Method for controlling movement of polynucleotide, and helicase variant

Abstract

A method for moving a polynucleotide through a transmembrane pore, comprising: under the control of an electric field force, moving a polynucleotide, which is linked to a helicase variant, through a transmembrane pore, wherein the helicase variant does not have active unwinding activity or translocation activity.

Description

Methods of controlling polynucleotide movement and helicase variants TECHNICAL FIELD

[0001] The present application relates to the field of gene sequencing, and in particular, the present application relates to methods of controlling polynucleotide movement and helicase variants. More particularly, the present application relates to a method of nanopore sequencing, a sequencing adapter complex, a nucleic acid molecule, an expression vector, a recombinant cell and a method of preparing a helicase variant. BACKGROUND

[0002] Nanopore sequencing is a single-molecule sequencing technology that identifies individual nucleotide molecules by using electrical signals in a nanopore, enabling rapid sequencing of DNA or RNA sequences. It is widely used in genomics, transcriptomics, biological research and clinical diagnosis.

[0003] The principle of nanopore sequencing technology is based on the change of electrical signal. A nanopore (protein pore or solid-state pore) inserted as a signal sensor in the membrane separates two electrolytic chambers filled with electrolyte. When a voltage is applied between the two electrolytic chambers, a stable current through the nanopore is generated. When the nucleic acid to be tested enters the nanopore, it will hinder the flow of ions and cause fluctuations in the current signal. Nucleotides with different bases have different effects on the current. Therefore, by detecting the current fluctuation signal of the nanopore in real time, and decoding the current signal with machine learning analysis, the sequence information of the nucleic acid molecule to be tested can be sequenced in real time.

[0004] The key step of nanopore sequencing is to ensure that the nucleic acid to be tested can pass through the nanopore uniformly and stably at single-base resolution. In the prior art, a method of using an adapter-motor protein (helicase) complex is used for nanopore sequencing. In the adapter-motor protein complex, the spacer prevents the motor protein from moving along the single-stranded polynucleotide and unwinding the double-stranded polynucleotide in the buffer; the spacer is disabled by the transmembrane protein under the action of the electric field force after the sequencing library is captured. The spacer used in the prior art is composed of multiple modified nucleotides, which has a high cost, and the speed of sequencing is also limited by the unwinding speed of the motor protein.

[0005] Therefore, there is an urgent need to develop a method of moving the nucleic acid to be tested, so that the speed of the nucleic acid to be tested is no longer limited and the cost is reduced.

[0006] SUMMARY

[0007] The present application aims to at least partially solve one of the technical problems in the related art.

[0008] The inventors found that the use of spacers to block the helicase in existing nanopore sequencing is costly, and the sequencing speed is limited by the unwinding speed of the helicase itself. To overcome this problem, the inventors constructed a helicase variant based on the existing helicase, so that the helicase changes from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force. In this process, there is no need to use spacers to block the helicase, reducing the cost; at the same time, the helicase changes to use the electric field force to control the speed of moving the polynucleotide, so that the speed of moving the polynucleotide is no longer limited by the helicase.

[0009] Based on this, the first aspect of the present application, the present application provides a method for controlling the movement of a polynucleotide. According to an embodiment of the present application, the method comprises: a polynucleotide connected with a helicase variant moves through a transmembrane pore under the control of an electric field force, wherein the helicase variant does not have active unwinding activity or translocation activity. According to the method of the embodiment of the present application, the helicase variant changes from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without the need to use spacers to block the helicase, reducing the cost; at the same time, the helicase changes to use the electric field force to control the speed of moving the polynucleotide, so that the speed of moving the polynucleotide is no longer limited by the helicase.

[0010] The second aspect of the present application, the present application provides a method for nanopore sequencing. According to an embodiment of the present application, the method comprises using the method of the first aspect of the present application, and the polynucleotide to be tested moves through the nanopore under the control of the electric field force;

[0011] Based on the current signal generated when the polynucleotide to be tested moves through the nanopore, the sequence of the polynucleotide to be tested is determined. According to the method of the embodiment of the present application, the helicase variant changes from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without the need to use spacers to block the helicase, reducing the cost; at the same time, the helicase changes to use the electric field force to control the speed of moving the polynucleotide, so that the speed of moving the polynucleotide is no longer limited by the helicase.

[0012] The third aspect of the present application, the present application provides a helicase variant. According to an embodiment of the present application, the helicase variant has at least one amino acid mutation in the region related to the unwinding activity compared with the amino acid sequence shown in SEQ ID NO: 2, and the helicase mutant does not have active unwinding activity or translocation activity. According to the helicase variant of the embodiment of the present application, the helicase does not have active unwinding activity or translocation activity.

[0013] In a fourth aspect, the present application provides a sequencing adaptor complex. According to an embodiment of the present application, the sequencing adaptor complex comprises: a first strand, a second strand and the helicase variant of the third aspect of the present application; wherein the first strand comprises, in order, a guide sequence, a helicase binding domain and a first complementary sequence, the helicase variant binds to the helicase binding domain;

[0014] the second strand comprises a second complementary sequence and a ligation sequence, the first complementary sequence and the second complementary sequence are at least partially complementary to each other; the 3' end of the first complementary sequence comprises a free thymine deoxynucleotide; and the ligation sequence is used to ligate the sequencing adaptor complex to a membrane.

[0015] In a fifth aspect, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the helicase variant of the third aspect of the present application.

[0016] In a sixth aspect, the present application provides an expression vector. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule of the fifth aspect of the present application.

[0017] In a seventh aspect, the present application provides a recombinant cell. According to an embodiment of the present application, the recombinant cell comprises the nucleic acid molecule of the fifth aspect of the present application or the expression vector of the sixth aspect of the present application.

[0018] In an eighth aspect, the present application provides a method for preparing a helicase variant. According to an embodiment of the present application, the method comprises: culturing the recombinant cell of the seventh aspect of the present application under conditions suitable for protein expression, so as to obtain the helicase variant. BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 is a purification diagram and SDS-PAGE gel electrophoresis result of BCH105-Mutl in Example 1.

[0020] FIG. 2 is a result diagram of ATPase enzyme activity of BCH105-Mutl and its mutants in Example 2.

[0021] FIG. 3 is a result diagram of dsDNA unwinding activity of BCH105-Mutl and its mutants in Example 3.

[0022] FIG. 4 is a schematic diagram of a sequencing adaptor structure in Example 4.

[0023] FIG. 5 is a schematic diagram of a sequencing adaptor complex in Example 4.

[0024] FIG. 6 is a non-denaturing gel electrophoresis diagram of BCH105-Mutl sequencing adaptor complex in Example 4.

[0025] Figure 7 is a non-denaturing gel electrophoresis plot of the sequencing adapter complex of BCH105-Mutl_l in Example 4.

[0026] Figure 8 is a non-denaturing gel electrophoresis plot of the sequencing adapter complex of BCH105-Mutl_2 in Example 4.

[0027] Figure 9 is a non-denaturing gel electrophoresis plot of the sequencing adapter complex of BCH105-Mutl_3 in Example 4.

[0028] Figure 10 is a non-denaturing gel electrophoresis plot of the sequencing adapter complex of BCH105-Mutl_4 in Example 4.

