the target polynucleotide through the pore.
  • the twelfth aspect of the present invention provides a kit for characterizing target polynucleotides, said kit comprising the Pif1-like helicase of the present invention, the construct or the nucleic acid, The expression vector or the host cell, and pores.
  • the thirteenth aspect of the present invention provides a device for characterizing target polynucleotides, said device comprising the Pif1-like helicase of the present invention, the construct or the nucleic acid, the The expression vector or the host cell, and the hole.
  • the device includes a sensor that supports the plurality of holes and can transmit signals for the interaction of the holes and the polynucleotide, and at least one memory for storing the target polynucleotide, and the necessary information during the characterization process. Solution.
  • the device includes a plurality of Pif1-like helicases and/or a plurality of constructs, and a plurality of holes.
  • the fourteenth aspect of the present invention provides a sensor for characterizing target polynucleotides, the sensor comprising a complex formed between the pore and the Pif1-like helicase of the present invention or the construct Things.
  • the pore is brought into contact with the helicase or construct in the presence of the target polynucleotide, and a potential is applied across the pore.
  • the potential is selected from voltage potential or chemical potential.
  • the fifteenth aspect of the present invention provides a method for forming a sensor for characterizing a target polynucleotide, comprising forming a complex between the pore and the Pif1-like helicase or the construct of the present invention , Thereby forming a sensor that characterizes the target polynucleotide.
  • the sixteenth aspect of the present invention provides two or more helicases linked to a polynucleotide, wherein at least one of the two or more helicases is the Pif1 of the present invention -like helicase.
  • the seventeenth aspect of the present invention provides a Pif1-like helicase oligomer.
  • the Pif1-like helicase oligomer comprises one or more Pif1-like helicases of the present invention. Enzyme.
  • the Pif1-like helicase oligomer may also include wild-type Pif1-like helicase or other types of helicase.
  • the other types of helicase can be Hel308 helicase, XPD helicase, Dda helicase, TraI helicase or TrwC helicase, etc.
  • the Pif1-like helicase oligomer contains more than two Pif1-like helicases of the present invention, wherein the Pif1-like helicases may be different or the same .
  • the "Pif1-like helicase" of the present invention is modified, and the modification is relative to the wild-type or natural helicase.
  • the "Pif1-like helicase", “construct” or “pore” of the present invention can be modified to facilitate identification or purification, for example, by adding histidine residues (His tag), aspartic acid Acid residues (asp tag), streptavidin tag, Flag tag, SUMO tag, GST tag or MBP tag, or by adding a signal sequence to promote their secretion from the cell, the polypeptide in the cell does not naturally contain the Signal sequence.
  • An alternative way to introduce genetic tags is to attach tags to natural or artificial sites on Pif1-like helicases, pores, or constructs through chemical reactions.
  • 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.
  • the "conservative amino acid substitutions" in the present invention include, but are not limited to: substitutions between alanine and serine, glycine, threonine, valine, proline or glutamic acid; and/or aspartame
  • substitutions between glycine and glycine, asparagine or glutamic acid and/or the substitution between serine and glycine, asparagine or threonine; and/or the substitution between leucine and isoleucine or The substitution between valine; and/or the substitution between valine and leucine, isoleucine; and/or the substitution between tyrosine and phenylalanine; and/or, Replacement between lysine and arginine.
  • the above-mentioned substitution basically does not change the activity of the amino acid sequence of the present invention.
  • the "two or more” mentioned in the present invention includes two, three, four, five, six, seven, eight or more and so on.
  • the "plurality” mentioned in the present invention includes but is not limited to two or more, three or more, four or more, five or more, six or more, seven or more, eight or more or more, and so on.
  • the "at least one" in the present invention includes but is not limited to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more or more, and so on.
  • non-natural amino acid in the present invention is an amino acid that is not naturally present in Pif1-like helicase.
  • it includes, but is not limited to, 4-azido-L-phenylalanine (Faz), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetyl Acetyl-L phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenoalkyl)-L-alanine, O-2-propyn-1-yl-L- Tyrosine, 4(dihydroxyboronyl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino-3- ⁇ 4-[(Propan-2-ylsulfanyl)carbonyl]phenyl ⁇ propionic acid, (2S)-2-amino-3- ⁇ 4-
  • FIG. 1 Schematic diagram of fluorescence analysis for detecting Pif1-like helicase enzyme activity.
  • the fluorescent substrate chain has a 5'-end ssDNA overhang, and a 50-base hybridized dsDNA portion.
  • the upper part of the main chain (B) has a black hole quencher (BHQ-1) base (E) at the 3'end, and the hybrid complementary strand (D) has a carboxyfluorescein at the 5'end (C). It also includes a 0.5 ⁇ M capture chain (F) complementary to the shorter chain (D) of the fluorescent substrate.
  • BHQ-1 black hole quencher
  • D hybrid complementary strand
  • C carboxyfluorescein
  • the helicase (200nM) added to the substrate is connected to the 5'end portion of the fluorescent substrate along the After the main strand moves and the complementary strand is unwound, the excess capture strand preferentially anneals to the complementary strand DNA to prevent the initial substrate from re-annealing with lost fluorescence.
  • the capture strand (A) after adding an excess of the capture strand (A) that is completely complementary to the main chain, part of the ununtwisted dsDNA will have a strand untwisting effect due to the presence of excess A, and finally all the dsDNA will be untwisted, and the fluorescence value will reach Highest.
  • Figure 2 Fluorescence analysis to detect the ability of Pif1-like helicase to unwind hybridized dsDNA, specifically the graph of the time-dependent dsDNA unwinding ratio in a buffer containing 400mM NaCl.
  • Figure 3 shows the results of gel assays for the ability of different Pif1-like helicases to bind DNA.
  • Lane 1 is the pre-built dsDNA (SEQ ID NO: 18 hybridized with SEQ ID NO: 19 with 5'FAM modification).
  • Lanes 2-6 respectively contain Aph Acj61, Aph PX29, Sph CBH8, PphPspYZU05, and Mph MP1 pre-linked to dsDNA.
  • Lanes 7-11 respectively contain Aph Acj61-D97C/A363C, Aph PX29-D96C/A371C, Sph CBH8-A94C/A361C/C136A, PphPspYZU05-D104C/A375C/C146A and Mph MP1-E105C/A362C pre-linked to dsDNA.
  • Band A corresponds to SEQ ID NO: 19 which hybridizes with SEQ ID NO: 18.
  • the regions marked 1A, 2A, 3A, 4A, and 5A correspond to SEQ ID NO: 19 that hybridizes with SEQ ID NO: 18 with 1, 2, 3, 4, and 5 helicases, respectively.
  • Figure 4 A diagram of the DNA construct used in the examples, in which SEQ ID NO: 13 (marked as B), its 5'end is connected to 20 iSpC3 spacers (marked A), and its 3'end is connected to 4 iSpC3 spacer (marked as C), the spacer (marked as C) is connected to the 5'end of SEQ ID NO: 14 (marked as D), and the 3'end of SEQ ID NO: 14 (marked as D) is connected To SEQ ID NO: 17 or SEQ ID NO: 24 (marked as E), the SEQ ID NO: 15 (marked as F) region of the construct and SEQ ID NO: 16 (marked as G, which has a 3'cholesterol series) Strand) hybridization.
  • Figure 10 shows the purified Mph MP1 (SEQ ID NO: 11) SDS-PAGE gel electrophoresis image. Among them, M is Marker (Kd), and lane 1 is the electrophoresis result of Mph MP1 helicase.
  • Figure 11 shows the correspondence between the amino acid sequences of SEQ Nos: 1 to 11.
  • the recombinant plasmid containing the Pif1-like helicase sequence (a variant of amino acid sequence SEQ ID NO: 1-11, and its corresponding nucleotide sequence SEQ ID NO: 25-35) was transformed into BL21 (DE3) sensing by heat shock After spreading the ampicillin-resistant solid LB plate with the resuscitated bacteria solution, culture it overnight at 37°C. Pick a single colony and inoculate it in 100ml of liquid LB medium containing ampicillin resistance and cultivate it at 37°C. The 1% inoculum was transferred to ampicillin-resistant LB liquid medium for expansion culture, cultured at 37°C and 200 rpm, and its OD600 value was continuously measured.
  • the culture solution in the LB medium was cooled to 18° C., and isopropyl thiogalactoside (IPTG) was added to induce expression, so that the final concentration reached 1 mM. After 12-16h, collect the bacteria at 18°C. The bacteria were crushed under high pressure, purified by the FPLC method, and samples were collected.
  • IPTG isopropyl thiogalactoside
  • Figure 10 shows an SDS-PAGE gel electrophoresis image of purified Mph MP1 (variant of SEQ ID NO: 11).
  • Fluorescence analysis was used to detect enzyme activity to illustrate the ability of Pif1-like helicase to untie hybrid dsDNA.
  • the fluorescent substrate strand (final concentration 100 nM) has a 5'-end ssDNA overhang, and a 50-base hybridized dsDNA portion.
  • Including the upper part of the main chain has a black hole quencher (BHQ-1) base (SEQ ID NO: 20--BHQ-3') at the 3'end, and the hybrid complementary strand has a carboxyfluorescein at the 5'end ( 5'FAM-SEQ ID NO: 21).
  • BHQ-1 black hole quencher
  • 5'FAM-SEQ ID NO: 21 carboxyfluorescein at the 5'end
  • a 0.5 ⁇ M capture strand (SEQ ID NO: 22) complementary to the shorter strand of the fluorescent substrate.
  • SEQ ID NO: 22 0.5 ⁇ M capture strand
  • the Pif1-like helicase (200nM) added to the substrate is connected to the 5'end portion of the fluorescent substrate along the After the main strand moves and the complementary strand is unwound, the excess capture strand preferentially anneals to the complementary strand DNA to prevent the initial substrate from re-annealing with lost fluorescence.
  • Figure 2 shows the time-dependent dsDNA unwinding ratio in a buffer containing 400 mM NaCl (10 mM Hepes pH 8.0, 5 mM ATP, 5 mM MgCl2, 100 nM fluorescent substrate DNA, 0.5 ⁇ M capture DNA).
  • An exemplary gel assay is used to measure the ability of the modified Pif1-like helicase of the present invention to bind DNA.
  • the annealed DNA complex (SEQ ID NO: 18 and SEQ ID NO: 19 with 5'FAM modification) were hybridized with Aph Acj61, Aph PX29, Sph CBH8, Pph at a ratio of (1:1, volume/volume) PspYZU05, Mph MP1, Aph Acj61-D97C/A363C, Aph PX29-D96C/A371C, Sph CBH8-A94C/A361C/C136A, Pph PspYZU05-D104C/A375C/C146A, Mph PE1-E105C/A362C at pH 10mM/A362C , 400mM potassium chloride mixed, the final concentration of Pif1-like helicase is (600nM) and DNA is (30nM).
  • Pif1-like helicase binds to DNA for 1 hour at room temperature.
  • TMAD was added to each sample to a final concentration of 5 ⁇ M, and incubated at room temperature for 1 hour. Load the sample on a 4-20% TBE gel and run the gel at 160V for 1.5 hours. Then the gel was observed under blue fluorescence for DNA bands.
  • Figure 3 shows the effect of Pif1-like helicase on DNA binding ability before and after modification.
  • Lanes 2 to 6 show that a large amount of DNA is not bound by Pif1-like helicase during electrophoresis and no obvious binding bands are observed.
  • Lanes 7-11 show the binding bands of different numbers of enzymes bound to DNA.
  • Lanes 9 and 10 show that up to 5 Pif1-like helicases can bind to the single-stranded part of SEQ ID NO: 18. This indicates that Pif1-like helicase significantly enhanced the firmness of binding to DNA after modification.
  • Mph MP1-E105C/A362C (SEQ ID NO: 11 with mutation E105C/A362C) as an example to verify how Pif1-like helicase controls the movement of the entire DNA chain through a single MspA nanopore (SEQ ID NO: 12).
  • SEQ ID NO: 13 Its 5'end is connected to 20 iSpC3 spacers, and its 3'end is connected to 4 iSpC3 spacers, which are connected to SEQ ID NO: At the 5'end of 14, the 3'end of SEQ ID NO: 14 is connected to SEQ ID NO: 17, and the SEQ ID NO: 15 region of the DNA construct and SEQ ID NO: 16 (it has a 3'cholesterol tether) Hybrid).
  • the DNA polynucleotide and Pif1-like helicase are added to 70 ⁇ L of buffer in the cis-compartment of the electrophysiology room to initiate the helicase-DNA complex in the Capture of nanopores. If necessary by adding the divalent metal to the cis compartment (5mM MgCl 2) and NTP (2.86 ⁇ M ATP) ATP activates helicase activity. The experiment was carried out at a constant potential of +180mV.
  • Sph CBH8-A94C/A361C/C136A (SEQ ID NO: 5 with A94C/A361C/C136A mutation) and Eph Pei26-D99C/A366C/C141A (SEQ ID NO: 6 with D99C/A366C/C141A mutation)
  • Pph PspYZU05-D104C/A375C/C146A (SEQ ID NO: 8 with D104C/A375C and C146A mutations) as examples to verify how Pif1-like helicase controls the movement of the entire DNA chain through a single MspA nanopore.
  • DNA construct B as shown in Figure 4: SEQ ID NO: 13 Its 5'end is connected to 20 iSpC3 spacers, and its 3'end is connected to 4 iSpC3 spacers, which is connected to SEQ ID NO: At the 5'end of 14, the 3'end of SEQ ID NO: 14 is connected to SEQ ID NO: 24, and the SEQ ID NO: 15 region of the DNA construct is connected to SEQ ID NO: 16 (which has a 3'cholesterol tether) Hybridization.
  • This DNA construct B is similar to the construct used in Example 4, except that the region marked E corresponds to SEQ ID NO: 24.
