Enzyme modification method for nanopore sequencing

Abstract

The invention relates to the field of molecular biology, in particular to modified helicase as well as a modification method and application thereof. The modified helicase disclosed by the invention can be stably combined with nucleic acid molecules, so that the helicase has relatively good continuous speed control capability in nanopore sequencing. Meanwhile, according to the modified helicase disclosed by the invention, other amino acids do not need to be additionally introduced into the structural domain of the helicase, but connection is formed between naturally existing amino acid residues, so that the difficulty of protein modification can be greatly reduced, and the production cost is reduced.

Description

Technical Field

[0001] The present application relates to the field of molecular biology, and specifically to a modified helicase and a preparation method thereof, a complex, and also specifically to the uses of the helicase and the complex. Background Art

[0002] 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 instantly and continuously. Its advantages are single-molecule sequencing, high throughput, fast speed, and the ability to detect long-fragment nucleic acid molecules.

[0003] Helicases are a useful tool for controlling the movement of nucleic acid molecules during nanopore sequencing, but current helicases have some problems that need to be solved. For example, the helicase may fall off the nucleic acid molecule during sequencing, causing the nucleic acid molecule to be pulled through the pore at an uncontrolled speed and in an uncontrolled manner, so that the current signal of a single nucleotide is too short to be distinguished.

[0004] To address this problem, the prior art CN105899678A discloses a modified helicase, specifically, at least one cysteine / unnatural amino acid is introduced into the tower / pin / 1A domain of the helicase, thereby improving the continuous rate control ability of the helicase in nanopore sequencing. However, this method of introducing amino acid mutations into the helicase has a relatively complex production process and high cost, so the art needs a simpler and more feasible method for modifying the helicase to improve its stability in controlling the movement of nucleic acid molecules through the nanopore.

[0005] It should be noted that the methods described in this section are not necessarily methods that have been previously conceived or adopted. Unless otherwise indicated, it should not be assumed that any method described in this section is considered to be prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art. Summary of the invention

[0006] Based on this, in order to solve the above problems, the present application provides a modified helicase, characterized in that the helicase includes a polynucleotide binding domain and two RecA-like domains, and there is a connection between the natural amino acid residues located in different RecA-like domains; the modified helicase has the ability to control the movement of polynucleotides; and compared with the absence of the connection, the presence of the connection enhances the ability of the modified helicase to control the movement of polynucleotides. The modified helicase in the present application can stably bind to nucleic acid molecules, thereby having a better continuous speed control ability in nanopore sequencing.

[0007] In some embodiments, the native amino acid residues with connections are naturally present in the helicase. The modified helicase in the present application does not need to introduce additional amino acids into the domain of the enzyme, but forms connections between naturally occurring amino acid residues, which can greatly reduce the difficulty of protein modification and reduce production costs.

[0008] According to one embodiment of the present application, a method for preparing the modified helicase described in the present application is also provided.

[0009] According to one embodiment of the present application, a complex is also provided, characterized in that the complex includes: the helicase described in the present application or the helicase prepared according to the method described in the present application; and a target polynucleotide, wherein the target polynucleotide binds to the polynucleotide binding domain of the helicase.

[0010] According to one embodiment of the present application, there is also provided the use of the helicase described in the present application, the helicase prepared according to the method described in the present application, or the complex described in the present application in controlling the movement of a target analyte or characterizing the characteristics of a target analyte by a nanopore.

[0011] According to one embodiment of the present application, there is also provided the use of the helicase described in the present application, the helicase prepared according to the method described in the present application, or the complex described in the present application in the preparation of a product for controlling the movement of a target analyte or characterizing the characteristics of a target analyte by a nanopore.

[0012] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings exemplarily illustrate the embodiments and constitute a part of the specification, and together with the text description of the specification, are used to explain the exemplary implementation of the embodiments. The embodiments shown are for illustrative purposes only and do not limit the scope of the claims. In all drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0014] Figure 1 The gel electrophoresis diagram of the nucleic acid-protein complex based on Tyr-Tyr cross-linking prepared in Example 1 is shown.

[0015] Figure 2 The gel electrophoresis diagram of the Lys-Glu cross-linked nucleic acid-protein complex prepared in Example 2 is shown.

[0016] Figure 3 The column purification elution profile of the nucleic acid-protein complex purified in Example 3 is shown.

[0017] Figure 4 The gel electrophoresis diagram of the nucleic acid-protein complex after elution in Example 3 is shown, wherein FT, E1, E2, and E3 represent Figure 3 The different main peaks in the elution process shown in FIG. 7 correspond to nucleic acid-protein complexes.

[0018] Figure 5 The gel electrophoresis diagram of the nucleic acid-protein complex after treatment with different reagents in Example 3 is shown.

