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 for controlling polynucleotide movement and helicase variants Technical Field

[0001] This application relates to the field of gene sequencing, specifically, to a method for controlling the movement of polynucleotides and helicase variants. More specifically, this invention relates to a nanopore sequencing method, sequencing adapter complexes, nucleic acid molecules, expression vectors, recombinant cells, and a method for preparing helicase variants. Background Technology

[0002] Nanopore sequencing is a single-molecule sequencing technology that uses electrical signals within nanopores to identify individual nucleotide molecules, enabling rapid sequencing of DNA or RNA sequences. It is widely used in genomics, transcriptomics, biological research, and clinical diagnostics.

[0003] Nanopore sequencing technology operates on the principle of electrical signal changes. A nanopore (protein pore or solid pore) inserted into a membrane acts as a signal sensor, separating two electrolyte chambers. When a voltage is applied between the two chambers, a stable current flows through the nanopore. When the nucleic acid to be tested enters the nanopore, it impedes the flow of ions, causing fluctuations in the current signal. Nucleotides with different bases have different effects on the current. Therefore, by real-time detection of the current fluctuation signal in the nanopore and using machine learning to analyze and decode the current signal, the sequence information of the nucleic acid molecule to be tested can be sequenced in real time.

[0004] A key step in nanopore sequencing is ensuring that the target nucleic acid can pass through the nanopore uniformly and stably at single-base resolution. Current techniques employ an adapter-motor protein (helicase) complex method for nanopore sequencing. In this method, the spacer in the adapter-motor protein complex prevents the motor protein from moving along single-stranded polynucleotides and unwinding double-stranded polynucleotides in the buffer solution; once the sequencing library is captured by transmembrane proteins, the spacer's blocking effect is neutralized under the influence of an electric field. The spacers used in current techniques consist of multiple modified nucleotides, resulting in high cost, and the sequencing speed is also limited by the unwinding rate of the motor protein.

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

[0006] Summary of the Invention

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

[0008] The inventors discovered that using spacers to block helicase in existing nanopore sequencing methods is costly, and the sequencing speed is limited by the helicase's own unwinding speed. To overcome this problem, the inventors constructed a helicase variant based on existing helicases, transforming the helicase's mechanism from actively unwinding polynucleotides to passively unwinding them under the control of an electric field. In this process, the use of spacers to block the helicase is eliminated, reducing costs; simultaneously, the speed at which the helicase moves the polynucleotides is controlled by an electric field, freeing the polynucleotide movement speed from the limitation imposed by the helicase.

[0009] Based on this, in a first aspect, the present invention proposes a method for controlling the movement of polynucleotides. According to an embodiment of the present invention, the method includes: a polynucleotide linked to a helicase variant moving through a transmembrane pore under the control of an electric field, wherein the helicase variant does not possess active unwinding activity or translocation activity. According to the method of the embodiment of the present invention, the helicase variant changes from actively unwinding the polynucleotide to passively unwinding the polynucleotide under the control of an electric field, eliminating the need for a spacer to block the helicase and reducing costs; simultaneously, it changes to using an electric field to control the speed at which the helicase moves the polynucleotide, so that the movement speed of the polynucleotide is no longer limited by the helicase.

[0010] In a second aspect, the present invention provides a method for nanopore sequencing. According to embodiments of the present invention, the method includes using the method of the first aspect of the present invention to move a polynucleotide to be sequenced through a nanopore under the control of an electric field.

[0011] The sequence of the polynucleotide to be tested is determined based on the current signal generated when the polynucleotide passes through the nanopore. According to the method of this embodiment, the helicase variant changes from actively unwinding and moving the polynucleotide to passively unwinding the polynucleotide under the control of an electric field, eliminating the need for a spacer to block the helicase and reducing costs; simultaneously, it changes to using an electric field to control the speed at which the helicase moves the polynucleotide, so that the movement speed of the polynucleotide is no longer limited by the helicase.

[0012] In a third aspect, the present invention provides a helicase variant. According to embodiments of the invention, the helicase variant, compared to the amino acid sequence shown in SEQ ID NO:2, has at least one amino acid mutation in the region related to helicase activity, and the helicase mutant does not possess active helicase activity or translocation activity. The helicase variant according to embodiments of the present invention does not possess active helicase activity or translocation activity.