  • DNA construct B For DNA construct B, observe the DNA movement controlled by Pif1-like helicase, and the DNA movement controlled by Sph CBH8-A94C/A361C/C136A, Eph Pei26-D99C/A366C/C141A or Pph PspYZU05-D104C/A375C/C146A The results are shown in Figure 7-9.

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Abstract

一种经修饰的Pif1-like解旋酶、包含Pif1-like解旋酶的构建体，以及其在表征目标多核苷酸或控制目标多核苷酸穿过孔的移动中的应用。还涉及一种表征目标多核苷酸或控制目标多核苷酸穿过孔的移动的方法。所述的Pif1-like解旋酶可以有效的控制多核苷酸穿过孔的移动，提高链测序过程中的精确度。

Description

一种Pif1-like解旋酶及其应用 技术领域
本发明涉及基因测序、分子检测以及临床检测技术领域，具体涉及一种经修饰的Pif1-like解旋酶、包含Pif1-like解旋酶的构建体，以及其在表征目标多核苷酸或控制目标多核苷酸穿过孔的移动中的应用。
背景技术
纳米孔测序技术指以单个核酸分子为测量单元，利用纳米孔对其序列信息进行即时连续读取的基因测序技术。
其利用了可以为离子电流提供通道的纳米孔。电泳驱动多核苷酸通过纳米孔，并且由于多核苷酸穿过纳米孔，因此可以降低了通过纳米孔的电流。每个通过的核苷酸或一系列核苷酸获得了特征电流，并且对电流电平的记录对应于多核苷酸序列。在“链测序”方法(如使用解旋酶控制多核苷酸穿过孔的移动)中，单个多核苷酸链穿过所述孔并能实现对核苷酸的鉴定。这种测序技术的优势在于建库简单，无需扩增；读取速度快，对单个分子可以达到每小时上万碱基的读取速度；读长非常长，通常在上千碱基；有可能可以对RNA和DNA甲基化进行直接测量。这些都是现有的二代测序技术所无法达到的。
但是，纳米孔测序技术也有棘手的问题，需要解决，例如多核苷酸通过纳米孔的易位过快以至于单个核苷酸的电流电平过短而难以分辨。尤其是在面对核苷酸序列很长的情况下，例如500个或更多个，控制多核苷酸移动的分子马达可能会从多核苷酸上解脱。这允许多核苷酸在施加的场的方向上以不受控的方式被迅速拉动穿过所述孔。
现有技术中，也有对此问题的解决，例如，专利WO2013057495A3公开了一种新的表征目标多核苷酸的方法，所述的方法包括通过Hel308解旋酶或分子马达控制目标多核苷酸穿过孔的移动。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，所述的方法包括通过XPD解旋酶控制目标多核苷酸穿过孔的移动。专利CN107109380A公开了一种经修饰的酶，该酶为可以控制目标多核苷酸穿过孔的移动的经 修饰的Dda解旋酶。上述方法均可以一定程度上控制目标多核苷酸穿过孔的移动。但是，仍然开发新的控制目标多核苷酸穿过孔的移动的解旋酶，且现有技术中没有公开本发明所述的Pif1-like解旋酶。
发明内容
为解决多核苷酸穿过纳米孔的易位过快的问题，本发明提供了一种新的经修饰的Pif1-like解旋酶，经过修饰后的Pif1-like解旋酶可以更长时间保持与多核苷酸的结合，并控制多核苷酸穿过孔的移动。本发明所述的Pif1-like解旋酶是链测序过程中控制多核苷酸移动的有用工具，可以使多核苷酸以可控和逐步的方式顺着或逆着由所施加的电压引起的电场进行运动，从而控制多核苷酸通过纳米孔的速度，获得可识别的电流电平。尤其当多核苷酸链长度增加的情况下，例如500个或更多个核苷酸，需要具有提高进行性的分子马达时，本发明所述的Pif1-like解旋酶依然不会从多核苷酸上解脱，即对于控制500、1000、5000、10000、20000、50000、100000或更多个多核苷酸的移动特别有效。基于本发明所述的Pif1-like解旋酶在链测序领域控制多核苷酸平稳从孔移动，减少滑移或不规则移动的优势，可以促进核苷酸更精确、读长更长。
本发明的第一方面，提供了一种Pif1-like解旋酶，包括在Pif1-like解旋酶的塔结构域、销结构域和/或1A结构域(RecA型马达)引入至少一个半胱氨酸残基和/或至少一个非天然氨基酸，其中所述解旋酶保留其控制多核苷酸移动的能力。
优选的，包括在下列任一组中引入至少一个半胱氨酸残基和/或至少一个非天然氨基酸：
(a)塔结构域；
(b)销结构域；
(c)1A结构域；
(d)塔结构域和销结构域；
(e)塔结构域和1A结构域；
(f)1A结构域和销结构域；
(g)塔结构域、销结构域和1A结构域。
优选的，可以在塔结构域、销结构域和/或1A结构域引入1、2、3、4、5、6、7、8、9、10、11或更多个半胱氨酸残基，或者引入1、2、3、4、5、6、7、8、9、10、11或更多个非天然氨基酸，或者引入1、2、3、4、5、6、7、8、9、10、11或更多个半胱氨酸残基和非天然氨基酸。
优选的，引入半胱氨酸残基和/或至少一个非天然氨基酸后使得该Pif1-like解旋酶与多核苷酸的结合更稳定，并增强了控制其移动的能力。
优选的，所述的Pif1-like解旋酶选自Pba-PM2、Aph-Acj61、Aph-PX29、Avi-Aeh1、Sph-CBH8、Eph-Pei26、Aph-AM101、PphPspYZU05、Eph-EcS1、Eph-Cronus2或Mph-MP1。野生型的Pif1-like解旋酶具体见表1。
表1野生型的Pif1-like解旋酶
Figure PCTCN2020097126-appb-000001
Figure PCTCN2020097126-appb-000002
所述的Pif1-like解旋酶的塔结构域、销结构域、钩结构域、1A结构域以及2A结构域的氨基酸残基位置见表2。
表2鉴定了构成每个Pif1-like解旋酶中每个结构域的残基
Pif1-like解旋酶 SEQ ID NO 1A结构域 2A结构域
Pba-PM2 1 M1-L88,M106-V181 E264-P278,N296-A394
Aph-Acj61 2 M1-L88,I106-M180 E265-P279,N297-A392
Aph-PX29 3 M1-L88,K110-V182 T266-P280,N298-S403
Avi-Aeh1 4 M1-L88,K110-V182 T266-P280,N298-S404
Sph-CBH8 5 M1-L84,M103-K177 E260-P274,N292-A391
Eph-Pei26 6 M1-L90,M108-M183 E266-P280,N298-A396
Aph-AM101 7 M1-L99,D117-M191 T276-P290,N308-P402
PphPspYZU05 8 M1-L95,I113-K187 D274-P288,N306-A404
Eph-EcS1 9 M1-L85,M103-K177 E260-P274,N292-A391
Eph-Cronus2 10 M1-L87,I105-K180 E265-P279,H297-A393
Mph-MP1 11 M1-L96,P114-K184 E264-P278,N296-P389
Figure PCTCN2020097126-appb-000003
在本发明的一个具体实施方式中，所述的Pif1-like解旋酶选自Mph-MP1、Sph-CBH8、Eph-Pei26或PphPspYZU05。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：11的变体，其中在塔结构域(残基E264-P278和N296-P389)和/或销结构域(残基K97-A113)和/或1A结构域(残基M1-L96和P114-K184)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：1的变体，其中在塔结构域(残基E264-P278和N296-A394)和/或销结构域(残基K89-E105)和/或1A结构域(残基M1-L88和M106-V181)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：2的变体，其中在塔结构域(残基E265-P279和N297-A392)和/或销结构域(残基K89-D105)和/或1A结构域(残基M1-L88和I106-M180)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：3的变体，其中在塔结构域(残基T266-P280和N298-S403)和/或销结构域(残基K89-A109)和/或1A结构域(残基 M1-L88和K110-V182)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：4的变体，其中在塔结构域(残基T266-P280和N298-S404)和/或销结构域(残基K89-A109)和/或1A结构域(残基M1-L88和K110-V182)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：5的变体，其中在塔结构域(残基E260-P274和N292-A391)和/或销结构域(残基K86-E102)和/或1A结构域(残基M1-L84和M103-K177)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：6的变体，其中在塔结构域(残基E266-P280和N298-A396)和/或销结构域(残基K91-E107)和/或1A结构域(残基M1-L90和M108-M183)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：7的变体，其中在塔结构域(残基T276-P290和N308-P402)和/或销结构域(残基K100-D116)和/或1A结构域(残基M1-L99和D117-M191)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：8的变体，其中在塔结构域(残基D274-P288和N306-A404)和/或销结构域(残基K95-E112)和/或1A结构域(残基M1-L95和I113-K187)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：9的变体，其中在塔结构域(残基E260-P274和N292-A391)和/或销结构域(残基K86-E102)和/或1A结构域(残基M1-L85和M103-K177)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：10的变体，其中在塔结构域(残基E265-P279和H297-A393)和/或销结构域(残基K88-E104)和/或1A结构域(残基M1-L87和I105-K180)中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：11的变体，其包括(i)E105C和/或A362C；(ii)E104C和/或K360C；(iii)E104C和/或A362C；(iv)E104C和/或Q363C；(v)E104C和/或K366C；(vi)E105C和/或M356C；(vii)E105C和/或K360C；(viii)E104C和/或M356C；(ix)E105C和/或Q363C；(x)E105C和/或K366C；(xi)F108C和/或M356C；(xii)F108C和/或K360C；(xiii)F108C和/或A362C；(xiv)F108C和/或Q363C；(xv)F108C和/或K366C；(xvi)K134C和/或M356C；(xvii)K134C和/或K360C；(xviii)K134C和/或A362C；(xix)K134C和/或Q363C；(xx)K134C和/或K366C；(xxi)(i)到(xx)任一个和G359C；(xxii)(i)到(xx)任一个和Q111C；(xxiii)(i)到(xx)任一个和I138C；(xxiv)(i) 到(xx)任一个和Q111C和I138C；(xxv)E105C和/或F377C；(xxvi)Y103L，E105Y，N352N，A362C和Y365N；(xxvii)E105Y和A362C；(xxviii)A362C；(xxix)Y103L，E105C，N352N，A362Y和Y365N；(xxx)Y103L，E105C和A362Y；(xxxi)E105C和/或A362C，和I280A；(xxxii)E105C和/或L358C；(xxxiii)E104C和/或G359C；(xxxiv)E104C和/或A362C；(xxxv)K106C和/或W378C；(xxxvi)T102C和/或N382C；(xxxvii)T102C和/或W378C；(xxxviii)E104C和/或Y355C；(xxxix)E104C和/或N382C；(xl)E104C和/或K381C；(xli)E104C和/或K379C；(xlii)E104C和/或D376C；(xliii)E104C和/或W378C；(xliv)E104C和/或W374C；(xlv)E105C和/或Y355C；(xlvi)E105C和/或N382C；(xlvii)E105C和/或K381C；(xlviii)E105C和/或K379C；(xlix)E105C和/或D376C；(l)E105C和/或W378C；(li)E105C和/或W374C；(lii)E105C和A362Y；(liii)E105C，G359C和A362C；或(liv)I2C，E105C和A362C。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：1-10中任一个的变体，其包括在SEQ ID NO：11中的(i)到(liv)的任一个所限定的位置对应的位置处包括一个半胱氨酸残基。
优选的，所述Pif1-like解旋酶的氨基酸序列为SEQ ID NO：1到11所示氨基酸序列或与SEQ ID NO：1到11所示氨基酸序列具有至少30％、至少40％、至少50％、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％的同源性并具有控制多核苷酸移动的能力。
在本发明的一个具体实施方式中，所述的与SEQ ID NO：11的第105位和/或第362位相应的氨基酸引入半胱氨酸残基，例如，SEQ ID NO：2的第97位和/或第363位，SEQ ID NO：3的第96位和/或第371位，SEQ ID NO：5的第94位和/或第361位，SEQ ID NO：6的第99位和/或第366位，SEQ ID NO：8的第104位和/或第375位等等。