[0019] Figure 6 The figure shows the results of ATP consumption detection of nucleic acid-protein complex in Example 4.

[0020] Figure 7 The sequencing signal diagram of the nucleic acid-protein complex in Example 5 is shown, wherein Figure A is a complete sequencing signal diagram of 10 kb, and Figure B is a local amplification signal step diagram. DETAILED DESCRIPTION

[0021] Unless otherwise stated, the terms used herein have the commonly understood meanings for those skilled in the art. Unless otherwise stated, all numbers used in this specification and claims to represent content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as modified by the term "about" or "roughly" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the attached claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through this application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art.

[0022] Although the numerical ranges and parameters setting forth the broad scope of the present application are approximate, the numerical values ​​set forth in the specific embodiments are provided as accurately as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include each narrower numerical range that falls within the broader numerical range, just as if these narrower numerical ranges were all clearly written herein.

[0023] In this application, "polypeptide" and "peptide" are used interchangeably to refer to amino acid polymers of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies and enzymes are all included in the definition of polypeptides.

[0024] "Helicase" as used herein refers to a polypeptide that catalyzes the unwinding of DNA or RNA. In some non-limiting embodiments, the helicase has a polynucleotide binding domain, and the target polynucleotide can bind to the helicase through the polynucleotide binding domain. In some non-limiting embodiments, the helicase is a SF1B (Superfamily 1B) subfamily helicase (The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012 Jul 3; 20 (7): 1189-200), examples of which include but are not limited to: Dda helicase, RecD2 helicase, Pif1 helicase, or human DNA helicase B. In some exemplary embodiments, the helicase may also be a member of superfamily 1 or superfamily 2: Pif1-like, Upf1-like, UvrD / Rep, Ski-like, Rad3 / XPD, NS3 / NPH-II, DEAD, DEAH / RHA, RecG-like, REcQ-like, T1R-like, Swi / Snf-like, and Rig-I-like. In some exemplary embodiments, the helicase may also be a member of one of the following subfamilies: RecD, Upf1, PcrA, Rep, UvrD, Hel308, Mtr4, XPD, NS3, Mss116, Prp43, RecG, RecQ, T1R, RapA, and Hef.

[0025] As used herein, "amino acid" refers to natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids.

[0026] The "natural amino acid" described in this application refers to the proteinogenic amino acids known to those skilled in the art, including amino acids encoded by the genetic code, and variants of post-translational modifications thereof. The amino acids encoded by the genetic code include 20 common amino acids: A (alanine), V (valine), L (leucine), I (isoleucine), F (phenylalanine), W (tryptophan), M (methionine), P (proline), G (glycine), T (threonine), S (serine), C (cysteine), N (asparagine), Q (glutamine), Y (tyrosine), K (lysine), R (arginine), H (histidine), D (aspartic acid), E (glutamic acid), B (aspartic acid or asparagine), Z (glutamine or glutamic acid), J (leucine or isoleucine), and the less common pyrrolysine and selenocysteine. Natural amino acids also include post-translationally modified variants of the amino acids encoded by the genetic code, non-limiting examples of which include prenylated amino acids, prenylated amino acids, myristylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids.

[0027] The “interaction” mentioned in the present application includes but is not limited to non-covalent bond interactions, and the “non-covalent bond interactions” include hydrogen bonds, π interactions, and electrostatic interactions.

[0028] The "electrostatic interaction" mentioned in this application refers to various electrostatic attractions between dipoles, induced dipoles and charged groups in molecules (The T4phage SF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012 Jul 3; 20(7): 1189-200). The charged groups or dipole groups may be located on the surface and inside of the protein molecules.

[0029] In the present application, "connection" between amino acids or amino acid residues includes but is not limited to connection by chemical bonds, and the "covalent connection" refers to connection by covalent bonds. In some embodiments, the covalent connection is formed by a chemical cross-linking agent or an enzyme.

[0030] The "ectopic bond" described in this application refers to a type of covalent bond formed by the reaction of amino acid side chains other than backbone peptide bonds that exist inside a protein. Ectopic bonds can be formed within a protein molecule or between protein molecules in the natural environment. Generally, isopeptide bonds formed within a molecule help the protein respond to environmental stress and improve the stability of the protein (Trends Biochem Sci, 2011, 36 (4): 229-237). In some non-limiting embodiments, ectopic bonds can be formed by enzyme catalysis or by chemical cross-linking agent catalysis.

[0031] Modified helicase

[0032] According to one embodiment of the present application, a modified helicase can be provided, characterized in that the helicase includes a polynucleotide binding domain and two RecA-like domains, and there is a connection between the natural amino acid residues located in different RecA-like domains; the modified helicase has the ability to control the movement of polynucleotides; and compared with the absence of the connection, the presence of the connection enhances the ability of the modified helicase to control the movement of polynucleotides.