[0029] Figure 11 is a non-denaturing gel electrophoresis plot of the sequencing adapter complex of BCH105-Mutl_5 in Example 4.

[0030] Figure 12 is a non-denaturing gel electrophoresis plot of the sequencing adapter complex of BCH105-Mutl_6 in Example 4.

[0031] Figure 13 is a schematic diagram of the library formed by ligation of the sequencing adapter complex to the double-stranded DNA to be sequenced in Example 5.

[0032] Figure 14 is a plot of the sequencing signal of BCH105-Mutl in Example 5.

[0033] Figure 15 is a plot of the sequencing signal of BCH105-Mutl_l in Example 5.

[0034] Figure 16 is a plot of the sequencing signal of BCH105-Mutl_2 in Example 5.

[0035] Figure 17 is a plot of the sequencing signal of BCH105-Mutl_3 in Example 5.

[0036] Figure 18 is a plot of the sequencing signal of BCH105-Mutl_4 in Example 5.

[0037] Figure 19 is a plot of the sequencing signal of BCH105-Mutl_5 in Example 5.

[0038] Figure 20 is a plot of the sequencing signal of BCH105-Mutl_6 in Example 5.

[0039] In Figures 14-20, the horizontal axis is the time for the DNA to enter and completely pass through the nanopore, and the vertical axis is the current value corresponding to the DNA passing through the nanopore at a voltage of 180 mV. DETAILED DESCRIPTION

[0040] Embodiments of the present application are described in detail below. The embodiments described below are exemplary in nature and are intended to explain the present application, and are not to be construed as limiting the same. In the embodiments, specific techniques or conditions are not mentioned unless otherwise specified, and are performed according to techniques or conditions described in the literature or according to the product manual. In the embodiments, the reagents or instruments not specified by the manufacturer are all commercially available conventional products.

[0041] Note that the terms "first" and "second" are used only for the purpose of description, and are not to be construed as indicating or implying relative importance or implying the number of the technical features indicated. Thus, the features defined with "first" and "second" can include one or more of the features explicitly or implicitly. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0042] In this context, the term "containing", "including" or "comprising" is an open expression, i.e., it includes the indicated content of the present application, but does not exclude other aspects.

[0043] In this context, the term "nucleic acid molecule" refers to a biological molecule composed of nucleotides, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These nucleic acid molecules carry genetic information and are involved in the transmission and expression of genetic information in living organisms.

[0044] In this context, the term "expression vector" refers to a medium for inserting a target gene (or nucleic acid sequence) into a host cell and promoting protein expression of the gene in the host cell. The expression vector generally contains a promoter, a DNA sequence encoding the target gene, and other necessary regulatory elements to ensure proper expression of the target gene.

[0045] In this context, the term "recombinant cell" refers to a cell that has been genetically engineered or modified. These cells have been genetically recombined to insert foreign genes or mutated genes, thereby allowing them to have new functions, express specific proteins, or produce desired products.

[0046] In this context, the term "recombinant strain" refers to a bacterial strain that has been genetically engineered or modified. These strains have been genetically recombined to insert foreign genes or mutated genes, thereby allowing them to have new functions, express specific proteins, or produce desired products.

[0047] In this context, the term "active unwinding activity" refers to the ability of an unwinding enzyme to actively reduce the activation energy of opening the double helix structure of DNA or RNA and converting it to a single-stranded form. This activity generally involves the enzymatic mechanism of the unwinding enzyme, which gradually unwinds the double helix structure of the nucleic acid substrate (polynucleotide in this context) through the interaction between the enzyme and the nucleic acid substrate and the energy consumption.

[0048] In the present context, the term "translocation activity" refers to the ability of a helicase to move along a nucleic acid substrate (polynucleotide in the present context) without necessarily involving the direct unwinding of a double helix structure. This activity can involve binding between the helicase and the nucleic acid substrate, complex formation, and allosteric changes in the enzyme itself, followed by a uniform movement along the nucleic acid substrate.

[0049] The present application provides a method for controlling movement of a polynucleotide, a method for nanopore sequencing, a helicase variant, a sequencing adapter complex, a nucleic acid molecule, an expression vector, a recombinant cell, and a method for preparing a helicase variant.

[0050] Method for controlling movement of a polynucleotide

[0051] According to a first aspect of the present application, the present application provides a method for controlling movement of a polynucleotide. According to an embodiment of the present application, the method comprises: moving a polynucleotide having a helicase variant connected thereto through a transmembrane pore under the control of an electric field force, wherein the helicase variant does not have active unwinding activity or translocation activity. According to the method of the embodiment of the present application, the helicase variant is converted from a mode of moving the polynucleotide by active unwinding to passive unwinding of the polynucleotide under the control of the electric field force, without using a spacer to block the helicase, thereby reducing the cost and ensuring that the speed of movement of the polynucleotide is no longer limited by the helicase.

[0052] According to an embodiment of the present application, the helicase variant is connected to the polynucleotide by binding to an adapter, and the adapter has or does not have a spacer.

[0053] According to an embodiment of the present application, the polynucleotide is a double-stranded polynucleotide, and the adapter comprises a first strand and a second strand, and at least part of sequences of the first strand and the second strand are complementary to each other.

[0054] The double-stranded polynucleotide is connected to a membrane by the adapter, and the transmembrane pore is located on the membrane.

[0055] According to an embodiment of the present application, the double-stranded polynucleotide is connected to the membrane through the linker, including: the second strand of the linker is directly connected to the membrane; or, an oligonucleotide strand complementary to at least part of the sequence of the second strand is connected to the membrane, and the double-stranded polynucleotide is connected to the membrane through the complementarity between the second strand and the oligonucleotide strand. It needs to be explained that the "oligonucleotide strand" in the present application refers to a single-stranded DNA or RNA molecule composed of a small number of nucleotides. It usually contains several to dozens of nucleotides. According to an embodiment of the present application, the oligonucleotide strand is connected to the membrane and has a sequence complementary to at least part of the sequence of the second strand. This means that the base sequence of the oligonucleotide strand can be combined with certain base sequences in the second strand through base complementary pairing. In this way, the double-stranded polynucleotide can be connected through the second strand and the oligonucleotide strand. This connection method takes advantage of the characteristics of base complementary pairing, so that the double-stranded polynucleotide can be tightly combined with the oligonucleotide strand on the membrane. Such a design can be used to guide the double-stranded polynucleotide to the transmembrane pore or the membrane in nanopore sequencing or other related applications, and ensure its stable sequencing or other processing through the nanopore. The complementary sequence of the oligonucleotide strand binds to the complementary sequence of the second strand, providing connection stability and directionality, thereby enhancing the interaction of the double-stranded polynucleotide with the membrane.