为提高本发明所述的Pif1-like解旋酶与多核苷酸结合的稳定性，降低从多核苷酸上解脱的能力，所述引入的半胱氨酸与半胱氨酸之间相互连接、非天然氨基酸与非天然氨基酸之间相互连接之间、引入的半胱氨酸与非天然氨基酸之间相互连接、引入的半胱氨酸与解旋酶上的天然氨基酸之间相互连接、或者引入的非天然氨基酸与解旋酶上的天然氨基酸之间相互连接。
可以使任何数目和组合的两个以上引入的半胱氨酸和/或非天然氨基酸相互连接。 例如，可以使3，4，5，6，7，8或更多个半胱氨酸和/或非天然氨基酸相互连接。一个或多个半胱氨酸可以与一个或多个半胱氨酸连接。一个或多个半胱氨酸可以与一个或多个非天然氨基酸诸如Faz连接。一个或多个非天然氨基酸诸如Faz可以与一个或多个非天然氨基酸诸如Faz连接。一个或多个半胱氨酸可以与一个或多个解旋酶上的天然氨基酸连接。一个或多个非天然氨基酸诸如Faz可以与一个或多个解旋酶上的天然氨基酸连接。
其中，所述的连接可以是任何方式，包括直接相连或者间接相连，优选的，所述的连接可以为短暂的接触，也可以为永久的连接。更进一步优选的，所述的连接可以为非共价连接或共价连接。
在本发明的一个具体实施方式中，所述的共价连接可以采用化学交联剂、线性分子或催化剂连接。所述的化学交联剂包括但不限于马来酰亚胺，活性酯，琥珀酰亚胺，叠氮化物，炔烃(诸如二苯并环辛炔醇(DIBO或DBCO)，二氟环炔烃和线性炔烃)等；化学交联剂的长度可以从一个碳(碳酰氯型连接器)到多个埃变化。所述的线性分子包括但不限于聚乙二醇(PEGs)，多肽，多糖，脱氧核糖核酸(DNA)，肽核酸(PNA)，苏糖核酸(TNA)，甘油核酸(GNA)，饱和的和不饱和的烃，聚酰胺。所述的催化剂包括但不限于TMAD等任何可以使得半胱氨酸残基之间、非天然氨基酸之间、半胱氨酸残基与非天然氨基酸之间、非天然氨基酸与天然氨基酸之间、或者半胱氨酸残基与天然氨基酸之间产生共价键的催化剂。
优选的，所述的Pif1-like解旋酶进一步被修饰去除一个或多个半胱氨酸残基。
优选的，所述Pif1-like解旋酶还包括至少一个或多个天然半胱氨酸被取代。进一步优选为丙氨酸、丝氨酸或缬氨酸取代半胱氨酸。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：5的变体，并且所述一个或多个被取代的天然半胱氨酸残基为C109、C114、C136或C414中的一个或多个。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：1、2、3、4、6、7、8、9、10和11中任一个的变体，并且所述一个或多个被取代的天然半胱氨酸残基对应于SEQ ID NO：5中C109、C114、C136或C414中的一个或多个。
表3示出了与SEQ ID NO:5中的C109,C114，C136和C414对应的SEQ ID NO:1、2、3、4、6、7、8、9、10和11中的氨基酸位置。
表3 SEQ ID NO:1-4、6-11中的相应氨基酸位置
Figure PCTCN2020097126-appb-000004
优选的，所述的Pif1-like解旋酶进一步被修饰降低其表面的负电荷。
优选的，所述的Pif1-like解旋酶还包括增加净正电荷的取代。进一步优选的，所述的增加净正电荷的取代包括对表面带负电的氨基酸、极性或非极性的氨基酸进行取代或修饰，或者在表面带负电的氨基酸、极性或非极性的氨基酸临近位置引入带正电的氨基酸。更进一步优选的，所述的增加净正电荷的取代包括带正电的氨基酸取代带负电的氨基酸、不带电的氨基酸、芳香族氨基酸、极性或非极性的氨基酸。更进一步优选的，所述的增加净正电荷的取代包括不带电的氨基酸取代带负电的氨基酸、芳香族氨基酸、极性或非极性的氨基酸。适合的带正电荷的氨基酸包括但不限于，组氨酸(H)，赖氨酸(K)和/或精氨酸(R)。不带电荷的氨基酸没有净电荷。适合的不带电荷的氨基酸包括但不限于，半胱氨酸(C)，丝氨酸(S)，苏氨酸(T)，蛋氨酸(M)，天冬酰胺(N)或谷氨酰胺(Q)。非极性氨基酸具有非极性侧链。非极性氨基酸包括但不限于，甘氨酸(G)，丙氨酸(A)，脯氨酸(P)，异亮氨酸(I)，亮氨酸(L)或缬氨酸(V)。芳香族氨基酸具有芳香族侧链。适合的芳香族氨基酸包括但不限于，组氨酸(H)，苯丙氨酸(F)，色氨酸(W)或酪氨酸(Y)。
其中，所述的带正电的氨基酸、不带电荷的氨基酸、极性、非极性氨基酸或芳香族氨基酸可以是天然的或非天然的氨基酸，其可以是人工合成的或者经过修饰的天然氨基酸。
优选的取代包括但不限于，用精氨酸(R)取代谷氨酸(E)，用赖氨酸(K)取代谷氨酸(E)，用天冬酰胺(N)取代谷氨酸(E)，用赖氨酸(K)取代天冬氨酸(D)，以及用精氨酸(R)取代天冬氨酸(D)。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：11的变体并且所述一个或多个带负电荷的氨基酸为D5,E9,E24,E87,I65,S58,D209或D216中的一个或多个。可以中和任意数量的这些氨基酸，诸如这些氨基酸中的1，2，3，4，5，6，7或8个。可以中和任意的组合。
优选的，所述的Pif1-like解旋酶包括SEQ ID NO：1到10中任一个的变体并且所述一个或多个带负电荷的氨基酸对应于SEQ ID NO：11中的D5,E9,E24,E87,I65,S58,D209或D216中的一个或多个。对应于SEQ ID NO：11中的D5,E9,E24,E87,I65,S58,D209和D216的SEQ ID NO：1到10中的氨基酸可以使用图11中的比对确定。
优选的取代还包括，SEQ ID NO：1的变体，并且所述一个或多个带负电荷的氨基酸还包括S171。
优选的取代还包括SEQ ID NO：9的变体，并且所述一个或多个带负电荷的氨基酸还包括S173。
优选的，所述非天然氨基酸选自4-叠氮基-L-苯丙氨酸(Faz)，4-乙酰基-L-苯丙氨酸，3-乙酰基-L-苯丙氨酸，4-乙酰乙酰基-L苯丙氨酸，O-烯丙基-L-酪氨酸，3-(苯基硒烷基)-L-丙氨酸，O-2-丙炔-1-基-L-酪氨酸，4(二羟基硼基)-L-苯丙氨酸，4-[(乙基硫烷基)羰基]-L-苯丙氨酸，(2S)-2-氨基-3-{4-[(丙烷-2-基硫烷基)羰基]苯基}丙酸，(2S)-2-氨基-3-{4-[(2-氨基-3-硫烷基丙酰基)氨基]苯基}丙酸，O-甲基-L-酪氨酸，4-氨基-L-苯丙氨酸，4-氰基-L-苯丙氨酸，3-氰基-L-苯丙氨酸，4-氟-L-苯丙氨酸，4-碘-L-苯丙氨酸，4-溴-L-苯丙氨酸，O-(三氟甲基)酪氨酸，4-硝基L-苯丙氨酸，3-羟基-L-酪氨酸，3-氨基-L-酪氨酸，3-碘-L-酪氨酸，4-异丙基-L-苯丙氨酸，3-(2-萘基)-L-丙氨酸，4-苯基-L-苯丙氨酸，(2S)-2-氨基-3-(萘-2-基氨基)丙酸，6-(甲基硫烷基)正亮氨酸，6-氧-L-赖氨酸，D-酪氨酸，(2R)-2-羟基-3-(4-羟基苯基)丙酸，(2R)-2氨基辛酸酯3-(2，2′-二吡啶-5-基)-D-丙氨酸，2-氨基-3-(8-羟基-3-喹啉基)丙酸，4-苯甲酰-L-苯丙氨酸，S-(2-硝基苄基)半胱氨酸，(2R)-2-氨基-3-[(2-硝基苄基)硫烷基]丙酸，(2S)-2-氨基-3-[(2-硝基苄基)氧基]丙酸，O-(4，5-二甲氧基-2-硝基苄基)-L-丝氨酸，(2S)-2-氨基-6-({[(2-硝基苄基)氧基]羰基}氨基)己酸，O-(2-硝基苄基)-L-酪氨酸，2-硝基苯丙氨酸，4-[(E)-苯基二氮烯基]-L-苯丙氨酸，4-[3-(三氟甲基)-3H-二吖丙啶基-3基]-D-苯丙氨酸，2- 氨基-3-[[5-(二甲基氨基)-1-萘基]磺酰基氨基]丙酸，(2S)-2-氨基4-(7-羟基-2-氧-2H-色烯-4-基)丁酸，(2S)-3-[(6-乙酰基萘-2-基)氨基]-2-氨基丙酸，4(羧基甲基)苯丙氨酸，3-硝基-L-酪氨酸，O-硫基-L-酪氨酸，(2R)-6-乙酰氨基-2-氨基己酸酯，1-甲基组氨酸，2-氨基壬酸，2-氨基癸酸，L-同质半胱氨酸，5-硫烷基正缬氨酸，6-硫烷基-L-正亮氨酸，5-(甲基硫烷基)-L-正缬氨酸，N6-{[(2R，3R)-3-甲基-3，4-二氢-2H-吡咯2-基]羰基}-L-赖氨酸，N6-[(苄基氧基)羰基]赖氨酸，(2S)-2-氨基-6-[(环戊基羰基)氨基]己酸，N6-[(环戊基氧基)羰基]-L-赖氨酸，(2S)-2-氨基-6-{[(2R)-四氢呋喃-2-基羰基]氨基}己酸，(2S)-2-氨基-8-[(2R，3S)-3-乙炔基四氢呋喃-2-基]-8-氧基辛酸，N6-(叔丁氧基羰基)-L-赖氨酸，(2S)-2-羟基-6-({[(2-甲基-2-丙烷基)氧基]羰基}氨基)己酸，N6-[(烯丙氧基)羰基]赖氨酸，(2S)-2-氨基-6-({[(2-叠氮苄基)氧基]羰基}氨基)己酸，N6L-脯氨酰基-L-赖氨酸，(2S)-2-氨基-6-{[(丙-2-炔-1-基氧基)羰基]氨基}己酸或N6-[(2叠氮乙氧基)羰基]-L-赖氨酸。
优选的，所述的Pif1-like解旋酶还包括：
(a)至少一个与单链DNA(ssDNA)或双链DNA(dsDNA)中一个或多个核苷酸相互作用的氨基酸被取代；和/或，
(b)至少一个与跨膜孔相互作用的氨基酸被取代，
其中，所述Pif1-like解旋酶具有控制多核苷酸移动的能力。
进一步优选的，在(a)中，用包含较大侧链(R基团)的氨基酸取代至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸。
优选的，所述的Pif1-like解旋酶包括，用包含较大侧链(R基团)的氨基酸取代至少一个与单链或双链DNA一个或多个核苷酸的糖和/或碱基相互作用的氨基酸。可以取代任何数量的氨基酸，例如，1个或更多个、2个或更多个、3个或更多个、4个或更多个、5个或更多个、或者6个或更多个氨基酸。每个氨基酸可以与碱基、糖、或碱基和糖相互作用。可以采用蛋白质建模来鉴定与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸。
优选的，所述的Pif1-like解旋酶包含SEQ ID NO:11的变体，其中，所述至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸是P73,H93,N99,F109,I280,A161,F130,D132,D162,D163,E277,K415,Q291,H396,Y244或P100中的至少一个。这些数字对应SEQ ID NO:11中的相关位点，并且相比SEQ ID NO:11可能需要在一个或多个氨基酸已经在变体中插入或删除的情况下进行改变。如 上所述，本领域技术人员可以确定变体中的相应位点。进一步优选包含SEQ ID NO:11的变体，其中，所述至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸是F109和一个或多个P73,H93,N99,F109,I280,A161,F130,D132,D162,D163,E277,K415,Q291,H396,Y244或P100。
优选的，所述的Pif1-like解旋酶为SEQ ID NO:1到10中任一的变体，其中，所述至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸是至少一个与SEQ ID NO:11中的P73,H93,N99,F109,I280,A161,F130,D132,D162,D163,E277,K415,Q291,H396,Y244或P100相对应的氨基酸。进一步优选包含SEQ ID NO:1到10中任一的变体，其中，所述至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸是与SEQ ID NO:11中F109对应的氨基酸和一个或多个与SEQ ID NO:11中P73,H93,N99,F109,I280,A161,F130,D132,D162,D163,E277,K415,Q291,H396,Y244或P100相对应的氨基酸。
表4示出了与SEQ ID NO:11中的P73,H93,N99,F109,I280,A161,F130,D132,D162,D163,E277,K415,Q291,H396,Y244和P100对应的SEQ ID NO:1至10中的氨基酸。
表4与SEQ ID NO:1至10对应的氨基酸位置
Figure PCTCN2020097126-appb-000005
较大侧链(R基团)优选地(a)包含增加数量的碳原子(b)具有增加的长度(c)具有增加的分子体积和/或(d)具有增加的范德华体积。较大侧链(R基团)优选(a)；(b)；(c)；(d)；(a)和(b)；(a)和(c)；(a)和(d)；(b)和(c)；(b)和(d)；(c)和(d)；(a)，(b)和(c)；(a)，(b)和(d)；(a)，(c)和(d)；(b)，(c)和(d)；或(a)，(b)，(c)和(d)。可以采用本领域中的标 准方法对(a)至(d)中的每一个进行测量。
进一步优选的，所述较大侧链(R基团)增加了所述至少一个氨基酸与所述单链或双链DNA中一个或多个核苷酸之间的(i)静电相互作用；(ii)氢键和/或(iii)阳离子-pi((阳离子-π))相互作用。例如，在(i)中，诸如精氨酸(R)、组氨酸(H)和赖氨酸(K)等带正电荷的氨基酸具有增加静电相互作用的R基团。例如，在(ii)中，诸如天冬酰胺(N)、丝氨酸(S)、谷氨酰胺(Q)、苏氨酸(T)和组氨酸(H)等氨基酸具有增加氢键的R基团。例如，在(iii)中，诸如苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)等芳族氨基酸具有增加阳离子-pi(阳离子-π)相互作用的R基团。
包含较大侧链(R)的氨基酸可以是非天然氨基酸。所述非天然氨基酸可以是以下讨论的任何一种。
在本发明的一个具体实施方式中，所述较大侧链(R基团)的氨基酸不是丙氨酸(A)、半胱氨酸(C)、甘氨酸(G)、硒代半胱氨酸(U)、甲硫氨酸(M)、天冬氨酸(D)或谷氨酸(E)。
在本发明的一个具体实施方式中，所述的Pif1-like解旋酶包含以下取代中的一个或多个：
A)组氨酸(H)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)或天冬酰胺(N)；或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。组氨酸(H)更优选被(a)N、Q或W或(b)Y、F、Q或K取代。
B)天冬酰胺(N)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)或组氨酸(H)；或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。天冬酰胺(N)更优选被R、H、W或Y取代。
