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WO2022126304A1 - Hélicase modifiée et son application - Google Patents

Hélicase modifiée et son application Download PDF

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Publication number
WO2022126304A1
WO2022126304A1 PCT/CN2020/136031 CN2020136031W WO2022126304A1 WO 2022126304 A1 WO2022126304 A1 WO 2022126304A1 CN 2020136031 W CN2020136031 W CN 2020136031W WO 2022126304 A1 WO2022126304 A1 WO 2022126304A1
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WO
WIPO (PCT)
Prior art keywords
helicase
amino
seq
polynucleotide
acid
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/136031
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English (en)
Chinese (zh)
Inventor
王慕旸
张周刚
吕伟丽
王艳双
陈呈尧
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Qitan Technology Ltd Beijing
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Qitan Technology Ltd Beijing
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Application filed by Qitan Technology Ltd Beijing filed Critical Qitan Technology Ltd Beijing
Priority to PCT/CN2020/136031 priority Critical patent/WO2022126304A1/fr
Priority to CN202080107912.2A priority patent/CN116601297A/zh
Publication of WO2022126304A1 publication Critical patent/WO2022126304A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • Nanopore sequencing utilizes nanopores that provide channels for ionic current. Electrophoresis drives the polynucleotide through the nanopore, and as the polynucleotide passes through the nanopore, it reduces the current through the nanopore. A characteristic current is obtained for each passing nucleotide or series of nucleotides, and the recording of the current level corresponds to the polynucleotide sequence.
  • strand sequencing a single polynucleotide strand passes through the pore and enables identification of nucleotides. Strand sequencing can include the use of a nucleotide processing protein, such as a helicase, to control the movement of the polynucleotide through the pore.
  • patent WO2013057495A3 discloses a new method for characterizing target polynucleotides, which includes controlling the movement of target polynucleotides through a pore by Hel308 helicase or molecular motors.
  • Patent US20150065354A1 discloses a method for characterizing target polynucleotides using XPD helicase, the method comprising controlling the movement of target polynucleotides through a pore by the XPD helicase.
  • Patent CN107109380A discloses a modified enzyme, which is a modified Dda helicase that can control the movement of a target polynucleotide through a pore.
  • the introduced cysteine and the cysteine are connected to each other, and the introduced cysteine Interconnection between unnatural amino acids and unnatural amino acids, between introduced cysteine and unnatural amino acids, between introduced cysteine and natural amino acids, or between introduced unnatural amino acids and natural amino acids connected to each other.
  • the linkage may be permanent, such as a covalent linkage.
  • Covalent attachment can be carried out using chemical crosslinkers, which can vary in length from one carbon (phosgene type linker) to multiple Angstroms.
  • chemical crosslinkers can vary in length from one carbon (phosgene type linker) to multiple Angstroms.
  • the F8813 helicase is further modified to reduce the negative charge on its surface.
  • the fifth aspect of the present invention provides a host cell, the host cell comprising the nucleic acid of the present invention or the expression vector of the present invention.
  • a tenth aspect of the present invention provides a method for characterizing a target polynucleotide, the method comprising:
  • the pores include but are not limited to those derived from M. smegmatis porin A, M. smegmatis porin B, M. smegmatis porin C, 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 and the like.
  • the rate of passage of the target polynucleotide through the pore is controlled by the F8813 helicase or construct, resulting in an identifiable stable current level for target determination Characterization of polynucleotides.
  • the target polynucleotide is single-stranded, double-stranded or at least partially double-stranded.
  • the product is selected from kits, devices or sensors.
  • the device includes a sensor that supports the plurality of pores and transmits a signal that the pores interact with the polynucleotide, and at least one memory for storing the polynucleotide of interest, and necessary for carrying out the characterization process. solution.
  • the seventeenth 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 F8813 of the present invention helicase.
  • between the F8813 helicase and the wild-type F8813 helicase between the F8813 helicase and the F8813 helicase, between the wild-type F8813 helicase and the wild-type F8813 helicase, and between the F8813 helicase
  • they can be connected or arranged in a head-to-head, tail-to-tail or head-to-tail manner.
  • the "F8813 helicase", “construct” or “pore” of the invention can be modified to aid in identification or purification, for example by adding histidine residues (His tag), aspartic acid residues base (asp tag), streptavidin tag, Flag tag, SUMO tag, GST tag or MBP tag, or by adding a signal sequence to facilitate their secretion from cells in which the polypeptide does not naturally contain the signal sequence .