[0056] According to an embodiment of the present application, the second strand is connected to the membrane through cholesterol modification; or, the oligonucleotide strand is connected to the membrane through cholesterol modification. According to an embodiment of the present application, the second strand or the oligonucleotide strand can be connected to the membrane through cholesterol modification. This means that a cholesterol molecule is introduced into the second strand or the oligonucleotide strand, thereby realizing the combination with the membrane. Cholesterol is a lipid molecule commonly found in cell membranes, with amphiphilic properties. Its hydrophobic part can interact with the hydrophobic region of the membrane, while its hydrophilic part interacts with the hydrophilic region of the membrane. This property makes cholesterol play an important role in regulating the fluidity and stability of the cell membrane. By introducing cholesterol modification into the second strand or the oligonucleotide strand, it can interact with the membrane and form a stable connection. This connection method can increase the interaction force between the double-stranded polynucleotide and the membrane, thereby helping to control the position and stability of the double-stranded polynucleotide on the membrane. The cholesterol-modified second strand or oligonucleotide strand can interact with the membrane through covalent bonds or non-covalent bonds. Covalent bond connection usually involves introducing a cholesterol group into the molecular structure, while non-covalent bond connection can be achieved through hydrophobic interaction, electrostatic interaction or hydrogen bonding, etc. Therefore, according to an embodiment of the present application, cholesterol modification can be used to connect the second strand or the oligonucleotide strand to the membrane to enhance the interaction and stability of the double-stranded polynucleotide with the membrane. This connection method helps to accurately guide and process the double-stranded polynucleotide in nanopore sequencing or other related applications.

[0057] According to an embodiment of the present application, the first strand comprises, in sequence, a guide sequence, a helicase binding domain, and a first complementary sequence, the helicase variant binding to the helicase binding domain; the second strand comprises a second complementary sequence and a ligation sequence, the first complementary sequence and the second complementary sequence at least partially complementary pairing; the 3' free end of the first complementary sequence contains a free thymine deoxynucleotide; the ligation sequence is used to connect the sequencing adapter complex to the membrane.

[0058] According to an embodiment of the present application, the sequence of the first strand is shown as SEQ ID NO: 21, the sequence of the second strand is shown as SEQ ID NO: 22, and the oligonucleotide strand is shown as SEQ ID NO: 23.

[0059] According to an embodiment of the present application, the transmembrane pore comprises a protein nanopore or a solid-state nanopore.

[0060] It needs to be explained that the "nanopore" in the present application refers to a small pore or channel with a diameter of only nanometers. Such a pore passes through a thin film, which is usually composed of artificially synthesized materials (such as silica gel or silicon nitride) or natural proteins (such as protein nanopores). When a double-stranded DNA molecule passes through the transmembrane nanopore, a voltage is applied across the transmembrane nanopore, creating an ionic current. The nucleotides of the DNA molecule will pass through the transmembrane nanopore one by one, and cause slight current changes during the passing process. These current changes can be detected and recorded, and used to infer the sequence of the DNA. The diameter of the transmembrane nanopore is usually comparable to the size of the DNA, so that the DNA molecule can pass through. In addition, the surface of the transmembrane nanopore usually has a charge characteristic to attract the passing of the DNA molecule.

[0061] According to an embodiment of the present application, the membrane is a biological membrane or a biomimetic membrane. Wherein, the "biological membrane" in the present application refers to a thin film structure existing in or on the body of an organism, composed of biological macromolecules such as lipids, proteins, etc. These membranes play important biological functions inside and outside the cell, such as cell membranes, organelle membranes, etc. The "biomimetic membrane" in the present application refers to an artificially synthesized thin film with similar structure and properties to biological membranes. These membranes can be prepared by artificial synthesis methods, such as using synthetic lipids or other synthetic materials. Biomimetic membranes are widely used in nanopore technology to control and regulate the properties and functions of transmembrane pores. Therefore, according to an embodiment of the present application, the membrane can be a biological membrane, i.e. a thin film structure with biological origin; or a biomimetic membrane, with similar structure and properties to biological membranes.

[0062] According to embodiments of the present application, the membrane is an amphiphilic bilayer. In the present application, an "amphiphilic bilayer" refers to a bilayer structure composed of two substances with different affinities. In biological membranes and biomimetic membranes, the most common amphiphilic bilayer is a phospholipid bilayer. Phospholipid molecules have two parts, a hydrophilic head and a hydrophobic hydrocarbon tail, allowing them to form a bilayer structure in an aqueous environment. This amphiphilic bilayer structure plays an important role in nanopore sequencing as a supporting structure for the transmembrane pore and provides a suitable environment to guide the passage of DNA molecules.

[0063] According to embodiments of the present application, the membrane is a phospholipid membrane.

[0064] According to embodiments of the present application, the helicase variant is obtained by mutating at least one amino acid in a region of the helicase associated with helicase activity. According to embodiments of the present application, the helicase variant is obtained by mutating at least one amino acid in a region of the helicase associated with helicase activity. Helicases are a class of enzymes that can unwind the double helix structure of DNA or RNA, converting it into a single-stranded form. The region of the helicase associated with helicase activity is crucial to the helicase activity of the helicase, and contains key amino acid residues that affect helicase activity. In the present application, at least one amino acid is mutated in the region of the helicase associated with helicase activity, which causes the helicase to lose active helicase activity or translocation activity. According to the method of embodiments of the present application, the helicase variant is converted from actively helicase moving the polynucleotide to passively helicase under the control of the electric field force, without the use of a spacer to block the helicase, reducing costs and ensuring that the speed of the polynucleotide is no longer limited by the helicase.

[0065] According to embodiments of the present application, the amino acid sequence of the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO: 2. According to the method of embodiments of the present application, the helicase variant is converted from actively helicase moving the polynucleotide to passively helicase under the control of the electric field force, without the use of a spacer to block the helicase, reducing costs and ensuring that the speed of the polynucleotide is no longer limited by the helicase.

[0066] According to an embodiment of the present application, the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO: 2 except for the amino acid sequences of V119, C133, C164, K199, C292, C323, C347, and L369. According to the method of the embodiment of the present application, the helicase variant is changed from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without using a spacer to block the helicase, reducing the cost, and being able to ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0067] According to an embodiment of the present application, the helicase variant includes at least one of the following first group of mutation sites compared with the amino acid sequence shown in SEQ ID NO: 2:

[0068] L106, L125, K105, F110, H108, D113, D117, S313, N314, W384, F388, R380, E392, Y378, V179, N270, H99, K407, P69, M147, Q175, and H406. According to the method of the embodiment of the present application, the helicase variant is changed from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without using a spacer to block the helicase, reducing the cost, and being able to ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0069] According to an embodiment of the present application, the helicase variant further includes at least one of the following mutation sites: F110, L125, V179, N314, L106, and M147. According to the method of the embodiment of the present application, the helicase variant is changed from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without using a spacer to block the helicase, reducing the cost, and being able to further ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0070] According to an embodiment of the present application, the helicase variant further includes at least one of the following mutations: F110A, L125M, V179W, N314A, L106A, and M147I. According to the method of the embodiment of the present application, the helicase variant is changed from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without using a spacer to block the helicase, and being able to further ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0071] According to an embodiment of the present application, the helicase variant further comprises a mutation of F110A; or, the helicase variant comprises a mutation of L125M; or, the helicase variant comprises a mutation of V179W; or, the helicase variant comprises a mutation of N314A; or, the helicase variant comprises a mutation of L106A, L125M; or, the helicase variant comprises a mutation of M147I, V179W. According to the method of the embodiment of the present application, the helicase variant is changed from the active helicase moving the polynucleotide to the passive helicase moving the polynucleotide under the control of the electric field force, without using the spacer to block the helicase, reducing the cost, and further ensuring that the moving speed of the polynucleotide is no longer limited by the helicase.