C)脯氨酸(P)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)、苏氨酸(T)或组氨酸(H)；(iii)酪氨酸(Y)、苯丙氨酸(F)或色氨酸(W)；或(iv)亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)取代。脯氨酸(P)更优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)、苏氨酸(T)或组氨酸(H)；(iii)苯丙氨酸(F)或色氨酸(W)或(iv)亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)取代。脯氨酸(P)更优选被(a)F，(b)L、V、I、T或F或(c)W、F、Y、H、I、L或V取代。
D)缬氨酸(V)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)；或(iv)异亮氨酸(I)或亮氨酸(L)取代。缬氨酸(V)更优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N) 或组氨酸(H)；(iii)酪氨酸(Y)或色氨酸(W)；或(iv)异亮氨酸(I)或亮氨酸(L)取代。缬氨酸(V)更优选被I或H或I、L、N、W或H取代。
E)苯丙氨酸(F)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)组氨酸(H)；或(iii)酪氨酸(Y)或色氨酸(W)取代。苯丙氨酸(F)更优选被(a)W，(b)W、Y或H，(c)W、R或K或(d)K、H、W或R取代。
F)谷氨酰胺(Q)优选被(i)精氨酸(R)或赖氨酸(K)或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。
G)丙氨酸(A)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)或(iv)异亮氨酸(I)或亮氨酸(L)取代。
H)丝氨酸(S)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)；或(iv)异亮氨酸(I)或亮氨酸(L)取代。丝氨酸(S)优选被K、R、W或F取代。
I)赖氨酸(K)优选被(i)精氨酸(R)或(iii)酪氨酸(Y)或色氨酸(W)取代。
J)精氨酸(R)优选被(iii)酪氨酸(Y)或色氨酸(W)取代。
K)甲硫氨酸(M)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。
L)亮氨酸(L)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)或天冬酰胺(N)或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。
M)天冬氨酸(D)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。天冬氨酸(D)更优选被H、Y或K取代。
N)谷氨酸(E)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代。
O)异亮氨酸(I)优选被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)或(iv)亮氨酸(L)取代。
P)酪氨酸(Y)优选被(i)精氨酸(R)或赖氨酸(K)；或(ii)色氨酸(W)取代。酪氨酸(Y)更优选被W或R取代。
在本发明的一个具体实施方式中，所述的Pif1-like解旋酶更优选包含SEQ ID NO:11的变体，其包含：
Figure PCTCN2020097126-appb-000006
Figure PCTCN2020097126-appb-000007
本发明的解旋酶优选是这样一种解旋酶，即，至少一个与ssDNA或dsDNA中一个或多个核苷酸的一个或多个磷酸基团相互作用的氨基酸被取代。可以取代任何数量的氨基酸，例如，1个或更多个、2个或更多个、3个或更多个、4个或更多个、5个或更多个或者6个或更多个氨基酸。ssDNA中的核苷酸各自包含三个磷酸基团。被取代的每个氨基酸每次可以与任何数量的磷酸基团相互作用，例如，每次一个、两个或三个磷酸基团。与一个或多个磷酸基团相互作用的氨基酸可以采用蛋白质建模来进行鉴定。
取代优选增加所述至少一个氨基酸和所述ssDNA或dsDNA中一个或多个磷酸基团之间的(i)静电相互作用；(ii)氢键和/或(iii)阳离子-pi(阳离子-π)相互作用。以下使用标记(i)、(ii)和(iii)对增加(i)、(ii)和(iii)的优选取代进行讨论。
取代优选增加位点的净正电荷。可以采用本领域已知的方法来测量任何位点处的净电荷。例如，可采用等电点来限定氨基酸的净电荷。通常在约7.5处测量净电荷。取代优选是用带正电荷、不带电荷、非极性或芳族的氨基酸取代带负电荷的氨基酸。带负电荷的氨基酸是带有净负电荷的氨基酸。带负电荷的氨基酸包括但不限于天冬氨酸(D)和谷氨酸(E)。带正电荷的氨基酸是带有净正电荷的氨基酸。带正电荷的氨基酸可以是天然存在的或非天然存在的。带正电的氨基酸可以是合成的或修饰的。例如，带有净正电荷的修饰氨基酸可以专门设计用于本发明。对氨基酸进行的多种不同类型的修饰是本领域公知的。优选的天然存在的带正电荷的氨基酸包括但不限于组氨酸(H)、赖氨酸(K)和精氨酸(R)。
不带电荷的氨基酸、非极性氨基酸或者芳族氨基酸可以是天然存在的或非天然存在的。其可以是合成的或修饰的。不带电荷的氨基酸没有净电荷。合适的不带电荷氨基酸包括但不限于半胱氨酸(C)、丝氨酸(S)、苏氨酸(T)、甲硫氨酸(M)、天冬酰胺(N)和谷氨酰胺(Q)。非极性氨基酸具有非极性侧链。合适的非极性氨基酸包括但不限于甘氨酸(G)、丙氨酸(A)、脯氨酸(P)、异亮氨酸(I)、亮氨酸(L)和缬氨酸(V)。芳族氨基 酸具有芳族侧链。合适的芳族氨基酸包括但不限于组氨酸(H)、苯丙氨酸(F)、色氨酸(W)和酪氨酸(Y)。
Pif1-like解旋酶优选包含SEQ ID NO:11的变体，其中，至少一个与ssDNA或dsDNA中一个或多个核苷酸的一个或多个磷酸基团相互作用的氨基酸是H75,T91,S94,K97,N246,N247,N284,K288,N297,T394或K397中的至少一个。这些数字对应SEQ ID NO:11中的相关位点，并且相比SEQ ID NO:11可能需要在一个或多个氨基酸已经在变体中插入或删除的情况下进行改变。本领域技术人员可以确定如上所述的变体中的相应位点。
Pif1-like解旋酶优选包含SEQ ID NO:1到10中任一的变体，其中，所述至少一个与ssDNA或dsDNA中一个或多个核苷酸的一个或多个磷酸基团相互作用的氨基酸是与SEQ ID NO:11中的H75,T91,S94,K97,N246,N247,N284,K288,N297,T394或K397相对应的至少一个氨基酸。
表5示出了与SEQ ID NO:11中的H75,T91,S94,K97,N246,N247,N284,K288,N297,T394和K397对应的SEQ ID NO:1至10中的氨基酸。
表5与SEQ ID NO:11相应的SEQ ID NO:1至10中的氨基酸的位置
Figure PCTCN2020097126-appb-000008
优选的，所述的Pif1-like解旋酶包含以下任一个或多个：
a)组氨酸(H)被(i)精氨酸(R)或赖氨酸(K)；(ii)天冬酰胺(N)、丝氨酸(S)、谷氨酰 胺(Q)或苏氨酸(T)；或(iii)苯丙氨酸(F)、色氨酸(W)或酪氨酸(Y)取代；
b)苏氨酸(T)被(i)精氨酸(R)、组氨酸(H)或赖氨酸(K)；(ii)天冬酰胺(N)、丝氨酸(S)、谷氨酰胺(Q)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代；
c)丝氨酸被(i)精氨酸(R)、组氨酸(H)或赖氨酸(K)；(ii)天冬酰胺(N)、谷氨酰胺(Q)、苏氨酸(T)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代；
d)天冬酰胺(N)被(i)精氨酸(R)、组氨酸(H)或赖氨酸(K)；(ii)丝氨酸(S)、谷氨酰胺(Q)、苏氨酸(T)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代；和/或，
e)赖氨酸(K)被(i)精氨酸(R)或组氨酸(H)；(ii)天冬酰胺(N)、丝氨酸(S)、谷氨酰胺(Q)、苏氨酸(T)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代。
在本发明的一个具体实施方式中，所述Pif1-like解旋酶是SEQ ID NO:11的变体，其包含(a)至(k)中的一个或多个，其中，
(a)H75N,H75Q,H75K或H75F；(b)T91K,T91Q或T91N；(c)S94H,S94N,S94K,S94T,S94R或S94Q；(d)K97Q,K97H或K97Y；(e)N246H或N246Q；(f)N247Q或N247H；(g)N284H或N284Q；(h)K288Q或K288H；(i)N297Q,N297K或N297H；(j)T394K,T394H或T394N；或(k)K397R,K397H或K397Y。
本发明的解旋酶还是这样一种解旋酶，其中，与跨膜孔相互作用的解旋酶的一部分包含一个或多个修饰，优选一个或多个取代，进一步优选所述解旋酶包括至少一个与跨膜孔相互作用的氨基酸被取代。所述与跨膜孔相互作用的解旋酶的一部分通常是当解旋酶用于控制多核苷酸穿过孔的移动时与跨膜孔相互作用的解旋酶的一部分，例如，以下进行更详细的讨论。当解旋酶用于控制多核苷酸穿过孔的移动时，所述一部分通常包含与孔相互作用或接触的氨基酸。在施加电势下，当解旋酶与正移动穿过孔的多核苷酸结合或连接时，所述一部分通常包含与孔相互作用或接触的氨基酸。
与跨膜孔相互作用的一部分优选包含SEQ ID NO:11的变体，其中的位点包含E196,W202,N199或G201上的一个或多个氨基酸被取代，例如，2个、3个、4个或5个。
与跨膜孔相互作用的一部分优选包含SEQ ID NO:1至10中任一的变体，其包含与SEQ ID NO:11在(a)E196；(b)W202；(c)；N199；或(d)G201位点对应的至少一个 或多个的取代。
表6示出了与SEQ ID NO:11中的E196，W202，N199，G201对应的SEQ ID NO:1至10中的氨基酸。
表6与SEQ ID NO:11对应的SEQ ID NO:1至10中的氨基酸位置
Figure PCTCN2020097126-appb-000009
在本发明的一个具体实施方式中，所述的Pif1-like解旋酶是SEQ ID NO.11的变体，其包含在以下位点上的取代：
-F109/E196/H75,例如，F109W/E196L/H75N,F109W/E196L/H75Q,F109W/E196L/H75K或者F109W/E196L/H75F；
-F109/E196/T91,例如，F109W/E196L/T91K,F109W/E196L/T91Q或者F109W/E196L/T91N；
-F109/S94/E196,例如，F109W/S94H/E196L,F109W/S94T/E196L,F109W/S94R/E196L,F109W/S94Q/E196L,F109W/S94N/E196L,或者F109W/S94K/E196L；
F109/N99/E196，例如，,F109W/N99R/E196L,F109W/N99H/E196L,F109W/N99W/E196L或者F109W/N99Y/E196L；
-F109/S94/E196/I280,例如，F109W/S94H/E196L/I280K；
-F109/P100/E196,例如，F109W/P100L/E196L,F109W/P100V/E196L,F109W/P100I/E196L或者
F109W/P100T/E196L；
-F109/D132/E196,例如，F109W/D132H/E196L,F109W/D132Y/E196L或者F109W/D132K/E196L；
-F109/A161/E196,例如，F109W/A161I/E196L,F109W/A161L/E196L,F109W/A161N/E196L,F109W/A161W/E196L或者F109W/A161H/E196L；
-F109/D163/E196,例如，F109W/D163W/E196L,F109W/D163F/E196L,F109W/D163Y/E196L,F109W/D163H/E196L,F109W/D163I/E196L,F109W/D163L/E196L或者F109W/D163V/E196L；
-F109/Y244/E196,例如，F109W/Y244W/E196L,F109W/Y244Y/E196L或者F109W/Y244H/E196L；
-F109/N246/E196,例如，F109W/N246H/E196L或者F109W/N246Q/E196L；
-F109/E196/I280,例如，F109W/E196L/I280K,F109W/E196L/I280H,F109W/E196L/I280W或者F109W/E196L/I280R；
-F109/E196/Q291,例如，F109W/E196L/Q291K,F109W/E196L/Q291R,F109W/E196L/Q291W或者F109W/E196L/Q291F；
-F109/N297/E196,例如，F109W/N297Q/E196L,F109W/N297K/E196L或者F109W/N297H/E196L；
-F109/T394/E196,例如，F109W/T394K/E196L,F109W/T394H/E196L或者F109W/T394N/E196L；
-F109/H396/E196,例如，F109W/H396Y/E196L,F109W/H396F/E196L,F109W/H396Q/E196L或者F109W/H396K/E196L；
-F109/K397/E196,例如，F109W/K397R/E196L,F109W/K397H/E196L或者F109W/K397Y/E196L；或者，
-F109/Y416/E196,例如，F109W/Y416W/E196L或者F109W/Y416R/E196L。