  • His tag histidine residues
  • asp tag aspartic acid residues base
  • streptavidin tag Flag tag
  • Flag tag SUMO tag
  • GST tag GST tag
  • MBP tag GST tag
  • An alternative way of introducing a genetic tag is to chemically link the tag to a natural or artificial site on the F8813 helicase, pore or construct.
  • Figure 3 shows the F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 reaction mixture (SEQ ID NO: 1, mutations C172V/C217A/D284C/S589C/C594A linked by bismaleimide PEG3 linker) and F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG4 reaction mixture (SEQ ID NO: 1, mutation C172V/C217A/D284C/S589C/C594A via bismaleimide PEG4 linker ligated) Coomassie-stained 4-20% SDS-PAGE gel.
  • Fig. 7 is a graph showing the time-dependent displacement ratio of dsDNA in a buffer containing 400 mM NaCl.
  • SEQ ID NO: 1 The coding sequence of SEQ ID NO: 1 of C172V/C217A/D284C/S589C/C594A was obtained and optimized to obtain SEQ ID NO:2.
  • the fluorogenic substrate strand (final concentration 100 nM) has a 3'-end ssDNA overhang, and a 50-base hybridized dsDNA portion.
  • the upper part of the main chain has carboxyfluorescein (5'FAM-SEQ ID NO: 9) at the 5' end, and the hybridized complementary strand has a black hole quencher (BHQ-1) base (SEQ ID NO: 9) at the 3' end. NO:10--BHQ-3').
  • BHQ-1 black hole quencher
  • the substrate is essentially non-fluorescent.
  • a 0.5 ⁇ M capture strand complementary to the shorter strand (SEQ ID NO: 11).
  • F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide-PEG3 (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A was linked to bismaleimide-PEG3) and F8813_C172V /C217A/D284C/S589C/C594A-bismaleimide-PEG4 (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A linked to bismaleimide-PEG4) buffer exchanged to 20 mM HEPES , 50 mM NaCl, 1 mM DTT, 50% glycerol, 0.1 mM EDTA, pH 8.0.
  • FIG. 4 The results show that the DNA construct is mobilized by F8813 helicase-controlled DNA, and the results of F8813 helicase-controlled DNA mobilization are shown in FIG. 4 .
  • the DNA movement controlled by the F8813 helicase was 24 seconds long and corresponded to the translocation of the DNA construct of nearly 200 bp through the nanopore.
  • Figure 5 shows an enlarged view of a partial region of DNA movement controlled by the F8813 helicase.
  • This example shows how a DNA-containing leader is linked to RNA to facilitate loading of a DNA helicase, namely F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (with mutation C172V/C217A/D284C/S589C/ SEQ ID NO: 1 of C594A linked to bismaleimide-PEG3) and then observed that helicases control the movement of RNA through the nanopore. 0.28kb RNA was obtained by in vitro transcription. The DNA-containing leader region is ligated to the 3' end of the RNA. The F8813 helicase was then loaded into the DNA ligation site in the leader and the substrate was analyzed through the nanopore.
  • a DNA helicase namely F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (with mutation C172V/C217A/D284C/S5
  • DNA-RNA construct B as shown in Figure 8: SEQ ID NO: 13 with 3' end linked to 20 iSpC3 spacers and its 5' end linked to 4 iSpC3 spacers linked to SEQ ID NO : the 3' end of SEQ ID NO: 14, the 5' end of this SEQ ID NO: 14 is linked by annealing to the RNA single strand SEQ ID NO: 15, the SEQ ID NO: 16 region of the construct is identical to SEQ ID NO: 7 (which has 3 'cholesterol tether) hybridization.
  • DNA-RNA construct B in buffer in 50 mM NaCl, 10 mM Tris pH 7.5 was combined with F8813_C172V/C217A/D284C/S589C/C594A-double in buffer (50 mM KCl, 10 mM HEPES, pH 8.0) Maleimide PEG3 was pre-incubated for 30 min at room temperature. Buffer (10 mM HEPES, 600 mM KCl, pH 8.0, 3 mM MgCl 2 ) and ATP were then added to the premix.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
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  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
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  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
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Abstract

La présente invention concerne une hélicase F8813 modifiée, une construction comprenant l'hélicase F8813 modifiée, et une application dans la caractérisation d'un polynucléotide cible ou la régulation du mouvement d'un polynucléotide cible traversant des pores. L'invention concerne également un procédé de régulation du mouvement d'un polynucléotide ou un procédé de caractérisation du polynucléotide cible. L'hélicase F8813 modifiée peut être utilisée pour réguler la translocation d'une chaîne d'ADN à travers les pores transmembranaires dans le séquençage de la chaîne d'ADN, et peut également réaliser le séquençage direct de l'ARN. De plus, l'hélicase modifiée peut rester liée au polynucléotide pendant un temps plus long, réduisant ainsi le phénomène de libération d'un polynucléotide d'un polynucléotide séquencé.