[0013] In a fourth aspect, the present invention provides a sequencing adapter complex. According to an embodiment of the invention, it comprises: a first strand, a second strand, and a helicase variant as described in the third aspect of the invention; wherein the first strand sequentially comprises a guide sequence, a helicase-binding domain, and a first complementary sequence, and the helicase variant binds to the helicase-binding domain;

[0014] The second strand includes a second complementary sequence and a linker sequence, wherein the first complementary sequence is at least partially complementary to the second complementary sequence; the 3' free end of the first complementary sequence contains a free thymine deoxynucleotide; and the linker sequence is used to link the sequencing adapter complex to the membrane.

[0015] In a fifth aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes a helicase variant described in the third aspect of the invention.

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

[0017] In a seventh aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the fifth aspect of the present invention or the expression vector described in the sixth aspect of the present invention.

[0018] In an eighth aspect, the present invention provides a method for preparing a helicase variant. According to an embodiment of the invention, the method includes: culturing recombinant cells according to the seventh aspect of the invention under conditions suitable for protein expression to obtain the helicase variant. Attached Figure Description

[0019] Figure 1 shows the purification diagram of BCH105-Mut1 in Example 1 and the SDS-PAGE gel electrophoresis results.

[0020] Figure 2 shows the enzyme activity results of BCH105-Mut1 and its mutant ATPase in Example 2.

[0021] Figure 3 shows the dsDNA melting activity results of BCH105-Mut1 and its mutant in Example 3.

[0022] Figure 4 is a schematic diagram of the sequencing adapter structure in Example 4.

[0023] Figure 5 is a schematic diagram of the sequencing adapter complex in Example 4.

[0024] Figure 6 is a non-denaturing gel electrophoresis diagram of the BCH105-Mut1 sequencing adapter complex in Example 4.

[0025] Figure 7 is a non-denaturing gel electrophoresis diagram of the BCH105-Mut1_1 sequencing adapter complex in Example 4.

[0026] Figure 8 is a non-denaturing gel electrophoresis image of the BCH105-Mut1_2 sequencing adapter complex in Example 4.

[0027] Figure 9 is a non-denaturing gel electrophoresis image of the BCH105-Mut1_3 sequencing adapter complex in Example 4.

[0028] Figure 10 is a non-denaturing gel electrophoresis diagram of the BCH105-Mut1_4 sequencing adapter complex in Example 4.

[0029] Figure 11 is a non-denaturing gel electrophoresis diagram of the BCH105-Mut1_5 sequencing adapter complex in Example 4.

[0030] Figure 12 is a non-denaturing gel electrophoresis image of the BCH105-Mut1_6 sequencing adapter complex in Example 4.

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

[0032] Figure 14 shows the sequencing signal of BCH105-Mut1 in Example 5.

[0033] Figure 15 shows the sequencing signal of BCH105-Mut1_1 in Example 5.

[0034] Figure 16 shows the sequencing signal of BCH105-Mut1_2 in Example 5.

[0035] Figure 17 shows the sequencing signal of BCH105-Mut1_3 in Example 5.

[0036] Figure 18 shows the sequencing signal of BCH105-Mut1_4 in Example 5.

[0037] Figure 19 shows the sequencing signal of BCH105-Mut1_5 in Example 5.

[0038] Figure 20 shows the sequencing signal of BCH105-Mut1_6 in Example 5.

[0039] In Figures 14-20, the horizontal axis represents the time it takes for DNA to completely exit the nanopore from its entry point, and the vertical axis represents the current value corresponding to the DNA passing through the nanopore at a voltage of 180mV. Detailed Implementation

[0040] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0041] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0042] In this document, the terms “containing,” “comprising,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0043] In this article, the term "nucleic acid molecule" refers to biomolecules composed of nucleotides, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These nucleic acid molecules carry genetic information and are responsible for the transmission and expression of this information within an organism.

[0044] In this paper, the term "expression vector" refers to a medium used to insert a target gene (or nucleic acid sequence) into a host cell and induce protein expression of that gene within the host cell. An expression vector typically 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 article, the term "recombinant cell" refers to a cell that has undergone genetic engineering or modification. These cells have undergone genetic recombination, inserting foreign or mutated genes to give them new functions, express specific proteins, or produce desired products.

[0046] In this article, the term "recombinant strain" refers to a bacterial strain that has undergone genetic engineering or modification. These strains have undergone gene recombination, inserting foreign or mutated genes to give them new functions, express specific proteins, or produce desired products.