Pif1-like解旋酶是具有下述氨基酸序列的酶：所述氨基酸序列从野生型解旋酶的氨基酸序列变化而来并保留多核苷酸结合活性。特别的，SEQ ID NO：1到11中任一 个的变体是具有下述氨基酸序列的酶：所述氨基酸序列从SEQ ID NO：1到11中任一个的氨基酸序列变化而来并保留多核苷酸结合活性。多核苷酸结合活性可使用本领域已知方法确定。合适的方法包括，但不限于荧光各向异性法，色氨酸荧光法和电泳迁移位移试验法(EMSA)。例如，变体结合单链多核苷酸的能力可以如实施例中所描述的进行确定。
基于氨基酸同一性，对于SEQ ID NO：1到11中任一个氨基酸序列的整个长度，变体优选与该序列具有至少20％的同一性。更优选的是，基于氨基酸同一性，所述变体多肽与SEQ ID NO：1到11中任一个氨基酸序列的整个序列，可以具有至少70％，至少75％，至少80％，至少85％，至少90％和更优选至少95％，97％或99％的同一性。在100或更多，例如150，200，300，400或500或更多个连续氨基酸长度上，可以具有至少为70％，例如至少80％，至少85％，至少90％或至少95％的氨基酸同一性(严格同源性)。
本发明的第二方面，提供了一种构建体，所述的构建体包含至少一个或多个本发明所述的Pif1-like解旋酶。
优选的，所述的构建体还包含多核苷酸结合部分。
优选的，所述的构建体具有控制多核苷酸移动的能力。
优选的，所述的多核苷酸结合部分可以与多核苷酸的碱基结合的部分，和/或与多核苷酸的糖结合的部分，和/或与多核苷酸的磷酸结合的部分。
优选的，组成所述构建体的Pif1-like解旋酶与多核苷酸结合部分可以单独制备，后直接连接。与可以通过遗传融合的方式直接制备构建体，例如将编码Pif1-like解旋酶与多核苷酸结合部分的核苷酸连接，后转入宿主细胞中表达、纯化获得。
进一步优选的，所述的多核苷酸结合部分为能够与多核苷酸结合的多肽，包括但不限于真核单链结合蛋白、细菌单链结合蛋白、古生菌单链结合蛋白、病毒单链结合蛋白或双链结合蛋白中的一种或两种以上的组合。
在本发明的一个具体实施方式中，所述的多核苷酸结合部分包括但不限于表7所示的任一种：
表7与多核苷酸结合的结合部分
Figure PCTCN2020097126-appb-000010
Figure PCTCN2020097126-appb-000011
本发明的第三方面，提供了一种核酸，所述的核酸编码本发明所述的Pif1-like解旋酶或所述的构建体。
本发明的第四方面，提供了一种表达载体，所述的表达载体包含本发明所述的核酸。
优选的，所述的核酸可操作的连接至表达载体中的调控元件，其中所述的调控元件优选为启动子。
在本发明的一个具体实施方式中，所述的启动子选自T7、trc、lac、ara或λ L。
优选的，所述的表达载体包括但不限于质粒、病毒或噬菌体。
本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞包含本发明所述的核酸或所述的表达载体。
优选的，所述的宿主细胞包括但不限于大肠杆菌。
在本发明的一个具体实施方式中，所述的宿主细胞选自BL21(DE3)、JM109(DE3)、B834(DE3)、TUNER、C41(DE3)、Rosetta2(DE3)、Origami、Origami B等等。
本发明的第六方面，提供了一种Pif1-like解旋酶的制备方法，所述的方法包括提供野生型Pif1-like解旋酶，然后对所述的野生型Pif1-like解旋酶进行修饰，获得本发明所述的Pif1-like解旋酶。
本发明的第七方面，提供了一种Pif1-like解旋酶的制备方法，所述的方法包括培养本发明所述的宿主细胞并进行诱导表达，纯化后获得Pif1-like解旋酶。
在本发明的一个具体实施方式中，所述的方法包括根据本发明所述Pif1-like解旋酶的氨基酸序列，获得编码Pif1-like解旋酶的核酸序列，酶切连接至表达载体后转化至大肠杆菌中，诱导表达和纯化，获得Pif1-like解旋酶。
本发明的第八方面，提供了一种控制多核苷酸移动的方法，所述的方法包括将本发明所述的Pif1-like解旋酶或所述的构建体与多核苷酸接触。
优选的，所述的控制多核苷酸移动为控制多核苷酸穿过孔的移动。所述的孔为纳米孔，所述的纳米孔为跨膜孔。该孔可以是天然的或人造的，包括但不限于蛋白孔、多核苷酸孔或固态孔。
在本发明的一个具体实施方式中，所述的跨膜孔选自生物孔、固态孔或生物与固态杂交的孔。
在本发明的一个具体实施方式中，所述的孔包括但不限于衍生自耻垢分枝杆菌孔蛋白A、耻垢分枝杆菌孔蛋白B、耻垢分枝杆菌孔蛋白C、耻垢分枝杆菌孔蛋白D、溶血素、胞溶素、白细胞介素、外膜孔蛋白F、外膜孔蛋白G、外膜磷脂酶A、WZA或奈瑟氏菌自转运脂蛋白等等。
优选的，所述的方法可以包含一个或多个的Pif1-like解旋酶共同控制多核苷酸的移动。
本发明的第九方面，提供了一种表征目标多核苷酸的方法，所述的方法包括：
I)将本发明所述的Pif1-like解旋酶或所述的构建体，与目标多核苷酸以及孔接触，使得Pif1-like解旋酶或构建体控制目标多核苷酸穿过孔的移动；
并II)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。
优选的，所述的方法中可以使用任意数量的本发明所述的Pif1-like解旋酶。优选可以为一个或多个，更优选为1、2、3、4、5、6、7、8、9个或更多个。其中，所述的两个以上本发明所述的Pif1-like解旋酶可以相同或不同。也可以包含野生型Pif1-like解旋酶或者其他类型的解旋酶。进一步的，两个以上个解旋酶之间可以连接或者只是通过分别结合在多核苷酸上而排列发挥控制多核苷酸移动的功能。
优选的，当在孔施加一种力(如电压)，目标多核苷酸通过孔的速率被Pif1-like解旋酶或构建体所控制，从而获得一种可识别的稳定的电流水平，用于确定目标多核苷酸的特征。
优选的，重复步骤I)和II)一次或多次。
优选的，所述的方法还包括横跨与所述解旋酶或构建体，和目标多核苷酸接触的孔施加势差的步骤。
优选的，所述的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。进一步优选的，所述的孔为纳米孔，所述的纳米孔为跨膜孔。所述跨膜孔为目标多核苷酸的移动提供了通道。
所述的膜可以为任何现有技术中存在的膜，优选为两性分子层，即一种由具有至少一个亲水性部分和至少一个亲脂性或疏水性部分的两性分子诸如磷脂质形成的层，两性分子可以是合成的或天然存在的。进一步优选的，所述的膜为脂质双层膜。
所述的目标多核苷酸可以使用任何已知的方法连接到膜上。如果膜是两性分子层，如脂质双分子层，所述多核苷酸优选通过在所述膜中存在的多肽或通过在所述膜中存在的疏水锚被连接到该膜上。其中，疏水锚优选为脂质、脂肪酸、甾醇、碳纳米管或氨基酸。
优选的，所述的孔选自生物孔、固态孔或生物与固态杂交的孔。
在本发明的一个具体实施方式中，所述的孔包括但不限于衍生自耻垢分枝杆菌孔蛋白A、耻垢分枝杆菌孔蛋白B、耻垢分枝杆菌孔蛋白C、耻垢分枝杆菌孔蛋白D、溶血素、胞溶素、白细胞介素、外膜孔蛋白F、外膜孔蛋白G、外膜磷脂酶A、WZA或奈瑟氏菌自转运脂蛋白等等。
当提供了促进移动的所有必要组分时，Pif1-like解旋酶沿着DNA以5’-3’的方向移动，但DNA在孔中的定向(取决于DNA的哪个末端被捕获)意味着酶可以用于逆 着所施加的场的方向将DNA移出孔，或顺着施加的场的方向将DNA移进孔。
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。
进一步优选的，所述的目标多核苷酸可以通过标签、间隔物、甲基化、氧化或损伤的方式进行修饰。
在本发明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。
进一步优选的，所述的目标多核苷酸的长度可以为10-100000个或更多个。
在本发明的一个具体实施方式中，所述的目标多核苷酸的长度可以为至少10个、至少50个、至少100个、至少200个、至少300个、至少400个、至少500个、至少1000个、至少2000个、至少5000个、至少10000个、至少50000个或至少100000个等等。
优选的，所述的解旋酶结合到单链多核苷酸的内部核苷酸中。
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。
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特征。
在本发明的一个具体实施方式中，所述的电测量包括但不限于电流测量、阻抗测量、隧道测量、风洞测量或场效应晶体管(FET)测量等等。
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，所述的表征还包括应用改进型维特比算法。
本发明的第十方面，提供了一种表征目标多核苷酸的产品，所述的产品包含本发明所述的Pif1-like解旋酶、所述的构建体、所述的核酸、所述的表达载体或所述的宿主细胞，和孔。
优选的，所述的产品中包含多个Pif1-like解旋酶和/或多个构建体。
优选的，所述的产品中包含多个孔。进一步优选的，所述的孔为纳米孔，所述的纳米孔为跨膜孔。
在本发明的一个具体实施方式中，所述的跨膜孔选自生物孔、固态孔或生物与固态杂交的孔。
在本发明的一个具体实施方式中，所述的孔包括但不限于衍生自耻垢分枝杆菌孔蛋白A、耻垢分枝杆菌孔蛋白B、耻垢分枝杆菌孔蛋白C、耻垢分枝杆菌孔蛋白D、溶血素、胞溶素、白细胞介素、外膜孔蛋白F、外膜孔蛋白G、外膜磷脂酶A、WZA或奈瑟氏菌自转运脂蛋白等等。
在本发明的一个具体实施方式中，所述的产品包含多个Pif1-like解旋酶或多个构建体，和多个孔。
优选的，所述的产品选自试剂盒、装置或传感器。
进一步优选的，所述的试剂盒中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。
本发明的第十一方面，提供了本发明所述的Pif1-like解旋酶、所述的构建体、所述的核酸、所述的表达载体、所述的宿主细胞或所述的产品在表征目标多核苷酸或控制目标多核苷酸穿过孔的移动中的应用。
本发明的第十二方面，提供了一种表征目标多核苷酸的试剂盒，所述的试剂盒包含本发明所述的Pif1-like解旋酶、所述的构建体或所述的核酸、所述的表达载体或所述的宿主细胞，和孔。
本发明的第十三方面，提供了一种表征目标多核苷酸的装置，所述的装置包含本发明所述的Pif1-like解旋酶、所述的构建体或所述的核酸、所述的表达载体或所述的宿主细胞，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，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Pif1-like解旋酶和/或多个构建体，和多个孔。
本发明的第十四方面，提供了一种表征目标多核苷酸的传感器，所述的传感器包含在所述孔和本发明所述Pif1-like解旋酶或所述的构建体之间形成复合物。
优选的，在所述目标多核苷酸存在下使所述孔和解旋酶或构建体接触，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。
本发明的第十五方面，提供了一种形成表征目标多核苷酸的传感器的方法，包括在所述孔和本发明所述Pif1-like解旋酶或所述的构建体之间形成复合物，从而形成表征目标多核苷酸的传感器。
本发明的第十六方面，提供了一种与多核苷酸连接的两个或多个解旋酶，其中，所述的两个或多个解旋酶中至少一个为本发明所述的Pif1-like解旋酶。
本发明的第十七方面，提供了一种Pif1-like解旋酶寡聚体，所述的Pif1-like解旋酶寡聚体包含一个或多个的本发明所述的Pif1-like解旋酶。
优选的，所述的Pif1-like解旋酶寡聚体还可以包含野生型Pif1-like解旋酶或其他类型的解旋酶。其中，所述的其他类型的解旋酶可以为Hel308解旋酶、XPD解旋酶、Dda解旋酶、TraI解旋酶或者TrwC解旋酶等等。
优选的，所述的Pif1-like解旋酶与野生型Pif1-like解旋酶之间、Pif1-like解旋酶与Pif1-like解旋酶之间、野生型Pif1-like解旋酶与野生型Pif1-like解旋酶、Pif1-like解旋酶与其他类型解旋酶之间或者野生型Pif1-like解旋酶与其他类型解旋酶之间，可以通过头对头、尾对尾或者头对尾的方式连接或排列。
优选的，所述的Pif1-like解旋酶寡聚体包含两个以上的本发明所述的Pif1-like解旋酶，其中，所述的Pif1-like解旋酶可以是不同的或者相同的。
本发明所述的“Pif1-like解旋酶”是经过修饰的，所述的修饰是相对于野生型或天然解旋酶是修饰的。
本发明所述的“Pif1-like解旋酶”、“构建体”或“孔”，均可以被修饰以助于鉴定或纯化，例如通过添加组氨酸残基(His标签)，天冬氨酸残基(asp标签)，链霉亲和素标签，Flag标签，SUMO标签，GST标签或MBP标签，或通过添加信号序列以促进它们从细胞中分泌，该细胞中的多肽不天然地含有该信号序列。引入遗传标签的替换方式是通过化学反应将标签连到Pif1-like解旋酶、孔或构建体上的天然或人工位点。
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“两个以上”包括两个、三个、四个、五个、六个、七个、八个或更多个等等。
本发明所述的“多个”包括但不限于两个以上、三个以上、四个以上、五个以上、六个以上、七个以上、八个以上或更多个等等。
本发明所述的“至少一个”包括但不限于一个以上、两个以上、三个以上、四个以上、五个以上、六个以上、七个以上、八个以上或更多个等等。