[0033] When sequencing a polynucleotide with a long sequence (e.g., 500 nucleotides or more), the helicase that controls the movement of the polynucleotide may fall off the polynucleotide, thereby affecting the sequencing process and accuracy. The modified helicase in the present application improves the binding stability of the helicase and the target polynucleotide by forming a connection between the natural amino acid residues located in different RecA-like domains, thereby improving the continuous rate control ability in nanopore sequencing.

[0034] In some embodiments, the natural amino acid residues that are connected have interactions and / or spatial distances less than 50 angstroms, 45 angstroms, 40 angstroms, 35 angstroms, 30 angstroms, 25 angstroms, 20 angstroms, 15 angstroms, 10 angstroms, 8 angstroms, 5 angstroms, 4 angstroms, 3 angstroms, 2 angstroms or 1 angstrom. In some embodiments, the interactions include non-covalent interactions, preferably, the non-covalent interactions include hydrogen bonds, π interactions, and electrostatic interactions. In some preferred embodiments, the interactions are electrostatic interactions. In some preferred embodiments, the electrostatic interactions are generated after the helicase binds to the target polynucleotide.

[0035] In the present application, when connecting the natural amino acid residues in the helicase, it is preferred to connect the natural amino acid residues that are close to each other or connect the natural amino acid residues that interact with each other, which can further reduce the difficulty of protein modification. In addition, if there is interaction, the natural amino acid residues that interact with each other can be directly covalently connected, and if there is no interaction, the natural amino acid residues can be covalently connected using a linker.

[0036] In some embodiments, the helicase belongs to the SF1B (Superfamily 1B) subfamily. In some embodiments, the helicase includes but is not limited to Dda helicase, RecD2 helicase, Pif1 helicase, or DNA helicase B. In some preferred embodiments, the helicase includes but is not limited to the Dda helicase of T4 phage (The T4phageSF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012Jul 3; 20(7): 1189-200), the RecD2 helicase of bacteria (Mechanistic basis of50-30translocation in SF1B helicases. Cell 137, 849-859), the Pif1 helicase of eukaryotic cells (Unwinding the functionsof the Pif1 family helicases. DNA Repair (Amst.) 9, 237-249), and the DNA helicase B of humans (Cell cycle-dependent regulation of a human DNA helicase that localizes in DNA damage foci. Mol. Biol. Cell 15, 3320-3332). In some more preferred embodiments, the helicase is the Dda helicase from T4 phage. Dda helicase includes the following five domains: 1A (RecA-like) domain, 2A (RecA-like) domain, tower domain, pin domain, and hook domain (Xiaoping He et al., 2012, Structure; 20: 1189-1200).

[0037] In some embodiments, the linked native amino acid residue is naturally occurring in the helicase.

[0038] The modified helicase in the present application does not require the introduction of additional amino acids into the enzyme domain, but instead forms connections between naturally occurring amino acid residues, which can greatly reduce the difficulty of protein modification and reduce production costs.

[0039] In some embodiments, the natural amino acids present in the connection include glutamic acid, lysine, tyrosine, arginine, aspartic acid, cysteine, methionine, tryptophan (Chem. Sci., 2019, 10, 4966–4972). In some embodiments, the natural amino acids present in the connection include any two of glutamic acid, lysine, tyrosine, arginine, aspartic acid, cysteine, methionine, and tryptophan, preferably, the natural amino acid residues present in the connection include the connection of tyrosine to tyrosine, the connection of lysine to glutamic acid, and the connection of cysteine ​​to methionine. In some preferred embodiments, the natural amino acids present in the connection include glutamic acid, lysine, and tyrosine.

[0040] In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected include E93, E94, K364, Y92, or Y363, and these natural amino acid residues have interactions and / or spatial distances less than 50 angstroms. The natural amino acid residues that interact and / or have spatial distances less than 50 angstroms in the Dda helicase are known in the art (The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012 Jul 3; 20 (7): 1189-200), the disclosure of which is incorporated herein by reference in its entirety. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected are selected from at least one of the following groups: E93 and K364, E94 and K364, or Y92 and Y363. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected are E93 and K364. In some preferred embodiments, the helicase is a Dda helicase, and the native amino acid residues present and connected are E94 and K364. In some preferred embodiments, the helicase is a Dda helicase, and the native amino acid residues present and connected are Y92 and Y363.

[0041] In some embodiments, the linked native amino acid residues are located around the polynucleotide binding domain.