[0072] According to an embodiment of the present application, the amino acid sequence of the helicase mutant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO: 2 at other amino acid sequences except at least one of the first site group, V119, C133, C164, K199, C292, C323, C347, L369; or the helicase variant has an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14. According to the method of the embodiment of the present application, the helicase variant is changed from the active helicase moving the polynucleotide to the passive helicase moving the polynucleotide under the control of the electric field force, without using the spacer to block the helicase, reducing the cost, and further ensuring that the moving speed of the polynucleotide is no longer limited by the helicase.

[0073] According to an embodiment of the present application, the helicase variant further comprises mutating at least one of the second site group to cysteine or introducing a non-natural amino acid compared with the amino acid sequence shown in SEQ ID NO: 2:

[0074] G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, Y119C, T120, T121, K122, A123, K124, L125, V126, V127, N128, K129, F130, N131, E132, A133, L134, P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392, and K393. According to the method of the embodiment of the present application, the helicase variant is changed from the way of actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of the electric field force, without using a spacer to block the helicase, reducing the cost, and further ensuring that the moving speed of the polynucleotide is no longer limited by the helicase.

[0075] According to the embodiment of the present application, the unnatural amino acid includes one of 4-azido-L-phenylalanine, 4-azido-L-phenylalanine, 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenyl)-L-alanine, O2-propargyl-L-tyrosine, 4-(dihydroxyboronyl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine.

[0076] According to the embodiment of the present application, the pin domain is connected with the tower domain.

[0077] According to the embodiment of the present application, the connection is formed by introducing a cross-linking agent, and the cross-linking agent includes one of a maleimide, an active ester, a succinimide, an azide, an alkyne, a phosgene-type reagent, a sulfonyl chloride reagent, an isothiocyanate, an acyl halide, a hydrazine, a disulfide, a vinyl sulfone, an aziridine, and a photosensitive reagent.

[0078] According to the embodiment of the present application, the connection includes at least one of a covalent connection, a covalent and non-covalent binding connection, and a non-covalent connection.

[0079] According to the embodiment of the present application, the covalent connection is performed by at least one of a cross-linking agent, a protein fusion, a polypeptide molecule, and a small molecule.

[0080] According to an embodiment of the present application, further comprising introducing a cross-linking agent comprising at least one of a maleimide, an active ester, a succinimide, an azide, an alkyne, a difluorocycloalkyne, a phosphine, a haloacetyl, a phosgene-type reagent, a sulfonyl chloride reagent, an isothiocyanate, an acyl halide, a hydrazine, a disulfide, a vinyl sulfone, an aziridine, and a photosensitive reagent.

[0081] According to an embodiment of the present application, G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, V119, T120, T121, K122, A123, K124, L125, V126, V127, N128, K129, F130, N131, E132, C133, L134 are derived from the pin domain of the helicase variant.

[0082] According to an embodiment of the present application, P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392, K393 are derived from the tower domain of the helicase variant.

[0083] Method of nanopore sequencing

[0084] In a second aspect, the present application provides a method of nanopore sequencing. According to an embodiment of the present application, the method comprises moving a polynucleotide to be sequenced, to which a helicase variant is attached, through a nanopore under the control of an electric field force by the method of the first aspect of the present application; and determining the sequence of the polynucleotide to be sequenced based on the current signal generated when the polynucleotide to be sequenced passes through the nanopore. According to the method of an embodiment of the present application, the helicase variant is changed from actively moving the polynucleotide to passively unwinding the polynucleotide under the control of an electric field force. In this process, the helicase does not need to be blocked by a spacer, reducing the cost of nanopore sequencing; at the same time, the speed of the helicase moving the polynucleotide is controlled by the electric field force, so that the speed of the polynucleotide is no longer limited by the helicase.

[0085] Helicase variant

[0086] According to a third aspect of the present application, a helicase variant is provided. According to embodiments of the present application, the helicase variant has at least one amino acid mutation in a region related to unwinding activity compared to the amino acid sequence set forth in SEQ ID NO: 2, and the helicase variant does not have active unwinding activity or translocation activity. According to embodiments of the present application, the helicase variant does not have active unwinding activity or translocation activity.

[0087] According to embodiments of the present application, the helicase variant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology in the amino acid sequence compared to the amino acid sequence set forth in SEQ ID NO: 2, except for V119, C133, C164, K199, C292, C323, C347, and L369.

[0088] According to embodiments of the present application, the helicase variant includes a mutation site in at least one of the following first site groups:

[0089] L106, L125, K105, F110, H108, D113, D117, S313, N314, W384, F388, R380, E392, Y378, V179, N270, H99, K407, P69, M147, Q175, and H406. According to embodiments of the present application, the helicase variant is changed from moving the polynucleotide by active unwinding to passively unwinding the polynucleotide under the control of an electric field force, without using a spacer to block the helicase variant, reducing costs, and ensuring that the moving speed of the polynucleotide is no longer limited by the helicase.

[0090] According to embodiments of the present application, the helicase variant includes a mutation site in at least one of the following: F110, L125, V179, N314, L106, and M147. According to embodiments of the present application, the helicase variant is changed from moving the polynucleotide by active unwinding to passively unwinding the polynucleotide under the control of an electric field force, without using a spacer to block the helicase variant, reducing costs, and ensuring that the moving speed of the polynucleotide is no longer limited by the helicase.

[0091] According to an embodiment of the present application, the helicase variant further comprises at least one of the following mutations: F110A, L125M, V179W, N314A, L106A and M147I. The helicase variant according to the embodiment of the present application changes the way of moving the polynucleotide from active unwinding to passive unwinding under the control of electric field force, without using a spacer to block the helicase variant, reduces the cost, and can ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0092] According to an embodiment of the present application, the helicase variant further comprises the following mutations: F110A; or, the helicase variant comprises the following mutations: L125M; or, the helicase variant comprises the following mutations: V179W; or, the helicase variant comprises the following mutations: N314A; or, the helicase variant comprises the following mutations: L106A, L125M; or, the helicase variant comprises the following mutations: M147I, V179W. The helicase variant according to the embodiment of the present application changes the way of moving the polynucleotide from active unwinding to passive unwinding under the control of electric field force, without using a spacer to block the helicase variant, reduces the cost, and can ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0093] According to an embodiment of the present application, the amino acid sequence of the helicase mutant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO: 2, except for the sequence of other amino acids at at least one site in the first site group and V119, C133, C164, K199, C292, C323, C347, L369; or the helicase variant has an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14. The helicase variant according to the embodiment of the present application changes the way of moving the polynucleotide from active unwinding to passive unwinding under the control of electric field force, without using a spacer to block the helicase variant, reduces the cost, and can ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0094] According to an embodiment of the present application, the helicase variant further comprises mutating at least one site in the second site group to cysteine or introducing a non-natural amino acid compared with the amino acid sequence shown in SEQ ID NO: 2:

[0095] G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, V119, T120, T121, K122, A123, K124, L125, V126, V127, N128, K129, F130, N131, E132, A133, L134, P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392, and K393. According to the helicase variant of the embodiments of the present application, the way of moving the polynucleotide by the active unwinding is changed to the passive unwinding of the polynucleotide under the control of the electric field force, without using the spacer to block the helicase variant, reducing the cost, and being able to ensure that the moving speed of the polynucleotide is no longer limited by the helicase.