PCT/CN2020/136031 2020-12-14 2020-12-14 Hélicase modifiée et son application Ceased WO2022126304A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2020/136031 WO2022126304A1 (fr) 2020-12-14 2020-12-14 Hélicase modifiée et son application
CN202080107912.2A CN116601297A (zh) 2020-12-14 2020-12-14 一种经修饰的解旋酶及其应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024056038A1 (fr) * 2022-09-16 2024-03-21 北京普译生物科技有限公司 Hélicase de capif1 modifiée et son utilisation
CN118256468A (zh) * 2024-02-22 2024-06-28 北京普译生物科技有限公司 一种修饰的ToPif1解旋酶及其应用
WO2024138635A1 (fr) * 2022-12-30 2024-07-04 深圳华大生命科学研究院 Hélicase et son procédé de préparation et son utilisation dans un séquençage à haut débit
CN119709690A (zh) * 2023-09-27 2025-03-28 北京齐碳科技有限公司 一种用于纳米孔测序的酶修饰方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119709689A (zh) * 2023-09-27 2025-03-28 北京齐碳科技有限公司 解旋酶及其应用
CN118126984A (zh) * 2024-02-22 2024-06-04 北京普译生物科技有限公司 一种修饰的Pae CasDinG解旋酶及其应用
CN118126983B (zh) * 2024-02-22 2025-09-02 北京普译生物科技有限公司 一种修饰的mpk2解旋酶及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014013260A1 (fr) * 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Hélicases modifiées
CN104039979A (zh) * 2011-10-21 2014-09-10 牛津纳米孔技术公司 使用孔和Hel308解旋酶表征目标多核苷酸的酶方法
US20150065354A1 (en) * 2011-12-29 2015-03-05 Oxford Nanopore Technologies Limited Method for characterising a polynucelotide by using a xpd helicase
CN106574300A (zh) * 2014-05-02 2017-04-19 牛津纳米孔技术公司 改善目标多核苷酸相对于跨膜孔移动的方法
CN107109380A (zh) * 2014-10-07 2017-08-29 牛津纳米孔技术公司 经修饰的酶
WO2020061997A1 (fr) * 2018-09-28 2020-04-02 北京齐碳科技有限公司 Hélicase et son utilisation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015055981A2 (fr) * 2013-10-18 2015-04-23 Oxford Nanopore Technologies Limited Enzymes modifiées
US20240301010A1 (en) * 2019-09-29 2024-09-12 Qitan Technology Ltd., Beijing Mnep monomer variant and application thereof
EP4036107A4 (fr) * 2019-09-29 2024-01-17 Qitan Technology Ltd., Beijing Variant de monomère mmup et son application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104039979A (zh) * 2011-10-21 2014-09-10 牛津纳米孔技术公司 使用孔和Hel308解旋酶表征目标多核苷酸的酶方法
US20150065354A1 (en) * 2011-12-29 2015-03-05 Oxford Nanopore Technologies Limited Method for characterising a polynucelotide by using a xpd helicase
WO2014013260A1 (fr) * 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Hélicases modifiées
CN106574300A (zh) * 2014-05-02 2017-04-19 牛津纳米孔技术公司 改善目标多核苷酸相对于跨膜孔移动的方法
CN107109380A (zh) * 2014-10-07 2017-08-29 牛津纳米孔技术公司 经修饰的酶
WO2020061997A1 (fr) * 2018-09-28 2020-04-02 北京齐碳科技有限公司 Hélicase et son utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 8 July 2018 (2018-07-08), ANONYMOUS: "MULTISPECIES: ATP-dependent DNA helicase [Methanosarcina]", XP055697339, retrieved from Genbank Database accession no. WP_048166958 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024056038A1 (fr) * 2022-09-16 2024-03-21 北京普译生物科技有限公司 Hélicase de capif1 modifiée et son utilisation
WO2024138635A1 (fr) * 2022-12-30 2024-07-04 深圳华大生命科学研究院 Hélicase et son procédé de préparation et son utilisation dans un séquençage à haut débit
CN119709690A (zh) * 2023-09-27 2025-03-28 北京齐碳科技有限公司 一种用于纳米孔测序的酶修饰方法
CN118256468A (zh) * 2024-02-22 2024-06-28 北京普译生物科技有限公司 一种修饰的ToPif1解旋酶及其应用

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