[0042] When the target polynucleotide is analyzed, the target polynucleotide is bound to the polynucleotide binding domain, and the binding of the target polynucleotide to the polynucleotide binding domain is strengthened by forming a connection between the natural amino acid residues located around the polynucleotide binding domain, thereby improving the binding stability of the helicase to the target polynucleotide.

[0043] The natural amino acids in the helicase that are connected can be connected in any suitable manner. In some embodiments, the connection is covalently connected. The method of covalent connection is known in the art. In some preferred embodiments, the connection is an ectopic bond connection. In some embodiments, the covalent connection can be formed by enzyme catalysis or by chemical cross-linking agent catalysis. In some embodiments, the chemical cross-linking agent includes EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), EDC / NHS (N-hydroxysuccinimide), EDC / s-NHS (N-hydroxysuccinimide sulfonic acid sodium salt), chlorpyrifos oxon, HRP (horseradish peroxidase), laccase, or tyrosinase.

[0044] Non-limiting examples of enzyme-catalyzed formation include: dityrosine or trityrosine covalent bonds formed by peroxidase (HRP, myeloperoxidase Tyrosinas, laccase, etc.) (ACS Biomater Sci Eng, 2016, 2 (12): 2108-2121), this cross-linking reaction can also be catalyzed by G4-hemin nuclease (Chemistry, 2011, 17 (51): 14475-84); Lys-Gln ectopic bonds formed by transglutaminase are also called isopeptide bonds (Int J Biol Macromol, 2018, 107 (Pt B): 2364-2374); Lys-Lys cross-linking catalyzed by Lysyl Oxidases (Biomacromolecules, 2011, 12 (1): 82-7).

[0045] Non-limiting examples of cross-linking catalyzed by chemical cross-linkers include: isopeptide bonds between the side chains of D / E acidic amino acids and K / R basic amino acids catalyzed by EDC / NHS / S-NHS; Tyr-Tyr covalent cross-linking catalyzed by H2O2 / Hemin, ruthenium; and cross-linking between D / E and D / E mediated by diazo-containing cross-linkers (Anal Chem, 201, 90(2): 1195-1201).

[0046] Methods for modifying helicases

[0047] According to one embodiment of the present application, a method for preparing a modified helicase is also provided, wherein the helicase comprises a polynucleotide binding domain and two RecA-like domains, and is characterized in that a connection exists between natural amino acid residues located in different RecA-like domains; the modified helicase has the ability to control the movement of polynucleotides; and compared with the absence of the connection, the presence of the connection enhances the ability of the modified helicase to control the movement of polynucleotides.

[0048] In some embodiments, the natural amino acid residues that are connected have interactions and / or spatial distances less than 50 angstroms, 45 angstroms, 40 angstroms, 35 angstroms, 30 angstroms, 25 angstroms, 20 angstroms, 15 angstroms, 10 angstroms, 8 angstroms, 5 angstroms, 4 angstroms, 3 angstroms, 2 angstroms or 1 angstrom. In some embodiments, the interactions include non-covalent interactions, and preferably, the non-covalent interactions include hydrogen bonds, π interactions, and electrostatic interactions. In some preferred embodiments, the interactions are electrostatic interactions. In some preferred embodiments, the electrostatic interactions are generated after the helicase binds to the target polynucleotide. In addition, if there is an interaction, the interacting natural amino acid residues can be directly covalently linked, and if there is no interaction, the natural amino acid residues can be covalently linked using a linker.

[0049] In some embodiments, the helicase belongs to the SF1B (Superfamily 1B) subfamily. In some embodiments, the helicase includes but is not limited to Dda helicase, RecD2 helicase, Pif1 helicase, or DNA helicase B. In some preferred embodiments, the helicase includes but is not limited to the Dda helicase of T4 phage (The T4phageSF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012Jul 3; 20(7): 1189-200), the RecD2 helicase of bacteria (Mechanistic basis of50-30translocation in SF1B helicases. Cell 137, 849-859), the Pif1 helicase of eukaryotic cells (Unwinding the functionsof the Pif1 family helicases. DNA Repair (Amst.) 9, 237-249), and the DNA helicase B of humans (Cell cycle-dependent regulation of a human DNA helicase that localizes in DNA damage foci. Mol. Biol. Cell 15, 3320-3332). In some more preferred embodiments, the helicase is the Dda helicase from T4 phage. Dda helicase includes the following five domains: 1A (RecA-like) domain, 2A (RecA-like) domain, tower domain, pin domain, and hook domain (Xiaoping He et al., 2012, Structure; 20: 1189-1200).