[0096] According to the embodiments of the present application, the unnatural amino acid includes one of 4-azido-L-phenylalanine (PAZF), 4-azido-L-phenylalanine (PAZF-HCl), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenyl)-L-alanine, O2-propargyl-L-tyrosine, 4-(dihydroxyboronyl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine.

[0097] According to the embodiments of the present application, the pin domain is connected with the tower domain.

[0098] According to the embodiments of the present application, the connection is formed by introducing a cross-linking agent, the cross-linking agent includes one of a maleimide, an active ester, a succinimide, an azide, an alkyne, a phosgene-type reagent, a sulfonyl chloride reagent, an isothiocyanate, an acyl halide, a hydrazine, a disulfide, a vinyl sulfone, an aziridine, and a photosensitive reagent.

[0099] According to the embodiments of the present application, the connection includes at least one of a covalent connection, a covalent and non-covalent binding connection, and a non-covalent connection.

[0100] According to embodiments of the present application, the covalent attachment is by at least one of a cross-linking agent, a protein fusion, a polypeptide molecule, a small molecule.

[0101] According to embodiments of the present application, further comprising introducing a cross-linking agent comprising at least one of a maleimide, an active ester, a succinimide, an azide, an alkyne, a difluorocycloalkyne, a phosphine, a haloacetyl, a phosgene-type reagent, a sulfonyl chloride reagent, an isothiocyanate, an acyl halide, a hydrazine, a disulfide, a vinyl sulfone, an aziridine, and a photosensitive reagent.

[0102] According to embodiments of the present application, G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, V119, T120, T121, K122, A123, K124, L125, V126, V127, N128, K129, F130, N131, E132, C133, L134 are derived from a pin domain of the helicase variant.

[0103] According to embodiments of the present application, P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392, K393 are derived from a tower domain of the helicase variant.

[0104] Sequencing adapter complex

[0105] According to a fourth aspect of the present application, the present application provides a sequencing adapter complex. According to embodiments of the present application, the sequencing adapter complex comprises: a first strand and a second strand and a helicase variant as described in the third aspect of the present application; wherein the first strand comprises, in order, a guide sequence, a helicase binding domain, and a first complementary sequence, the helicase variant is bound to the helicase binding domain; the second strand comprises a second complementary sequence and a ligation sequence, the first complementary sequence and the second complementary sequence at least partially complementarily pair; a 3’ overhang of the first complementary sequence contains a free thymine deoxynucleotide; the ligation sequence is used to ligate the sequencing adapter complex to a membrane.

[0106] According to an embodiment of the present application, the sequence of the first strand is shown as SEQ ID NO: 21, and the sequence of the second strand is shown as SEQ ID NO: 22.

[0107] In the present application, the meaning of "sequencing adapter complex" is consistent with that of "adapter complex".

[0108] In which, it needs to be explained that the 3' end of the first complementary sequence contains a free thymine deoxyribonucleotide (dT) for TA ligation with the polynucleotide, so as to be applied to subsequent nanopore sequencing.

[0109] According to an embodiment of the present application, a spacer is further included between the helicase binding domain and the first complementary sequence.

[0110] According to an embodiment of the present application, an oligonucleotide chain is further included, the ligation sequence is complementary to at least part of the sequence of the oligonucleotide chain, and the 5' end of the oligonucleotide chain is connected with a cholesterol molecule.

[0111] According to an embodiment of the present application, the second strand is connected with a cholesterol molecule.

[0112] Nucleic acid molecule

[0113] In a fifth aspect of the present application, a nucleic acid molecule is provided. According to an embodiment of the present application, the nucleic acid molecule encodes the helicase variant of the third aspect of the present application.

[0114] Expression vector

[0115] In a sixth aspect of the present application, an expression vector is provided. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule of the fifth aspect of the present application. According to an embodiment of the present application, the expression vector is a prokaryotic expression vector.

[0116] Recombinant cell

[0117] In a seventh aspect of the present application, a recombinant cell is provided. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule of the fifth aspect of the present application or the expression vector of the sixth aspect of the present application. According to an embodiment of the present application, the recombinant cell includes but is not limited to a recombinant strain, a recombinant unicellular organism and a recombinant multicellular organism.

[0118] According to an embodiment of the present application, the recombinant strain includes but is not limited to Escherichia coli.

[0119] According to an embodiment of the present application, the recombinant unicellular organism includes but is not limited to yeast.

[0120] According to embodiments of the present application, the recombinant multicellular organism includes, but is not limited to, a mammalian cell.

[0121] Method for preparing a helicase variant

[0122] According to an eighth aspect of the present application, the present application provides a method for preparing a helicase variant. According to embodiments of the present application, the method comprises: culturing the recombinant cell of the seventh aspect of the present application under conditions suitable for protein expression, so as to obtain the helicase variant.

[0123] The sequence listing of the present application is shown as follows:

[0124] Sequence listing

[0125] Wherein, it needs to be explained that X in SEQ ID NO: 17 refers to iSPC3.

[0126] The schemes of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or conditions are not specified in the examples, the technology or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by market purchase.

[0127] Example 1: Expression of BCH105 helicase variants

[0128] In this example, BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, BCH105-Mut1_6 helicase variants were constructed and purified respectively, and the specific experimental methods are as follows:

[0129] (1) Construction: The sequences of BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, BCH105-Mut1_6 shown in Table 1 were connected into the PET.28a(+) plasmid using double enzyme digestion sites Nde1 and Xho1, and the N-terminal of the expressed BCH105-Mut1 helicase has a 6×His tag and a thrombin cleavage site.

[0130] Table 1

[0131] (2) Purification: The recombinant plasmids containing BCH105-Mutl, BCH105-Mutl_l, BCH105-Mutl_2, BCH105-Mutl_3, BCH105-Mutl_4, BCH105-Mutl_5, BCH105-Mutl_6 were transformed into E. coli expression strain Arctic Express (DE3) or its derivative, respectively. Single colony was picked and inoculated into 20 mL LB medium containing kanamycin, and incubated at 37°C overnight. Then, it was inoculated into 2 L LB medium containing kanamycin, and incubated at 37°C until OD600=0.6-0.8. The temperature was decreased to 10°C, and 500 μM IPTG was added to induce expression for 40-46 hours. The bacterial cells were collected.