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“非天然氨基酸”为不是天然存在于Pif1-like解旋酶中的氨基酸。优选的，包括但不限于4-叠氮基-L-苯丙氨酸(Faz)，4-乙酰基-L-苯丙氨酸，3-乙酰基-L-苯丙氨酸，4-乙酰乙酰基-L苯丙氨酸，O-烯丙基-L-酪氨酸，3-(苯基硒烷基)-L-丙氨酸，O-2-丙炔-1-基-L-酪氨酸，4(二羟基硼基)-L-苯丙氨酸，4-[(乙基硫烷基)羰基]-L-苯丙氨酸，(2S)-2-氨基-3-{4-[(丙烷-2-基硫烷基)羰基]苯基}丙酸，(2S)-2-氨基-3-{4-[(2-氨基-3-硫烷基丙酰基) 氨基]苯基}丙酸，O-甲基-L-酪氨酸，4-氨基-L-苯丙氨酸，4-氰基-L-苯丙氨酸，3-氰基-L-苯丙氨酸，4-氟-L-苯丙氨酸，4-碘-L-苯丙氨酸，4-溴-L-苯丙氨酸，O-(三氟甲基)酪氨酸，4-硝基L-苯丙氨酸，3-羟基-L-酪氨酸，3-氨基-L-酪氨酸，3-碘-L-酪氨酸，4-异丙基-L-苯丙氨酸，3-(2-萘基)-L-丙氨酸，4-苯基-L-苯丙氨酸，(2S)-2-氨基-3-(萘-2-基氨基)丙酸，6-(甲基硫烷基)正亮氨酸，6-氧-L-赖氨酸，D-酪氨酸，(2R)-2-羟基-3-(4-羟基苯基)丙酸，(2R)-2氨基辛酸酯3-(2，2′-二吡啶-5-基)-D-丙氨酸，2-氨基-3-(8-羟基-3-喹啉基)丙酸，4-苯甲酰-L-苯丙氨酸，S-(2-硝基苄基)半胱氨酸，(2R)-2-氨基-3-[(2-硝基苄基)硫烷基]丙酸，(2S)-2-氨基-3-[(2-硝基苄基)氧基]丙酸，O-(4，5-二甲氧基-2-硝基苄基)-L-丝氨酸，(2S)-2-氨基-6-({[(2-硝基苄基)氧基]羰基}氨基)己酸，O-(2-硝基苄基)-L-酪氨酸，2-硝基苯丙氨酸，4-[(E)-苯基二氮烯基]-L-苯丙氨酸，4-[3-(三氟甲基)-3H-二吖丙啶基-3基]-D-苯丙氨酸，2-氨基-3-[[5-(二甲基氨基)-1-萘基]磺酰基氨基]丙酸，(2S)-2-氨基4-(7-羟基-2-氧-2H-色烯-4-基)丁酸，(2S)-3-[(6-乙酰基萘-2-基)氨基]-2-氨基丙酸，4(羧基甲基)苯丙氨酸，3-硝基-L-酪氨酸，O-硫基-L-酪氨酸，(2R)-6-乙酰氨基-2-氨基己酸酯，1-甲基组氨酸，2-氨基壬酸，2-氨基癸酸，L-同质半胱氨酸，5-硫烷基正缬氨酸，6-硫烷基-L-正亮氨酸，5-(甲基硫烷基)-L-正缬氨酸，N6-{[(2R，3R)-3-甲基-3，4-二氢-2H-吡咯2-基]羰基}-L-赖氨酸，N6-[(苄基氧基)羰基]赖氨酸，(2S)-2-氨基-6-[(环戊基羰基)氨基]己酸，N6-[(环戊基氧基)羰基]-L-赖氨酸，(2S)-2-氨基-6-{[(2R)-四氢呋喃-2-基羰基]氨基}己酸，(2S)-2-氨基-8-[(2R，3S)-3-乙炔基四氢呋喃-2-基]-8-氧基辛酸，N6-(叔丁氧基羰基)-L-赖氨酸，(2S)-2-羟基-6-({[(2-甲基-2-丙烷基)氧基]羰基}氨基)己酸，N6-[(烯丙氧基)羰基]赖氨酸，(2S)-2-氨基-6-({[(2-叠氮苄基)氧基]羰基}氨基)己酸，N6L-脯氨酰基-L-赖氨酸，(2S)-2-氨基-6-{[(丙-2-炔-1-基氧基)羰基]氨基}己酸或N6-[(2叠氮乙氧基)羰基]-L-赖氨酸。
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：检测Pif1-like解旋酶酶活性的荧光分析示意图。其中，荧光底物链具有5’端 ssDNA突出部分，以及50个碱基的杂交的dsDNA部分。a)所示，包括主链上部(B)在3’端具有黑洞淬灭剂(BHQ-1)碱基(E)，并且所述杂交的互补链(D)在5’端具有羧基荧光素(C)。还包括，与荧光底物的较短链(D)互补的0.5μM的捕获链(F)。如b)所示，在ATP(5mM)和MgCl2(5mM)存在下，添加到所述底物中的解旋酶(200nM)连接到所述荧光底物的5’端部分，沿着所述主链移动，并解开所述互补链后，过量的捕获链优先与互补链DNA退火以防止初始底物与丢失荧光重新退火。如c)所示，加入过量与主链完全互补的捕获链(A)后，部分未被解旋的dsDNA由于过量A的存在而产生链解旋效果，最终所有dsDNA被解旋，荧光值达到最高。
图2：荧光分析检测Pif1-like解旋酶的解开杂交dsDNA的能力，具体为含有400mM NaCl的缓冲液中的时间依赖型dsDNA被解旋比例的变化图。
图3：显示了针对不同Pif1-like解旋酶结合DNA能力进行的凝胶测定结果。泳道1为预先构建的dsDNA(SEQ ID NO：18与含5’FAM修饰的SEQ ID NO：19杂交)。泳道2-6分别包含预先连接到dsDNA的Aph Acj61、Aph PX29、Sph CBH8、PphPspYZU05和Mph MP1。泳道7-11分别包含预先连接到dsDNA的Aph Acj61-D97C/A363C、Aph PX29-D96C/A371C、Sph CBH8-A94C/A361C/C136A、PphPspYZU05-D104C/A375C/C146A和Mph MP1-E105C/A362C。条带A对应于与SEQ ID NO：18杂交的SEQ ID NO：19。标记为1A、2A、3A、4A、5A的区域分别对应于1、2、3、4、5个解旋酶结合与SEQ ID NO：18杂交的SEQ ID NO：19。
图4：实施例中使用的DNA构建体的图，其中SEQ ID NO：13(标记为B)其5’末端连接到20个iSpC3间隔区(标记为A)，其3’末端连接到4个iSpC3间隔区(标记为C)，该间隔区(标记为C)连接到SEQ ID NO：14(标记为D)的5’末端，该SEQ ID NO：14(标记为D)的3’末端连接到SEQ ID NO：17或SEQ ID NO：24(标记为E)，该构建体的SEQ ID NO：15(标记为F)区域与SEQ ID NO：16(标记为G，其具有3’胆固醇系链)杂交。
图5：当Pif1-like解旋酶(Mph MP1-E105C/A362C(具有突变E105C/A362C的SEQ ID NO：11))控制DNA构建体A穿过纳米孔8MspA(SEQ ID NO：12)移位时的示例电流轨迹(y轴坐标＝电流(pA，0到250)，x轴坐标＝采样频率(hz，0到3.5*105))。
图6：图5电流轨迹图中所示的Pif1-like解旋酶控制的DNA移动的区域放大图(y轴坐标＝电流(pA，30到100)，x轴坐标＝采样频率(hz，2.346到2.366*105))。
图7：当Pif1-like解旋酶(Sph CBH8-A94C/A361C/C136A(具有A94C/A361C/C136A突变的SEQ ID NO：5)控制DNA构建体B穿过MspA纳米孔移位时的示例电流轨迹(y轴坐标＝电流(pA)，x轴坐标＝时间(s))。
图8：当解旋酶(Eph Pei26-D99C/A366C/C141A(具有D99C/A366C/C141A突变的SEQ ID NO：6))控制DNA构建体B穿过MspA纳米孔移位时的示例电流轨迹(y轴坐标＝电流(pA)，x轴坐标＝时间(s))。
图9：当解旋酶(Pph PspYZU05-D104C/A375C/C146A(具有D104C/A375C和C146A突变的SEQ ID NO：8))控制DNA构建体B穿过MspA纳米孔移位时的示例电流轨迹(y轴坐标＝电流(pA)，x轴坐标＝时间(s))。
图10：显示了经纯化后的Mph MP1(SEQ ID NO:11)SDS-PAGE凝胶电泳图。其中，M是Marker(Kd)，1道为Mph MP1解旋酶的电泳结果图。
图11：显示了SEQ NO：1至11的氨基酸序列对应关系。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。各实施例中所用的设备和试剂均常规市售可得。
实施例1 Pif1-like解旋酶的制备
1、材料和方法
将含有Pif1-like解旋酶序列的重组质粒(氨基酸序列SEQ ID NO:1-11的变体，其对应核苷酸序列SEQ ID NO:25-35)通过热击转化到BL21(DE3)感受态细胞，复苏菌液涂布氨苄抗性固体LB平板后在37℃过夜培养，挑取单克隆菌落，接种至100ml含有氨苄抗性的液体LB培养基中37℃培养。按1％的接种量转接至氨苄抗性的LB液体培养基中进行扩大培养，37℃、200rpm条件下培养，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(Isopropylβ-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FPLC方法进行纯化，收集样品。
2、结果
图10显示了经纯化后的Mph MP1(SEQ ID NO:11的变体)SDS-PAGE凝胶电泳图。
实施例2
使用荧光分析检测酶活性来阐述Pif1-like解旋酶的解开杂交dsDNA的能力。
1、材料和方法
如图1中的a)所示，荧光底物链(终浓度100nM)具有5’端ssDNA突出部分，以及50个碱基的杂交的dsDNA部分。包括主链上部在3’端具有黑洞淬灭剂(BHQ-1)碱基(SEQ ID NO:20--BHQ-3’)，并且所述杂交的互补链在5’端具有羧基荧光素(5’FAM-SEQ ID NO:21)。当杂交的来自荧光素的荧光被局部的BHQ-1淬灭时，底物基本上是无荧光的。还包括与荧光底物的较短链互补的0.5μM的捕获链(SEQ ID NO:22)。如b)所示，在ATP(5mM)和MgCl 2(5mM)存在下，添加到底物中的Pif1-like解旋酶(200nM)连接到所述荧光底物的5’端部分，沿着所述主链移动，并解开所述互补链后，过量的捕获链优先与互补链DNA退火以防止初始底物与丢失荧光重新退火。同时，体系内仍存在一定量的杂交dsDNA未被Pif1-like解旋酶解旋。如c)所示，加入过量与主链完全互补的捕获链A(SEQ ID NO:23)后，部分未被解旋的dsDNA由于过量A的存在而产生链解旋效果，最终所有 dsDNA被解旋，荧光值达到最高。
2、结果
图2显示了含有400mMNaCl的缓冲液中的时间依赖型dsDNA被解旋比例的变化图(10mM Hepes pH8.0,5mM ATP,5mM MgCl2,100nM荧光底物DNA,0.5μM捕获DNA)。
实施例3
示例性的采用凝胶测定来测量本发明经修饰的Pif1-like解旋酶结合DNA的能力。
具体的，测量Aph Acj61-D97C/A363C(具有D97C/A363C突变的SEQ ID NO：2)，Aph PX29-D96C/A371C(具有D96C/A371C突变的SEQ ID NO：3)，Sph CBH8-A94C/A361C/C136A(具有A94C/A361C和C136A突变的SEQ ID NO：5)和Pph PspYZU05-D104C/A375C/C146A(具有D104C/A375C和C146A突变的SEQ ID NO：8)以及Mph MP1-E105C/A362C(具有E105C/A362C突变的SEQ ID NO：11)结合DNA的能力。
1、材料和方法
将退火的DNA复合物(SEQ ID NO：18与含5’FAM修饰的SEQ ID NO：19杂交)以(1：1，体积/体积)的比例分别与Aph Acj61，Aph PX29，Sph CBH8，Pph PspYZU05，Mph MP1，Aph Acj61-D97C/A363C，Aph PX29-D96C/A371C，Sph CBH8-A94C/A361C/C136A，Pph PspYZU05-D104C/A375C/C146A，Mph MP1-E105C/A362C在10mM HEPE，pH值8.0，400mM氯化钾中混合，得到Pif1-like解旋酶的最终浓度为(600nM)和DNA为(30nM)。Pif1-like解旋酶在室温下结合DNA进行1小时。将TMAD加入各样品中至终浓度5μM，在室温下培育1小时。将样品上样到4-20％TBE凝胶上，并在160V跑胶1.5小时。然后将该凝胶在蓝色荧光下观察DNA条带。
2、结果
图3显示了Pif1-like解旋酶在修饰前后对DNA结合能力的影响。泳道2至6显示大量DNA在电泳过程中未被Pif1-like解旋酶结合而观察不到明显的结合条带。而泳道7-11 显示了不同个数酶结合至DNA的结合条带，其中泳道9和10显示了高达5个Pif1-like解旋酶可以结合到SEQ ID NO：18的单链部分。这表明，Pif1-like解旋酶在修饰后明显增强了与DNA结合的牢固程度。
实施例4
本实施例以Mph MP1-E105C/A362C(具有突变E105C/A362C的SEQ ID NO：11)为例，验证Pif1-like解旋酶如何控制整个DNA链移动穿过单个MspA纳米孔(SEQ ID NO：12)。1、材料和方法
制备如图4所示的DNA构建体A：SEQ ID NO：13其5’末端连接到20个iSpC3间隔区，其3’末端连接到4个iSpC3间隔区，该间隔区连接到SEQ ID NO：14的5’末端，该SEQ ID NO：14的3’末端连接到SEQ ID NO：17，该DNA构建体的SEQ ID NO：15区域与SEQ ID NO：16(其具有3’胆固醇系链)杂交)。
将制备的DNA构建体和Mph MP1-E105C/A362C在25℃的缓冲液(10mM HEPES，pH 8.0，400mM NaCl，5％甘油，2mM DTT)中一起预孵育30分钟。
由嵌入1,2-二乙醇酰基-甘油-3-胆碱磷酸脂质双分子层的MspA纳米孔获得电测量。通过Montal-Mueller技术，在PTFE膜上的～25μm直径孔穴形成双分子层，隔开两个约100μL的缓冲溶液。所有实验在所述缓冲液中进行。使用装配有数字转换器的放大器测定单通道电流。将Ag/AgCl电极连接到所述缓冲液中使得顺式隔间连接到放大器的接地端，并且反式隔间连接到活性电极。
在所述双分子层实现单孔之后，将DNA多核苷酸和Pif1-like解旋酶添加到电生理学室的顺式隔间的70μL缓冲液中以引发解旋酶-DNA复合体在所述纳米孔的捕获。根据需要通过向所述顺式隔间添加二价金属(5mM MgCl 2)和NTP(2.86μM ATP)激活解旋酶ATP酶活性。实验在+180mV的恒定电势下实施。
2、结果和讨论
结果观察到了DNA构建体被Pif1-like解旋酶控制的DNA移动，Pif1-like解旋酶控 制的DNA移动的结果见图5。Pif1-like解旋酶控制的DNA移动为50秒长并对应于接近10kB的DNA构建体穿过所述纳米孔的移位。其中，图6显示了Pif1-like解旋酶控制的DNA移动的部分区域的放大图。
实施例5
本实施例以Sph CBH8-A94C/A361C/C136A(具有A94C/A361C/C136A突变的SEQ ID NO：5)和Eph Pei26-D99C/A366C/C141A(具有D99C/A366C/C141A突变的SEQ ID NO：6)以及Pph PspYZU05-D104C/A375C/C146A(具有D104C/A375C和C146A突变的SEQ ID NO：8)为例，验证Pif1-like解旋酶如何控制整个DNA链移动穿过单个MspA纳米孔。1、材料和方法
制备如图4所示的DNA构建体B：SEQ ID NO：13其5’末端连接到20个iSpC3间隔区，其3’末端连接到4个iSpC3间隔区，该间隔区连接到SEQ ID NO：14的5’末端，该SEQ ID NO：14的3’末端连接到SEQ ID NO：24，该DNA构建体的SEQ ID NO：15区域与SEQ ID NO：16(其具有3’胆固醇系链)杂交。该DNA构建体B与实施例4使用的构建体相似，不同在于标记为E的区域对应于SEQ ID NO：24。
将在缓冲液(在50mM NaCl中，10mM Tris pH7.5)中的DNA构建体B与在缓冲液(50mM KCl，10mMHEPES，pH 8.0)中的Sph CBH8-A94C/A361C/C136A，Eph Pei26-D99C/A366C/C141A或Pph PspYZU05-D104C/A375C/C146A在室温下预孵育30分钟。然后向DNA/酶预混合物中添加TMAD并进一步孵育30分钟。最后，向所述预混合物中添加缓冲液(10mMHEPES，600mM KCl，pH 8.0，3mM MgCl2)和ATP。