[0050] In some embodiments, the natural amino acid residues connected to the helicase are naturally present. In some embodiments, the natural amino acids connected to the helicase include glutamic acid, lysine, tyrosine, arginine, aspartic acid, cysteine, methionine, tryptophan (Chem. Sci., 2019, 10, 4966–4972). In some embodiments, the natural amino acids connected to the helicase include any two of glutamic acid, lysine, tyrosine, arginine, aspartic acid, cysteine, methionine, and tryptophan. Preferably, the natural amino acid residues connected to the helicase include the connection of tyrosine to tyrosine, the connection of lysine to glutamic acid, and the connection of cysteine ​​to methionine. In some preferred embodiments, the natural amino acids connected to the helicase include glutamic acid, lysine, and tyrosine. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues connected to the helicase include E93, E94, K364, Y92, or Y363, and these natural amino acid residues have interactions and / or spatial distances less than 50 angstroms. Other natural amino acid residues in Dda helicase that interact and / or have a spatial distance of less than 50 angstroms are known in the art (The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012 Jul 3; 20 (7): 1189-200), the disclosure of which is incorporated herein by reference in its entirety. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected are selected from at least one of the following groups: E93 and K364, E94 and K364, or Y92 and Y363. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected are E93 and K364. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected are E94 and K364. In some preferred embodiments, the helicase is a Dda helicase, and the natural amino acid residues that are connected are Y92 and Y363.

[0051] In some embodiments, the linked native amino acid residues are located around the polynucleotide binding domain.

[0052] The natural amino acids in the helicase that are connected can be connected in any suitable manner. In some embodiments, the connection is covalently connected. The method of covalent connection is known in the art. In some preferred embodiments, the connection is an ectopic bond connection. In some embodiments, the covalent connection can be formed by enzyme catalysis or by chemical cross-linking agent catalysis. In some embodiments, the chemical cross-linking agent includes EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), EDC / NHS (N-hydroxysuccinimide), EDC / s-NHS (N-hydroxysuccinimide sulfonic acid sodium salt), chlorpyrifos oxon, HRP (horseradish peroxidase), laccase, or tyrosinase.

[0053] Non-limiting examples of enzyme-catalyzed formation include: dityrosine or trityrosine covalent bonds formed by peroxidase (HRP, myeloperoxidase Tyrosinas, laccase, etc.) (ACS Biomater Sci Eng, 2016, 2 (12): 2108-2121), this cross-linking reaction can also be catalyzed by G4-hemin nuclease (Chemistry, 2011, 17 (51): 14475-84); Lys-Gln ectopic bonds formed by transglutaminase are also called isopeptide bonds (Int J Biol Macromol, 2018, 107 (Pt B): 2364-2374); Lys-Lys cross-linking catalyzed by Lysyl Oxidases (Biomacromolecules, 2011, 12 (1): 82-7).

[0054] Non-limiting examples of cross-linking catalyzed by chemical cross-linkers include: isopeptide bonds between the side chains of D / E acidic amino acids and K / R basic amino acids catalyzed by EDC / NHS / S-NHS; Tyr-Tyr covalent cross-linking catalyzed by H2O2 / Hemin, ruthenium; and cross-linking between D / E and D / E mediated by diazo-containing cross-linkers (Anal Chem, 201, 90(2): 1195-1201).

[0055] Compounds, uses

[0056] According to one embodiment of the present application, a complex is also provided, characterized in that the complex includes: the helicase described in the present application or the helicase prepared according to the method described in the present application; and a target polynucleotide, wherein the target polynucleotide binds to the polynucleotide binding domain of the helicase.

[0057] In some embodiments, there is a connection structure formed between the connected natural amino acid residues in the helicase, and the target polynucleotide is surrounded by the connection structure. When the target polynucleotide is analyzed, the connection structure strengthens the binding of the target polynucleotide to the polynucleotide binding domain, thereby improving the binding stability of the helicase and the target polynucleotide.

[0058] In some embodiments, the complex further comprises an adapter, and the target polynucleotide is linked to the adapter.

[0059] According to one embodiment of the present application, there is also provided the use of the helicase described in the present application, the helicase prepared according to the method described in the present application, or the complex described in the present application in controlling the movement of a target analyte or characterizing the characteristics of a target analyte by a nanopore.

[0060] According to one embodiment of the present application, there is also provided the use of the helicase described in the present application, the helicase prepared according to the method described in the present application, or the complex described in the present application in the preparation of a product for controlling the movement of a target analyte or characterizing the characteristics of a target analyte by a nanopore.

[0061] In some embodiments, the target analyte comprises a polynucleotide, a polypeptide, a polysaccharide, and / or a lipid. In some preferred embodiments, the target analyte comprises a polynucleotide, a polypeptide connected to a polynucleotide, a polysaccharide, and / or a lipid. In some preferred embodiments, the target analyte is selected from at least one of a single-stranded polynucleotide, a double-stranded polynucleotide, and / or a partially double-stranded polynucleotide.