[0132] After the bacteria expressing the BChl05-Mutl, BChl05-Mutl_l, BChl05-Mutl_2, BChl05-Mutl_3, BChl05-Mutl_4, BChl05-Mutl_5, BChl05-Mutl_6 helicase variants were collected using a centrifuge at 5000 rpm at 4°C, the bacteria were resuspended using Buffer A and the bacteria were broken using a cell disrupter. The supernatant was collected after high speed centrifugation. The supernatant was mixed with Ni-NTA resin equilibrated with Buffer A (formula: 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM Imidazole) and incubated at 4°C for 1 hour. The resin was then washed with 5-10 column volumes of Buffer B (formula: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM Imidazole) until no impurities were eluted. Then, 10 mL of Buffer B and 50 μΐ of thrombin were added to the resin and incubated at 4°C overnight for 14-16 hours. After that, the flow-through of the overnight enzyme digestion was collected and 10 mL of Buffer B was added again for washing and the flow-through was collected. The eluted protein of interest was diluted to a salt concentration of 50 mM using Buffer C (formula: 20 mM Tris-HCl pH 8.0) and loaded onto a Hitrap Q HP (Cytiva) column, and then eluted using Buffer D (formula: 20 mM Tris-HCl pH 8.0, 50 mM NaCl) and Buffer E (formula: 20 mM Tris-HCl pH 8.0, 1000 mM NaCl) with a gradient. The eluted protein of interest was then concentrated to 1 mL and further purified using a size exclusion column Superdex 200 increase 10 / 300 GL (Cytiva) with Buffer F (formula: 20 mM Tris-HCl pH 8.0, 80 mM NaCl). The eluted peak of the protein of interest (BChl05-Mutl, BChl05-Mutl_l, BChl05-Mutl_2, BChl05-Mutl_3, BChl05-Mutl_4, BChl05-Mutl_5, BChl05-Mutl_6 helicase variants) was collected and concentrated according to the elution curve of the size exclusion column and SDS-PAGE. The final protein of interest was stored at -80°C.

[0133] The purification results of the BCH105-Mutl helicase variant are exemplarily shown in this example, and the results are shown in Figure 1. The final obtained BCH105-Mutl helicase variant protein has good purity and high expression amount. The other unshown BCH105-Mutl_l, BCH105-Mutl_2, BCH105-Mutl_3, BCH105-Mutl_4, BCH105-Mutl_5, BCH105-Mutl_6 helicase variants have similar purification conditions as the BCH105-Mutl helicase variant, and the protein purities are all good and the expression amounts are all high.

[0134] Example 2: Detection of ATPase activity of helicase variants

[0135] The ATPase activities of the BCH105-Mutl, BCH105-Mutl_l, BCH105-Mutl_2, BCH105-Mutl_3, BCH105-Mutl_4, BCH105-Mutl_5, BCH105-Mutl_6 helicase variants were detected respectively, and the specific experimental methods are as follows:

[0136] (1) Preparation of double-stranded DNA (overhang DNA-1, referred to as ovDNA-1) and single-stranded DNA (ssDNA):

[0137] SEQ ID NO: 16 and SEQ ID NO: 17 sequences (six harmonious China) were synthesized, and SEQ ID NO: 16 and SEQ ID NO: 17 were annealed to 5' ovDNA-1 with 20 T overhangs. The annealing process was incubation at 95°C for 5 minutes, cooling at a rate of 0.1°C / s to 25°C, and then incubation at 25°C for 30 minutes. The annealing formula is shown in Table 2. 100 mM of SEQ ID NO: 17 was diluted to 10 mM with TE buffer (pH = 8) as ssDNA.

[0138] Table 2: ovDNA-1 annealing formula

[0139] (2) ATP hydrolysis reaction: The reaction system in Table 2 was added with corresponding reagents, and the ATP hydrolysis reaction was tested by incubation at 30°C for 30 min, and inactivation at 80°C for 5 min. Among them, ① and ② are experimental groups, and ③, ④, ⑤ and ⑥ are corresponding control groups, each group has 3 repeats. Among them, the high-salt reaction buffer (2x) in Table 3 is as follows: 20 mM HEPES (pH 8.0), 4 mM ATP, 4 mM MgCl2, 300 mM KCl dilute protein: BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, BCH105-Mut1_6 helicase were diluted to 10 μM with 1x PBS respectively.

[0140] Table 3: ATP hydrolysis reaction system

[0141] (3) Detection of residual ATP in the reaction: ATP detection kit (Bi Yun Tian, S0026B) was used to determine the residual ATP concentration according to the manufacturer's operation instruction.

[0142] This example exemplarily shows the results of BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, and the results are shown in Figure 2. BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4 have equivalent ATP hydrolysis activity.

[0143] Example 3: Detection of dsDNA unwinding activity of helicase variants

[0144] This example detects the dsDNA unwinding activity of BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, BCH105-Mut1_6 helicase variants, respectively, and the specific experimental method is as follows:

[0145] (1) Preparation of double-stranded DNA (ovDNA-2):

[0146] Synthesis of SEQ ID NO: 18 and SEQ ID NO: 19 sequences (Huada six combination), annealing SEQ ID NO: 18 and SEQ ID NO: 19 to 5' ovDNA-2 with 20 T overhangs, the annealing process is incubated at 95°C for 5 minutes, the temperature is reduced to 25°C at a rate of 0.1°C / s, and incubated for 30 minutes. The annealing formula of ovDNA-2 is shown in Table 4.

[0147] Table 4: ovDNA-2 annealing formula

[0148] (2) Preparation of melting reaction:

[0149] First, 3 μL of 10 μM ovDNA-2, 6 μL of 100 μM SEQ ID NO: 20 (20-fold competition DNA, which can pair with its complementary DNA sequence during the annealing process to prevent reannealing of the initial substrate and loss of fluorescence), 6 μL of 100 mM ATP were added to 585 μL of reaction buffer (formula: 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 150 mM KCl) to prepare the experimental reaction solution.

[0150] 1 μL of 10 μM SEQ ID NO: 19, 2 μL of 100 μM SEQ ID NO: 20 (20-fold competition DNA), 2 μL of 100 mM ATP were added to 195 μL of reaction buffer (formula: 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 150 mM KCl) to prepare the positive control solution.

[0151] Then, the helicase variants BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, BCH105-Mut1_6 were diluted to 4.8 μM with 1 × PBS, respectively.

[0152] Subsequently, the corresponding reagents prepared above were added according to Table 5, a was the experimental group, b was the negative control group, and c was the positive control group. The kinetic changes of fluorescence intensity within 30 min were detected at 30°C using a microplate reader, and each group was repeated three times. Within the error range and instrument fluctuation allowed, the experimental results were statistically analyzed by calculating the ratio of the fluorescence value of the experimental group to the fluorescence value of the positive control group, and the ratio of the fluorescence value of the negative control group to the fluorescence value of the positive control group.

[0153] Table 5: Melting reaction

[0154] The results of BCH105-Mutl, BCH105-Mutl_l, BCH105-Mutl_2, BCH105-Mutl_3, BCH105-Mutl_4 are shown in Figure 3. The negative control group remained unchanged during the determination process, while the fluorescence value of the experimental group gradually increased with the increase of reaction time. Among them, BCH105-Mutl has the highest activity, and the helicase activity of BCH105-Mutl_l, BCH105-Mutl_2, BCH105-Mutl_3, BCH105-Mutl_4 has different degrees of decline.

[0155] Example 4: Preparation of helicase variant-linker complex

[0156] In this example, BCH105-Mutl, BCH105-Mutl_l, BCH105-Mutl_2, BCH105-Mutl_3, BCH105-Mutl_4, BCH105-Mutl_5, BCH105-Mutl_6 helicase variant-linker complexes were prepared respectively, and the specific experimental methods are as follows:

[0157] (1) Preparation of linker: three partially complementary DNA strands (first strand: SEQ ID NO: 21, second strand: SEQ ID NO: 22, oligonucleotide strand: SEQ ID NO: 23) were annealed to form a linker, and the schematic diagram of the linker is shown in Figure 5. The linker does not have a spacer to block the progress of the helicase variant. The single-stranded complementary DNA with cholesterol is added to prevent the helicase variant from binding to the second strand and affecting sequencing.