在室温下由嵌入到在缓冲液(10mM HEPES，400mM KCl，pH 8.0)中的嵌段共聚物中的单个MspA纳米孔获得电测量值。在实现单个孔插入到嵌段共聚物中后，将Pif1-like解旋酶(Sph CBH8-A94C/A361C/C136A，Eph Pei26-D99C/A366C/C141A或Pph PspYZU05-D104C/A375C/C146A(1nM终浓度))，DNA(0.3nM终浓度)，燃料(ATP 3mM终浓度)的预混合物添加到单个纳米孔实验系统中。每个实验在保持电势180mV下进行2 小时，并监控Pif1-like解旋酶控制的DNA移动。
2、结果
对DNA构建体B，观察Pif1-like解旋酶控制的DNA移动，其Sph CBH8-A94C/A361C/C136A，Eph Pei26-D99C/A366C/C141A或Pph PspYZU05-D104C/A375C/C146A控制的DNA移动的结果分别参见图7-9。
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。

Claims (38)

  1. 一种Pif1-like解旋酶，其特征在于，包括在Pif1-like解旋酶的塔结构域、销结构域和/或1A结构域引入至少一个半胱氨酸残基和/或至少一个非天然氨基酸，其中所述Pif1-like解旋酶保留其控制多核苷酸移动的能力。
  2. 根据权利要求1所述的Pif1-like解旋酶，其中所述Pif1-like解旋酶包括：
    (a)SEQ ID NO：1的变体，其中在塔结构域的E264-P278和N296-A394，和/或销结构域的K89-E105，和/或1A结构域的M1-L88和M106-V181中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (b)SEQ ID NO：2的变体，其中在塔结构域的E265-P279和N297-A392，和/或销结构域的K89-D105，和/或1A结构域的M1-L88和I106-M180中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (c)SEQ ID NO：3的变体，其中在塔结构域的T266-P280和N298-S403，和/或销结构域的K89-A109，和/或1A结构域的M1-L88和K110-V182中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (d)SEQ ID NO：4的变体，其中在塔结构域的T266-P280和N298-S404，和/或销结构域的K89-A109，和/或1A结构域的M1-L88和K110-V182中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (e)SEQ ID NO：5的变体，其中在塔结构域的E260-P274和N292-A391，和/或销结构域的K86-E102，和/或1A结构域的M1-L84和M103-K177中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (f)SEQ ID NO：6的变体，其中在塔结构域的E266-P280和N298-A396，和/或销结构域的K91-E107，和/或1A结构域的M1-L90和M108-M183中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (g)SEQ ID NO：7的变体，其中在塔结构域的T276-P290和N308-P402，和/或销结构域的K100-D116，和/或1A结构域的M1-L99和D117-M191中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (h)SEQ ID NO：8的变体，其中在塔结构域的D274-P288和N306-A404，和/或销结构域的K95-E112，和/或1A结构域的M1-L95和I113-K187中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (i)SEQ ID NO：9的变体，其中在塔结构域的E260-P274和N292-A391，和/或销结构域的K86-E102，和/或1A结构域的M1-L85和M103-K177中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；
    (j)SEQ ID NO：10的变体，其中在塔结构域的E265-P279和H297-A393，和/或销结构域的K88-E104，和/或1A结构域的M1-L87和I105-K180中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸；或，
    (k)SEQ ID NO：11的变体，其中在塔结构域的E264-P278和N296-P389，和/或销结构域的K97-A113，和/或1A结构域的M1-L96和P114-K184中引入了至少一个半胱氨酸残基和/或至少一个非天然氨基酸。
  3. 根据权利要求2所述的Pif1-like解旋酶，其中所述解旋酶包括：
    (a)SEQ ID NO：11的变体，其包括(i)E105C和/或A362C；(ii)E104C和/或K360C；(iii)E104C和/或A362C；(iv)E104C和/或Q363C；(v)E104C和/或K366C；(vi)E105C和/或M356C；(vii)E105C和/或K360C；(viii)E104C和/或M356C；(ix)E105C和/或Q363C；(x)E105C和/或K366C；(xi)F108C和/或M356C；(xii)F108C和/或K360C；(xiii)F108C和/或A362C；(xiv)F108C和/或Q363C；(xv)F108C和/或K366C；(xvi)K134C和/或M356C；(xvii)K134C和/或K360C；(xviii)K134C和/或A362C；(xix)K134C和/或Q363C；(xx)K134C和/或K366C；(xxi)(i)到(xx)任一个和G359C；(xxii)(i)到(xx)任一个和Q111C；(xxiii)(i)到(xx)任一个和I138C；(xxiv)(i)到(xx)任一个和Q111C和I138C；(xxv)E105C和/或F377C；(xxvi)Y103L，E105Y，N352N，A362C和Y365N；(xxvii)E105Y和A362C；(xxviii)A362C；(xxix)Y103L，E105C，N352N，A362Y和Y365N；(xxx)Y103L，E105C和A362Y；(xxxi)E105C和/或A362C，和I280A；(xxxii)E105C和/或L358C；(xxxiii)E104C和/或G359C；(xxxiv)E104C和/或A362C；(xxxv)K106C和/或W378C；(xxxvi)T102C和/或N382C；(xxxvii)T102C和/或W378C；(xxxviii)E104C和/或Y355C；(xxxix)E104C和/或N382C；(xl)E104C和/或K381C；(xli)E104C和/或K379C；(xlii)E104C和/或D376C； (xliii)E104C和/或W378C；(xliv)E104C和/或W374C；(xlv)E105C和/或Y355C；(xlvi)E105C和/或N382C；(xlvii)E105C和/或K381C；(xlviii)E105C和/或K379C；(xlix)E105C和/或D376C；(l)E105C和/或W378C；(li)E105C和/或W374C；(lii)E105C和A362Y；(liii)E105C，G359C和A362C；或(liv)I2C，E105C和A362C；或者，
    (b)SEQ ID NO：1到10中任一个的变体，其包括在对应于SEQ ID NO：11的(i)到(liv)中任一个所限定的位置处的半胱氨酸残基。
  4. 根据权利要求1-3任一所述的Pif1-like解旋酶，所述Pif1-like解旋酶的氨基酸序列为SEQ ID NO：1到11所示氨基酸序列或与SEQ ID NO：1到11所示氨基酸序列具有至少30％、至少40％、至少50％、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％的同源性并具有控制多核苷酸移动的能力。
  5. 根据权利要求1-4任一所述的Pif1-like解旋酶，其特征在于，所述引入的半胱氨酸与半胱氨酸之间相互连接，引入的非天然氨基酸与非天然氨基酸之间相互连接，引入的半胱氨酸与非天然氨基酸之间相互连接，引入的半胱氨酸与天然氨基酸之间相互连接，或者引入的非天然氨基酸与天然氨基酸之间相互连接。
  6. 根据权利要求1-5任一所述的Pif1-like解旋酶，其特征在于，所述Pif1-like解旋酶还包括至少一个或多个的半胱氨酸被取代；优选为丙氨酸、丝氨酸或缬氨酸取代半胱氨酸。
  7. 根据权利要求1-6任一所述的Pif1-like解旋酶，其特征在于，所述的Pif1-like解旋酶包括：
    (a)SEQ ID NO：5的变体，并且所述一个或多个被取代的天然半胱氨酸残基为C109、C114、C136或C414中的一个或多个；或者，
    (b)SEQ ID NO：1、2、3、4、6、7、8、9、10和11中任一个的变体，并且所述一个或多个被取代的天然半胱氨酸残基对应于SEQ ID NO：5中C109、C114、C136或C414中的一个或多个。
  8. 根据权利要求1-7任一所述的Pif1-like解旋酶，其特征在于，所述的Pif1-like解旋酶还包括对表面带负电的氨基酸、极性或非极性氨基酸进行取代或修饰；进一步优选的，所述的取代包括带正电的氨基酸、不带电荷的氨基酸取代带负电的氨基酸、不带电荷的氨基酸、芳香族氨基酸、极性或非极性氨基酸。
  9. 根据权利要求8所述的Pif1-like解旋酶，其特征在于，其中所述Pif1-like解旋 酶包括：
    a)SEQ ID NO：11的变体并且所述一个或多个带负电荷的氨基酸为D5，E9，E24，E87，I65，S58，D209或D216中的一个或多个；或者，
    b)SEQ ID NO:1到10中任一个的变体，其中，所述一个或多个带负电荷的氨基酸对应于SEQ ID NO：11中D5，E9，E24，E87，I65，S58，D209或D216中的一个或多个。
  10. 根据权利要求9所述的Pif1-like解旋酶，其特征在于，其中所述Pif1-like解旋酶还包括：
    a)SEQ ID NO：1的变体，并且所述一个或多个带负电荷的氨基酸还包括S171；或者，
    b)SEQ ID NO：9的变体，并且所述一个或多个带负电荷的氨基酸还包括S173。
  11. 根据权利要求1-10任一所述的Pif1-like解旋酶，其特征在于，其中所述非天然氨基酸选自4-叠氮基-L-苯丙氨酸(Faz)，4-乙酰基-L-苯丙氨酸，3-乙酰基-L-苯丙氨酸，4-乙酰乙酰基-L苯丙氨酸，O-烯丙基-L-酪氨酸，3-(苯基硒烷基)-L-丙氨酸，O-2-丙炔-1-基-L-酪氨酸，4(二羟基硼基)-L-苯丙氨酸，4-[(乙基硫烷基)羰基]-L-苯丙氨酸，(2S)-2-氨基-3-{4-[(丙烷-2-基硫烷基)羰基]苯基}丙酸，(2S)-2-氨基-3-{4-[(2-氨基-3-硫烷基丙酰基)氨基]苯基}丙酸，O-甲基-L-酪氨酸，4-氨基-L-苯丙氨酸，4-氰基-L-苯丙氨酸，3-氰基-L-苯丙氨酸，4-氟-L-苯丙氨酸，4-碘-L-苯丙氨酸，4-溴-L-苯丙氨酸，O-(三氟甲基)酪氨酸，4-硝基L-苯丙氨酸，3-羟基-L-酪氨酸，3-氨基-L-酪氨酸，3-碘-L-酪氨酸，4-异丙基-L-苯丙氨酸，3-(2-萘基)-L-丙氨酸，4-苯基-L-苯丙氨酸，(2S)-2-氨基-3-(萘-2-基氨基)丙酸，6-(甲基硫烷基)正亮氨酸，6-氧-L-赖氨酸，D-酪氨酸，(2R)-2-羟基-3-(4-羟基苯基)丙酸，(2R)-2氨基辛酸酯3-(2，2′-二吡啶-5-基)-D-丙氨酸，2-氨基-3-(8-羟基-3-喹啉基)丙酸，4-苯甲酰-L-苯丙氨酸，S-(2-硝基苄基)半胱氨酸，(2R)-2-氨基-3-[(2-硝基苄基)硫烷基]丙酸，(2S)-2-氨基-3-[(2-硝基苄基)氧基]丙酸，O-(4，5-二甲氧基-2-硝基苄基)-L-丝氨酸，(2S)-2-氨基-6-({[(2-硝基苄基)氧基]羰基}氨基)己酸，O-(2-硝基苄基)-L-酪氨酸，2-硝基苯丙氨酸，4-[(E)-苯基二氮烯基]-L-苯丙氨酸，4-[3-(三氟甲基)-3H-二吖丙啶基-3基]-D-苯丙氨酸，2-氨基-3-[[5-(二甲基氨基)-1-萘基]磺酰基氨基]丙酸，(2S)-2-氨基4-(7-羟基-2-氧-2H-色烯-4-基)丁酸，(2S)-3-[(6-乙酰基萘-2-基)氨基]-2-氨基丙酸，4(羧基甲基)苯丙氨酸，3-硝基-L-酪氨酸，O-硫基-L-酪氨酸，(2R)-6-乙酰氨基-2-氨基己酸酯，1-甲基组氨酸，2-氨基壬酸，2-氨基癸酸，L-同质半胱氨酸，5-硫烷基正 缬氨酸，6-硫烷基-L-正亮氨酸，5-(甲基硫烷基)-L-正缬氨酸，N6-{[(2R，3R)-3-甲基-3，4-二氢-2H-吡咯2-基]羰基}-L-赖氨酸，N6-[(苄基氧基)羰基]赖氨酸，(2S)-2-氨基-6-[(环戊基羰基)氨基]己酸，N6-[(环戊基氧基)羰基]-L-赖氨酸，(2S)-2-氨基-6-{[(2R)-四氢呋喃-2-基羰基]氨基}己酸，(2S)-2-氨基-8-[(2R，3S)-3-乙炔基四氢呋喃-2-基]-8-氧基辛酸，N6-(叔丁氧基羰基)-L-赖氨酸，(2S)-2-羟基-6-({[(2-甲基-2-丙烷基)氧基]羰基}氨基)己酸，N6-[(烯丙氧基)羰基]赖氨酸，(2S)-2-氨基-6-({[(2-叠氮苄基)氧基]羰基}氨基)己酸，N6L-脯氨酰基-L-赖氨酸，(2S)-2-氨基-6-{[(丙-2-炔-1-基氧基)羰基]氨基}己酸或N6-[(2叠氮乙氧基)羰基]-L-赖氨酸。
  12. 根据权利要求1-11任一所述的Pif1-like解旋酶，其特征在于，所述的Pif1-like解旋酶还包括：
    (a)至少一个与单链DNA或双链DNA中一个或多个核苷酸相互作用的氨基酸被取代；和/或，
    (b)至少一个与跨膜孔相互作用的氨基酸被取代，
    其中，所述Pif1-like解旋酶具有控制多核苷酸移动的能力。