[0062] In some embodiments, the target analyte is a polynucleotide, and the characteristic is selected from one or more of groups (i)-(v): (i) the length of the polynucleotide; (ii) the identity of the polynucleotide; (iii) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide; and / or (v) whether the polynucleotide is modified.

[0063] According to one embodiment of the present application, the use of the helicase described in the present application, or the helicase obtained according to the method described in the present application in gene sequencing can be provided.

[0064] The modified helicases in the present application and the modified helicases obtained by the methods in the present application can be applied to any sequencing analysis, including but not limited to nanopore sequencing, and can also be applied to any sequencing methods that will appear in the future. In some preferred embodiments, the gene sequencing is nanopore sequencing. Methods for nanopore sequencing using helicases are well known in the art.

[0065] The various embodiments and preferences for the present application described above can be combined with each other (as long as they are not inherently contradictory to each other and are applicable to the purpose of the present application), and the various embodiments formed by such combination are all considered as part of the present application.

[0066] Example

[0067] The following is a description of exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered to be merely exemplary and are by no means intended to limit the scope of protection of the present application. The scope of protection of the present application is limited only by the claims. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0068] Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available conventional products. Unless otherwise specified, the experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0069] Example 1: Preparation of nucleic acid-protein complex based on Tyr-Tyr linkage

[0070] The main strand S1, antisense strand S2 and connecting strand YB were synthesized respectively, and their specific sequences are shown below:

[0071] Main strand S1: 5'-3333333333333333333333333333333-GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCTGACT-3'

[0072] Antisense strand S2: 5'-Ru-CGACTTCTACCGTTTGACTCCGC-3'

[0073] Connection chain YB: 5'-GTCAGCAGGACCGAATGA / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeA / / i2OMeG / / i2OMeU / / i2O MeC / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeC / / i2OMeC / / i2OMeG / / i2OMeA / / i2OMeC / / i2OMeC / -3'

[0074] in:

[0075] The main strand S1 sequentially comprises the leader sequence iSpC3 (i.e., a nucleotide lacking sugar and base, indicated as 3), which is connected to the 5' end of SEQ ID NO: 1, and the 3' end of SEQ ID NO: 1 is sequentially connected to the blocking strand iSpC18 (indicated as 8888) and the 5' end of SEQ ID NO: 2. iSpC3 and iSpC18 were purchased from Integrated DNA Technologies.

[0076] The sequence of the antisense strand S2 is shown in SEQ ID NO:3.

[0077] The connecting chain YB comprises the sequence shown in SEQ ID NO: 4, and a plurality of methoxy modifications including i2OMeA, i2OMeC, i2OMeG, and i2OMeU are connected to its 3' end.

[0078] The main strand S1, the antisense strand S2 and the connecting strand YB are mixed and annealed at a ratio of 1:1.1:1.1 to form an adapter. The annealing treatment is specifically to slowly cool from 95°C to 25°C, during which the cooling amplitude does not exceed 0.1°C / s.

[0079] Take 2 μM adapter, mix it with 4 times the amount of T4 Dda protein (SEQ ID NO: 5), and add 1 mM 1,3-propanediamine and 1,4-butanediamine respectively, and incubate at 30°C for 0.5 h, then add 2.5 mM sodium persulfate and place under a blue LED 450 nm light source (distance 15 cm, radiation power 50 mW / cm 2 ) for 1 min, then run TBE PAGE gel at 160 V for 40 min and detect the enzyme cross-linking. The results are as follows Figure 1 The results showed that tyrosine can be oxidized under the catalysis of Ru to form an effective nucleic acid-protein complex with an amino linker.

[0080] Example 2: Preparation of nucleic acid-protein complex based on Lys-Glu linkage

[0081] The main strand S1, antisense strand S2 and connecting strand YB were synthesized respectively, and their specific sequences are shown below:

[0082] Main strand S1: 5'-3333333333333333333333333333333-GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCG GTCCTGCTGACT-3'

[0083] Antisense strand S2: 5'-CGACTTCTACCGTTTGACTCCGC-3'

[0084] Connection chain YB: 5'-GTCAGCAGGACCGAATGA / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeA / / i2OMeG / / i2OMeU / / i2O MeC / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeC / / i2OMeC / / i2OMeG / / i2OMeA / / i2OMeC / / i2OMeC / -3'

[0085] in:

[0086] The main strand S1 sequentially comprises the leader sequence iSpC3 (i.e., a nucleotide lacking sugar and base, indicated as 3), which is connected to the 5' end of SEQ ID NO: 1, and the 3' end of SEQ ID NO: 1 is sequentially connected to the blocking strand iSpC18 (indicated as 8888) and the 5' end of SEQ ID NO: 2. iSpC3 and iSpC18 were purchased from Integrated DNA Technologies.