[0158] (2) Preparation of helicase variant-linker complex: The total system of helicase variant and linker is 200 μL, and the corresponding volume X of helicase variant is added at a final concentration of 10 μM. Buffer G (formula: 40 mM Hepes-Na, pH 7.5, 100 mM NaCl) and the linker prepared in step (1) of this example are added to a 1.5 mL DNA LoBind Tube Microcentrifuge Tube tube according to Table 6, and the remaining volume is added with Nuclease-Free water. Put it in a preheated metal bath at 28°C for 30 minutes.

[0159] Table 6: Preparation of helicase variant and linker reaction system

[0160] To the incubated helicase variant-linker complex, add 11 μL of 400 μM of BMOE, pipette 6-8 times to mix, place the 1.5 mL DNA LoBind Tube Microcentrifuge Tube in a 28 °C metal bath for 30 min. To the cross-linked helicase variant-linker complex, add 0.22 μL of 1 M DTT solution, pipette 6-8 times to mix, incubate at room temperature for 15 min.

[0161] Mix the cross-linked helicase variant-linker complex with Buffer H (formula: 50 mM Hepes-Na, pH 7.5, 1 M NaCl, 4 mM ATP, 20 mM MgCl2) at a 1:1 volume ratio, pipette 6-8 times to mix, place the mixed sample in a 30 °C pre-heated metal bath for 1 h.

[0162] Take out the magnetic bead DNA Clean Beads in a DNA LoBind Tube Microcentrifuge Tube and place it on a magnetic stand, let it stand for 2-5 min until the liquid is clear, carefully aspirate and discard the supernatant with a pipette. Wash the magnetic beads with Buffer I (formula: 50 mM Hepes-Na, pH 7.5, 2.5 M NaCl) for 2 times, each time use the “magnetic bead swimming method” to make the magnetic beads swim in the solution for more than 5 times to mix well. Each time the tube is moved, the magnetic beads need to be allowed to stand for 2-5 min until the liquid is completely clear, finally carefully aspirate and discard the supernatant with a pipette. Add Buffer J (formula: 50 mM Hepes-Na, pH 7.5, 2.5 M NaCl, 24% PEG8000, 0.05% TWEEN-20) to the washed magnetic beads, use the “magnetic bead swimming method” to make the magnetic beads swim in the solution for more than 5 times to mix well, after standing, carefully aspirate and discard the supernatant with a pipette, then repeat the Buffer J equilibration of the magnetic beads once. Finally, resuspend the magnetic beads with an appropriate volume of Buffer J.

[0163] The ATP-treated helicase variant-linker complex system was mixed with the equilibrated magnetic bead DNA Clean Beads at a volume ratio of 1:2, and incubated at room temperature for 30 minutes in a rotating mixer. The incubated helicase variant-linker complex was placed in a magnetic stand and left to stand until the liquid was completely clarified (about 5-10 min), and the supernatant was carefully aspirated and discarded with a pipette. The helicase variant-linker complex was removed from the magnetic stand, washed with Buffer K (formula: 50 mM Hepes-Na, pH 7.5, 2.5 M NaCl, 20% PEG8000), and the magnetic beads were allowed to swim in the solution for more than 5 times to mix thoroughly. Each time the test tube was moved, the magnetic beads were allowed to stand for 2-5 min until the liquid was completely clarified, and finally the supernatant was carefully aspirated and discarded with a pipette. Buffer K was used for washing once, and the liquid in the tube was aspirated as much as possible to remove the Buffer K adhering to the surface of the magnetic beads. Then 40 μL of Buffer L (formula: 50 mM Hepes-Na, pH 7.5, 20 mM NaCl) was added for elution, and after mixing, it was incubated at room temperature for 5 min. The incubated helicase variant-linker complex was placed in a magnetic stand and left to stand for 2-5 min until the liquid was clarified, and the supernatant was transferred to a new 1.5 mL DNA LoBind Tube Microcentrifuge Tube. The helicase variant-linker complex was subjected to TBE non-denaturing gel electrophoresis.

[0164] The results are shown in Figures 6 to 12. BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 can all successfully construct spacer-free helicase variant-linker complex, which can be used for library construction for nanopore sequencing.

[0165] Example 5: Application of helicase variant-linker complex preparation in nanopore sequencing

[0166] The helicase variant-linker complex prepared in Example 4 was connected with the double-stranded DNA (SEQ ID NO: 24) to be tested using fast T4 DNA ligase (NEB, E6057AVIAL) and purified to obtain a sequencing library containing the helicase variant-linker complex, as shown in Figure 13.

[0167] First, the current signal was collected using a patch-clamp amplifier, Ag / AgCl electrodes were immersed in the sequencing buffer (formula: 0.47M KCl, 25mM HEPES, 1mM EDTA, 30mM ATP, 25mM MgCl2, pH 8) and the electrodes were located in the cis and trans regions of the electrolytic cell, respectively, after a layer of bilayer phospholipid membrane was formed at the micropores of the two chambers, the nanopore protein was added; after the single nanopore protein was inserted into the phospholipid membrane, the electrical measurement was obtained.

[0168] Subsequently, the sequencing library containing the helicase variant-linker complex prepared above was added, respectively, and a voltage of 180mV was applied. It was observed that the double-stranded DNA to be tested in the sequencing library was captured by the nanopore, generating a characteristic block current amplitude value.

[0169] As shown in FIG. 14, the helicase variant BCH105-Mut1 appeared more current change signals, indicating that when the linker does not contain a spacer, the helicase variant BCH105-Mut1 will unwind the double-stranded DNA in the solution, resulting in a signal of direct perforation or blockage of single-stranded nucleic acid.

[0170] As shown in FIGS. 15-20, the helicase variants BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 appeared fewer current change signals and could normally perform sequencing, indicating that weakening the enzyme activity can inhibit the helicase to unwind the double-stranded DNA in the sequencing solution containing ATP, and under the action of the electric field force, the DNA can be controlled to pass through the nanopore at a uniform speed.

[0171] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method of controlling movement of a polynucleotide, comprising, The application relates to a polynucleotide connected with a helicase variant moving through a transmembrane pore under the control of an electric field force, wherein the helicase variant does not have active unwinding activity or translocation activity. The helicase variant is connected to the polynucleotide through a linker, and a spacer is present or absent on the linker. The polynucleotide is a double-stranded polynucleotide, and the linker comprises a first strand and a second strand, and at least part of the sequences of the first strand and the second strand are complementary.

2. The method of claim 1, wherein, The double-stranded polynucleotide is connected to the membrane through the linker, and the transmembrane pore is located on the membrane.

3. The method of claim 2, wherein, The double-stranded polynucleotide is connected to the membrane through the linker, which comprises that the second strand of the linker is directly connected to the membrane, or an oligonucleotide strand complementary to at least part of the sequence of the second strand is connected to the membrane, and the double-stranded polynucleotide is connected to the membrane through the complementary second strand and the oligonucleotide strand. Preferably, the second strand is connected to the membrane through a cholesterol modification, or the oligonucleotide strand is connected to the membrane through a cholesterol modification.