  13. 根据权利要求12所述的Pif1-like解旋酶，其中，在(a)中，用包含较大侧链的氨基酸取代至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸。
  14. 根据权利要求13所述的Pif1-like解旋酶，其中，所述Pif1-like解旋酶包括：
    (a)SEQ ID NO:11的变体，其中，所述至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸是P73，H93，N99，F109，I280，A161，F130，D132，D162，D163，E277，K415，Q291，H396，Y244或P100中的至少一个；或者，
    (b)SEQ ID NO:1到10中任一的变体，其中，所述至少一个与单链或双链DNA中一个或多个核苷酸的糖和/或碱基相互作用的氨基酸对应于SEQ ID NO:11中的P73，H93，N99，F109，I280，A161，F130，D132，D162，D163，E277，K415，Q291，H396，Y244或P100中的至少一个。
  15. 根据权利要求13或14所述的Pif1-like解旋酶，其中，所述较大侧链包括增加数目的碳原子，具有增加的长度，增加的分子体积和/或具有增加的范德华体积。
  16. 根据权利要求13-15任一所述的Pif1-like解旋酶，其中，所述较大侧链增加了所述至少一个氨基酸与所述单链或双链DNA中一个或多个核苷酸之间的(i)静电相互作用；(ii)氢键和/或(iii)阳离子-pi相互作用。
  17. 根据权利要求13-16任一所述的Pif1-like解旋酶，其中，所述较大侧链的氨基酸不是丙氨酸(A)、半胱氨酸(C)、甘氨酸(G)、硒代半胱氨酸(U)、甲硫氨酸(M)、天冬氨酸(D)或谷氨酸(E)。
  18. 根据权利要求12-17任一所述的Pif1-like解旋酶，其中，
    A)组氨酸(H)被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)或天冬酰胺(N)；或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代；
    B)天冬酰胺(N)被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)或组氨酸(H)；或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代；
    C)脯氨酸(P)被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)、苏氨酸(T)或组氨酸(H)；(iii)酪氨酸(Y)、苯丙氨酸(F)或色氨酸(W)；或(iv)亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)取代；
    D)苯丙氨酸(F)被(i)精氨酸(R)或赖氨酸(K)；(ii)组氨酸(H)；或(iii)酪氨酸(Y)或色氨酸(W)取代；
    E)天冬氨酸(D)被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；或(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)取代；
    F)缬氨酸(V)被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)；或(iv)异亮氨酸(I)或亮氨酸(L)取代；
    G)丝氨酸(S)被(i)精氨酸(R)或赖氨酸(K)；(ii)谷氨酰胺(Q)、天冬酰胺(N)或组氨酸(H)；(iii)苯丙氨酸(F)、酪氨酸(Y)或色氨酸(W)；或(iv)异亮氨酸(I)或亮氨酸(L)取代；和/或，
    H)酪氨酸(Y)被(i)精氨酸(R)或赖氨酸(K)；或(ii)色氨酸(W)取代。
  19. 根据权利要求12-18任一所述的Pif1-like解旋酶，其特征在于，所述Pif1-like解旋酶是SEQ ID NO:11的变体，并且包含：
    Figure PCTCN2020097126-appb-100001
    Figure PCTCN2020097126-appb-100002
  20. 根据权利要求12-19任一所述的Pif1-like解旋酶，其中，在(a)中，至少一个与单链DNA或双链DNA中一个或多个核苷酸的一个或多个磷酸基团相互作用的氨基 酸被取代。
  21. 根据权利要求20所述的Pif1-like解旋酶，其中，所述Pif1-like解旋酶包括：
    (a)SEQ ID NO:11的变体，其中，所述至少一个与ssDNA或dsDNA中一个或多个核苷酸的一个或多个磷酸基团相互作用的氨基酸是H75,T91,S94,K97,N246,N247,N284,K288,N297,T394或K397中的至少一个；或者，
    (b)SEQ ID NO:1到10中任一的变体，其中，所述至少一个与ssDNA或dsDNA中一个或多个核苷酸的一个或多个磷酸基团相互作用的氨基酸是与SEQID NO:11中的H75,T91,S94,K97,N246,N247,N284,K288,N297,T394或K397相对应的至少一个氨基酸。
  22. 根据权利要求21所述的Pif1-like解旋酶，其中，
    a)组氨酸(H)被(i)精氨酸(R)或赖氨酸(K)；(ii)天冬酰胺(N)、丝氨酸(S)、谷氨酰胺(Q)或苏氨酸(T)；或(iii)苯丙氨酸(F)、色氨酸(W)或酪氨酸(Y)取代；
    b)苏氨酸(T)被(i)精氨酸(R)、组氨酸(H)或赖氨酸(K)；(ii)天冬酰胺(N)、丝氨酸(S)、谷氨酰胺(Q)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代；
    c)丝氨酸被(i)精氨酸(R)、组氨酸(H)或赖氨酸(K)；(ii)天冬酰胺(N)、谷氨酰胺(Q)、苏氨酸(T)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代；
    d)天冬酰胺(N)被(i)精氨酸(R)、组氨酸(H)或赖氨酸(K)；(ii)丝氨酸(S)、谷氨酰胺(Q)、苏氨酸(T)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代；和/或，
    e)赖氨酸(K)被(i)精氨酸(R)或组氨酸(H)；(ii)天冬酰胺(N)、丝氨酸(S)、谷氨酰胺(Q)、苏氨酸(T)或组氨酸(H)；或(iii)苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)或组氨酸(H)取代。
  23. 根据权利要求20-22任一所述的Pif1-like解旋酶，其中，所述Pif1-like解旋酶是SEQ ID NO:11的变体，其包含(a)至(k)中的一个或多个，其中，
    (a)H75N,H75Q,H75K或H75F；(b)T91K,T91Q或T91N；(c)S94H,S94N,S94K,S94T,S94R或S94Q；(d)K97Q,K97H或K97Y；(e)N246H或N246Q；(f)N247Q或N247H；(g)N284H或N284Q；(h)K288Q或K288H；(i)N297Q,N297K或N297H；(j)T394K,T394H或T394N；或(k)K397R,K397H或K397Y。
  24. 根据权利要求12-23任一所述的Pif1-like解旋酶，其中，所述解旋酶包括至 少一个与跨膜孔相互作用的氨基酸被取代：
    (a)SEQ ID NO:11的变体，其中，所述Pif1-like解旋酶包含在(a)E196(b)W202(c)N199或(d)G201上的一个或多个取代；或者，
    (b)SEQ ID NO:1到10中任一的变体，其中，所述Pif1-like解旋酶包含在(a)E196(b)W202(c)N199或(d)G201相对应的至少一个取代。
  25. 根据权利要求1-24任一所述的Pif1-like解旋酶，其中，所述Pif1-like解旋酶是SEQ ID NO:11的变体，其包含在以下位点上的取代：
    -F109/E196/H75,例如，F109W/E196L/H75N,F109W/E196L/H75Q,F109W/E196L/H75K或者F109W/E196L/H75F；
    -F109/E196/T91,例如，F109W/E196L/T91K,F109W/E196L/T91Q或者F109W/E196L/T91N；
    -F109/S94/E196,例如，F109W/S94H/E196L,F109W/S94T/E196L,F109W/S94R/E196L,F109W/S94Q/E196L,F109W/S94N/E196L或者F109W/S94K/E196L；
    F109/N99/E196，例如，F109W/N99R/E196L,F109W/N99H/E196L,F109W/N99W/E196L或者F109W/N99Y/E196L；
    -F109/S94/E196/I280,例如，F109W/S94H/E196L/I280K；
    -F109/P100/E196,例如，F109W/P100L/E196L,F109W/P100V/E196L,F109W/P100I/E196L或者
    F109W/P100T/E196L；
    -F109/D132/E196,例如，F109W/D132H/E196L,F109W/D132Y/E196L或者F109W/D132K/E196L；
    -F109/A161/E196,例如，F109W/A161I/E196L,F109W/A161L/E196L,F109W/A161N/E196L,F109W/A161W/E196L或者F109W/A161H/E196L；
    -F109/D163/E196,例如，F109W/D163W/E196L,F109W/D163F/E196L,F109W/D163Y/E196L,F109W/D163H/E196L,F109W/D163I/E196L,F109W/D163L/E196L或者F109W/D163V/E196L；
    -F109/Y244/E196,例如，F109W/Y244W/E196L,F109W/Y244Y/E196L或者F109W/Y244H/E196L；
    -F109/N246/E196,例如，F109W/N246H/E196L或者F109W/N246Q/E196L；
    -F109/E196/I280,例如，F109W/E196L/I280K,F109W/E196L/I280H,F109W/E196L/I280W或者F109W/E196L/I280R；
    -F109/E196/Q291,例如，F109W/E196L/Q291K,F109W/E196L/Q291R,F109W/E196L/Q291W或者F109W/E196L/Q291F；
    -F109/N297/E196,例如，F109W/N297Q/E196L,F109W/N297K/E196L或者F109W/N297H/E196L；
    -F109/T394/E196,例如，F109W/T394K/E196L,F109W/T394H/E196L或者F109W/T394N/E196L；
    -F109/H396/E196,例如，F109W/H396Y/E196L,F109W/H396F/E196L,F109W/H396Q/E196L或者F109W/H396K/E196L；
    -F109/K397/E196,例如，F109W/K397R/E196L,F109W/K397H/E196L或者F109W/K397Y/E196L；或者，
    -F109/Y416/E196,例如，F109W/Y416W/E196L或者F109W/Y416R/E196L。
  26. 一种构建体，其特征在于，所述的构建体包含至少一个权利要求1-25任一所述的Pif1-like解旋酶。
  27. 根据权利要求26所述的构建体，其特征在于，所述的构建体还包含多核苷酸结合部分。
  28. 一种核酸，其特征在于，所述的核酸编码权利要求1-25任一所述的Pif1-like解旋酶或权利要求26-27任一所述的构建体。
  29. 一种表达载体，其特征在于，所述的表达载体包含权利要求28所述的核酸。
  30. 一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求28所述的核酸或权利要求29所述的表达载体。
  31. 一种控制多核苷酸移动的方法，其特征在于，所述的方法包括将权利要求1-25任一所述的Pif1-like解旋酶或权利要求26-27任一所述的构建体与多核苷酸接触。
  32. 一种表征目标多核苷酸的方法，其特征在于，所述的方法包括：
    I)将权利要求1-25任一所述的Pif1-like解旋酶或权利要求26-27任一所述的构建体，与目标多核苷酸以及孔接触，使得Pif1-like解旋酶或构建体控制目标多核苷酸穿过孔的移动；
    并II)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。
  33. 根据权利要求32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构建体，和目标多核苷酸接触的孔施加势差的步骤；优选的，所述的孔选自生物孔、固态孔或生物与固态杂交的孔。
  34. 根据权利要求32或33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。
  35. 根据权利要求32-34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；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。
  36. 一种表征目标多核苷酸的产品，其特征在于，所述的产品包含权利要求1-25任一所述的Pif1-like解旋酶、权利要求26-27任一所述的构建体、权利要求28所述的核酸、权利要求29所述的表达载体或权利要求30所述的宿主细胞，和孔。
  37. 根据权利要求36所述的产品，其特征在于，所述的产品选自试剂盒、装置或传感器。
  38. 权利要求1-25任一所述的Pif1-like解旋酶、权利要求26-27任一所述的构建体、权利要求28所述的核酸、权利要求29所述的表达载体、权利要求30所述的宿主细胞或权利要求36-37任一所述的产品在表征目标多核苷酸或控制目标多核苷酸穿过孔的移动中的应用。
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