[0087] The sequence of the antisense strand S2 is shown in SEQ ID NO:3.

[0088] The connecting chain YB comprises the sequence shown in SEQ ID NO: 4, and a plurality of methoxy modifications including i2OMeA, i2OMeC, i2OMeG, and i2OMeU are connected to its 3' end.

[0089] The main strand S1, the antisense strand S2 and the connecting strand YB are mixed and annealed at a ratio of 1:1.1:1.1 to form an adapter. The annealing treatment is specifically to slowly cool from 95°C to 25°C, during which the cooling amplitude does not exceed 0.1°C / s.

[0090] 1 μM adapter was taken, and 0, 1, 2, and 5 times the amount of T4 Dda protein (SEQ ID NO: 5) and 5 mM EDC were mixed and incubated at 30°C for 4 h. 20 mM β-ME was added to terminate the reaction. Then, TBE PAGE gel was run at 160 V for 40 min to detect the enzyme cross-linking. The results are shown in FIG. Figure 2 The results show that as the concentration of added protein increases, the proportion of nucleic acid-protein complexes gradually increases, and effective nucleic acid-protein complexes can be formed.

[0091] Example 3: Purification and quality control of nucleic acid-protein complexes

[0092] The incubation binding product with 5 times the amount of the substance added in Example 2 was added to the DNAPac PA200 column and purified with buffer A and buffer B to remove the enzyme that was not bound to the adapter and impurities such as EDC that was not completely reacted from the column.

[0093] The nucleic acid-protein complex was then eluted with a mixture of 10 column volumes of buffer A and buffer B. The main elution peaks were then pooled, their concentrations were measured, and TBE PAGE gel was run at 160 V for 40 min.

[0094] in,

[0095] The composition of buffer A was: 20 mM Na-CHES, 250 mM NaCl, 4% (W / V) glycerol, pH 8.6;

[0096] The composition of buffer B is: 20 mM Na-CHES, 1 M NaCl, 4% (W / V) glycerol, pH 8.6.

[0097] The elution process is as follows Figure 3 As shown, each peak in the elution process was collected and detected by TBE PAGE gel running at 160V for 40min. The results are shown in Figure 4 As shown, E2 is the required nucleic acid-protein complex.

[0098] The main peak eluted was quantified by Qubit, and 10 ng was taken for quality control. 1*Seqbuffer (10 mM HEPES7.0, 600 mM KCl, 25 mM ATP, 25 mM MgCl2), NF-H20 (control group, a certain amount of ultrapure water was added to supplement the reaction system), or 0.1% SDS was added and treated at 35°C for 30 minutes. Then, TBE PAGE gel was run at 160V for 40 minutes for detection. The results are shown in the figure. Figure 5The results showed that the spacer could successfully block the helicase in the complex, and the amount of nucleic acid produced by the detachment of the quality control enzyme was small; while no nucleic acid-protein complex remained in the SDS treatment group, indicating that the constructed lock-enzyme complex did not form a covalent cross-link with the nucleic acid.

[0099] Example 4: ATP consumption detection of nucleic acid-protein complexes

[0100] About 1 ng of the purified nucleic acid-protein complex in Example 3 was taken and added to 200 μL 1*Seq Buffer (10 mM HEPES7.0, 600 mM KCl, 25 mM ATP, 25 mM MgCl2). The nucleic acid-protein complex was not added and enzyme-free water was added as a control. Then, 0 h, 4 h, and 8 h were taken and the ADP content was detected by HPLC according to the peak time of elution on the C18 column. The results are shown in FIG. Figure 6 The results showed that under this locked enzyme mode, the nucleic acid-protein complex still has ATPase activity and the protein can still work normally.

[0101] Example 5: Sequencing function test of nucleic acid-protein complex

[0102] A 10 kb library was prepared by end repair, and the nucleic acid protein complex prepared in Example 2 was connected to the library for library construction according to the library construction kit QLK-V1.1.1 of Qi Carbon Technology. Finally, sequencing was performed on the QNome-3841 sequencer of Qi Carbon Technology Co., Ltd., and the actual sequencing signal is as follows Figure 7 The results showed that the nucleic acid-protein complex can generate sequencing signals normally, and the signal steps are clear for subsequent base inference.

[0103] Comparative Example 1: Sequencing function test of nucleic acid-protein complex without cross-linking

[0104] The method for preparing the nucleic acid-protein complex without forming cross-links is similar to that in Example 2, except that no EDC is added during the preparation process, that is, the enzyme does not form cross-links.