4. The method of claim 3, wherein, The first strand comprises a guide sequence, a helicase binding domain and a first complementary sequence in sequence, and the helicase variant is bound to the helicase binding domain. The second strand comprises a second complementary sequence and a connection sequence, the first complementary sequence and the second complementary sequence are at least partially complementary and paired, the 3' free end of the first complementary sequence contains a free thymine deoxyribonucleotide, and the connection sequence is used for connecting a sequencing adapter complex to the membrane.

5. The method of claim 4, wherein, The sequence of the first strand is shown in SEQ ID NO: 21, the sequence of the second strand is shown in SEQ ID NO: 22, and the sequence of the oligonucleotide strand is shown in SEQ ID NO:

23. The transmembrane pore comprises a protein nanopore or a solid-state nanopore.

6. The method of claim 5, wherein, Optionally, the membrane is a biological membrane or a biomimetic membrane.

7. The method of claim 3, wherein, The helicase variant is obtained by at least one amino acid mutation in a region of the helicase related to unwinding activity. The amino acid sequence of the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology compared with the amino acid sequence shown in SEQ ID NO:

2.

8. The method according to any one of claims 1 to 7, characterized in that, The amino acid sequence of the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology compared with the amino acid sequence shown in SEQ ID NO: 2, except for V119, C133, C164, K199, C292, C323, C347 and L369.

9. The method of claim 8, wherein, The helicase variant comprises at least one of the following first site groups of mutation sites compared with the amino acid sequence shown in SEQ ID NO: 2:

10. The method of claim 9, wherein, ​ 11. The method of claim 10, wherein, ​ L106, L125, K105, F110, H108, D113, D117, S313, N314, W384, F388, R380, E392, Y378, V179, N270, H99, K407, P69, M147, Q175, and H406; Preferably, the helicase variant comprises a mutation site of at least one of F110, L125, V179, N314, L106, and M147. Preferably, the helicase variant comprises a mutation of at least one of F110A, L125M, V179W, N314A, L106A, and M147I.

12. The method of claim 11, wherein, The helicase variant comprises a mutation of F110A; or, The helicase variant comprises a mutation of L125M; or, The helicase variant comprises a mutation of V179W; or, The helicase variant comprises a mutation of N314A; or, The helicase variant comprises a mutation of L106A, L125M; or, The helicase variant comprises a mutation of M147I, V179W.

13. The method of claim 11, wherein, The amino acid sequence of the helicase mutant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the amino acid sequence shown in SEQ ID NO: 2, except for at least one site in the first group of sites and other amino acid sequences of V119, C133, C164, K199, C292, C323, C347, and L369; or The helicase variant has an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:

14.

14. The method of claim 11, wherein, The helicase variant further comprises mutating at least one site in the second group of sites to cysteine or introducing a non-natural amino acid compared to the amino acid sequence shown in SEQ ID NO: 2: G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, V119C, T120, T121, K122, A123, K124, L125, V126, V127, N128, K129, F130, N131, E132, A133, L134, P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392, and K393; Preferably, the unnatural amino acid comprises one of 4-azido-L-phenylalanine, 4-azido-L-phenylalanine, 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenyl)-L-alanine, O2-propargyl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, and 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine.

15. The method of claim 14, wherein, The pin domain is linked to the tower domain; Preferably, the linking is formed by introducing a cross-linking agent, which comprises one of a maleimide, an active ester, a succinimide, an azide, an alkyne, a phosgene-type reagent, a sulfonyl chloride reagent, an isothiocyanate, an acyl halide, a hydrazine, a disulfide, a vinyl sulfone, an aziridine, and a photoactive reagent.

16. A method of nanopore sequencing, characterized in that, The method comprises: The method comprises: The method comprises:

17. A helicase variant, characterized in that, The method comprises:

18. The helicase variant of claim 17, wherein, The method comprises:

19. The helicase variant of claim 18, wherein, ​ L106, L125, K105, F110, H108, D113, D117, S313, N314, W384, F388, R380, E392, Y378, V179, N270, H99, K407, P69, M147, Q175, and H406; Preferably, the helicase variant comprises a mutation site of at least one of F110, L125, V179, N314, L106, and M147. Preferably, the helicase variant comprises a mutation of at least one of F110A, L125M, V179W, N314A, L106A, and M147I.

20. The helicase variant of claim 19, wherein, The helicase variant comprises a mutation of F110A; or, The helicase variant comprises a mutation of L125M; or, The helicase variant comprises a mutation of V179W; or, The helicase variant comprises a mutation of N314A; or, The helicase variant comprises a mutation of L106A, L125M; or, The helicase variant comprises a mutation of M147I, V179W.

21. The helicase variant of claim 19, wherein, The amino acid sequence of the helicase mutant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the amino acid sequence shown in SEQ ID NO: 2, except for the sequence of other amino acids at at least one site in the first group of sites and V119, C133, C164, K199, C292, C323, C347, and L369; or The helicase variant has an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:

14.

22. The helicase variant according to claim 19, wherein, The helicase variant further comprises mutating at least one site in the second group of sites to cysteine or introducing a non-natural amino acid compared to the amino acid sequence shown in SEQ ID NO: 2: G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, Y119C, T120, T121, K122, A123, K124, L125, V126, V127, N128, K129, F130, N131, E132, A133, L134, P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392, and K393; Preferably, the unnatural amino acid comprises one of 4-azido L-phenylalanine (PAZF), 4-azido L-phenylalanine (PAZF-HCl), 4-acetyl L-phenylalanine, 3-acetyl L-phenylalanine, 4-acetoacetyl L-phenylalanine, O-allyl L-tyrosine, 3-(phenylselenyl) L-alanine, O2-propargyl 1 L-tyrosine, 4-(dihydroxyboryl) L-phenylalanine, 4-[(ethylsulfanyl)carbonyl] L-phenylalanine.

23. A sequencing adaptor complex, characterized in that, comprising: a first strand, a second strand, and the helicase variant of any one of claims 17-22; wherein the first strand comprises, in order, a guide sequence, a helicase binding domain, and a first complementary sequence, the helicase variant binds to the helicase binding domain; the second strand comprises a second complementary sequence and a linker sequence, the first complementary sequence at least partially base pairs with the second complementary sequence; a 3' overhang of the first complementary sequence contains a free thymine deoxynucleotide; and the linker sequence is used to attach a sequencing adaptor complex to a membrane; Preferably, the sequence of the first strand is set forth in SEQ ID NO: 21, and the sequence of the second strand is set forth in SEQ ID NO:

22.

24. A nucleic acid molecule, wherein, The nucleic acid molecule encodes the helicase variant of any one of claims 17-22.

25. An expression vector comprising the nucleic acid of claim 24. comprising the nucleic acid molecule of claim 24.

26. A recombinant cell, wherein, carrying the nucleic acid molecule of claim 24 or the expression vector of claim 25.

27. A method of making a helicase variant, comprising, comprising: culturing the recombinant cell of claim 26 under conditions suitable for protein expression, so as to obtain the helicase variant. comprising: culturing the recombinant cell of claim 26 under conditions suitable for protein expression, so as to obtain the helicase variant.

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