[0105] The library construction and sequencing were performed in the same manner as in Example 5, and the results are shown in Table 1. The results showed that compared with Example 5, the enzyme in the complex of the non-cross-linked enzyme was easily detached during sequencing, and no effective sequencing signal could be obtained, let alone a complete sequencing signal of 10 kb.

[0106] Table 1 Test results of Example 5 and Comparative Example 1

[0107]

[0108] Note: A valid signal is a signal that can be subsequently identified by basecalling and converted into a sequence.

[0109] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementations have been described, for the applicant or other skilled personnel, there may be or are currently no alternatives, modifications, changes, improvements, and substantial equivalents of the above implementations that cannot be foreseen. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such alternatives, modifications, changes, improvements, and substantial equivalents. It is important that with the evolution of technology, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A modified helicase, characterized in that The helicase comprises a polynucleotide binding domain and two RecA-like domains, and there is a connection between the natural amino acid residues located in different RecA-like domains; The modified helicase has the ability to control the movement of a polynucleotide; and the presence of the connection enhances the ability of the modified helicase to control the movement of a polynucleotide compared to the absence of the connection.

2. The helicase according to claim 1, wherein the naturally-connected amino acid residues interact with each other and / or have a spatial distance of less than 50 angstroms.

3. According to the helicase according to claim 2, the interaction includes electrostatic interaction, and preferably, the interaction is generated after the helicase binds to the target polynucleotide.

4. The helicase according to claim 1, wherein the linked natural amino acid residues are naturally present in the helicase, preferably, the linked natural amino acid residues include glutamic acid, lysine, tyrosine, arginine, aspartic acid, cysteine, methionine, and tryptophan.

5. The helicase according to claim 1, wherein the linked native amino acid residues are located around the polynucleotide binding domain.

6. The helicase according to claim 1, wherein the connection is a covalent connection, preferably, the connection is an ectopic bond connection.

7. The helicase of claim 6, wherein the covalent linkage comprises a linkage formed by a chemical cross-linking agent or a linkage formed by an enzyme.

8. The helicase according to claim 7, wherein the chemical cross-linking agent comprises EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), EDC / NHS (N-hydroxysuccinimide), EDC / s-NHS (N-hydroxysuccinimide sulfonic acid sodium salt), Chlorpyrifos oxon, HRP (horseradish peroxidase), laccase, or tyrosinase.

9. The helicase according to any one of claims 1 to 8, which belongs to the SF1B (Superfamily 1B) subfamily.

10. The helicase according to claim 9, wherein the helicase comprises Dda helicase, RecD2 helicase, Pif1 helicase, or DNA helicase B. The helicase according to claim 10 , which is the Dda helicase from T4 bacteriophage.

12. The helicase of claim 11, wherein the linked native amino acid residues include E93, E94, K364, Y92, or Y363.

13. The helicase according to claim 12, wherein the linked natural amino acid residues are selected from at least one of the following groups: E93 and K364; E94 and K364; or Y92 and Y363.

14. A method for preparing the modified helicase according to any one of claims 1-13.

15. A composite, characterized in that The compound comprises: The helicase according to any one of claims 1 to 13 or the helicase prepared by the method according to claim 14; and A target polynucleotide that binds to the polynucleotide binding domain of the helicase. 16 . The complex according to claim 15 , wherein a connection structure is formed between the linked natural amino acid residues in the helicase, and the target polynucleotide is surrounded by the connection structure. The complex according to claim 15 , further comprising an adapter, and the target polynucleotide is connected to the adapter.

18. Use of the helicase according to any one of claims 1 to 13, the helicase prepared according to the method of claim 14, or the complex according to any one of claims 15 to 17 in controlling the movement of a target analyte or characterizing the characteristics of a target analyte by a nanopore.

19. Use of the helicase according to any one of claims 1 to 13, the helicase prepared according to the method of claim 14, or the complex according to any one of claims 15 to 17 in the preparation of a product for controlling the movement of a target analyte or characterizing the characteristics of a target analyte through a nanopore.

20. The use according to claim 18 or 19, wherein the target analyte comprises a polynucleotide, a polypeptide, a polysaccharide, and / or a lipid. Preferably, the target analyte comprises a polynucleotide, a polypeptide, a polysaccharide, and / or a lipid connected to a polynucleotide. Further preferably, the target analyte is selected from at least one of a single-stranded polynucleotide, a double-stranded polynucleotide, and / or a partially double-stranded polynucleotide.

21. The use according to claim 18 or 19, wherein the target analyte is a polynucleotide, and the characteristic is selected from one or more of groups (i)-(v): (i) the length of the polynucleotide; (ii) the identity of the polynucleotide; (iii) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide; and / or (v) whether the polynucleotide is modified.

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