[0047] In this paper, the term "active unwinding activity" refers to the ability of a helicase to actively lower the activation energy that opens the double helix structure of DNA or RNA and convert it into a single-stranded form. This activity typically involves the enzymatic mechanism of helicases, through the interaction and energy expenditure between the enzyme and the nucleic acid substrate (polynucleotides in this paper), gradually unwinding the double helix structure of the nucleic acid substrate.

[0048] In this paper, the term "translocation activity" refers to the relative movement of a helicase over a nucleic acid substrate (polynucleotides in this paper) without necessarily involving the direct unwinding of the double helix. This activity may involve the binding of the helicase to the nucleic acid substrate, the formation of a complex, and the allosteric changes of the enzyme itself, followed by uniform movement over the nucleic acid substrate.

[0049] This invention proposes a method for controlling the movement of polynucleotides, a method for nanopore sequencing, helicase variants, sequencing adapter complexes, nucleic acid molecules, expression vectors, recombinant cells, and a method for preparing helicase variants.

[0050] Methods for controlling polynucleotide migration

[0051] In a first aspect, the present invention provides a method for controlling the movement of polynucleotides. According to an embodiment of the invention, the method includes: moving a polynucleotide linked to a helicase variant through a transmembrane pore under the control of an electric field, wherein the helicase variant does not possess active unwinding activity or translocation activity. According to the method of the embodiment of the invention, the helicase variant changes the active unwinding movement of the polynucleotide to passive unwinding under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and ensuring that the movement speed of the polynucleotide is no longer limited by the helicase.

[0052] According to an embodiment of the invention, the helicase variant is attached to a polynucleotide by binding to a linker, which may or may not have a spacer.

[0053] According to an embodiment of the present invention, the polynucleotide is a double-stranded polynucleotide, and the linker includes a first strand and a second strand, wherein at least a portion of the sequences of the first strand and the second strand are complementary;

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

[0055] According to embodiments of the present invention, the double-stranded polynucleotide is linked to a membrane via the adapter, comprising: the second strand of the adapter being directly linked to the membrane; or, an oligonucleotide chain complementary to at least a portion of the sequence of the second strand being linked to the membrane, the double-stranded polynucleotide being linked to the membrane through the complementarity of the second strand and the oligonucleotide chain. It should be noted that, in this invention, "oligonucleotide chain" refers to a single-stranded DNA or RNA molecule composed of a small number of nucleotides. It typically contains several to tens of nucleotides. According to embodiments of the present invention, the oligonucleotide chain is linked to the membrane and has a sequence complementary to at least a portion of the sequence of the second strand. This means that the base sequence of the oligonucleotide chain and certain base sequences in the second strand can bind to each other through base complementary pairing. In this way, the double-stranded polynucleotide can be linked to the oligonucleotide chain through the second strand. This linkage method utilizes the property of base complementary pairing, enabling the double-stranded polynucleotide to bind tightly to the oligonucleotide chain on the membrane. Such a design can be used in nanopore sequencing or other related applications to guide the double-stranded polynucleotide to a transmembrane pore or membrane, and ensure its stable passage through the nanopore for sequencing or other processing. The binding of the complementary sequence of the oligonucleotide chain to the complementary sequence of the second chain provides linkage stability and directionality, thereby enhancing the interaction between the double-stranded polynucleotide and the membrane.

[0056] According to embodiments of the present invention, the second chain is connected to the membrane via cholesterol modification; or, the oligonucleotide chain is connected to the membrane via cholesterol modification. According to embodiments of the present invention, the second chain or oligonucleotide chain can be connected to the membrane via cholesterol modification. This means that cholesterol molecules are introduced into the second chain or oligonucleotide chain, thereby achieving binding to the membrane. Cholesterol is a common lipid molecule in cell membranes and possesses amphoteric properties. Its hydrophobic portion can interact with the hydrophobic regions of the membrane, while its hydrophilic portion interacts with the hydrophilic regions of the membrane. This property makes cholesterol play an important role in regulating the fluidity and stability of cell membranes. By introducing cholesterol modification into the second chain or oligonucleotide chain, 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 chain or oligonucleotide chain can interact with the membrane via covalent or non-covalent bonds. Covalent bonding typically involves introducing cholesterol groups into the molecular structure, while non-covalent bonding can be achieved through hydrophobic interactions, electrostatic interactions, or hydrogen bonds. Therefore, according to embodiments of the present invention, cholesterol modification can be used to link a second-stranded or oligonucleotide chain to a membrane to enhance the interaction and stability of the double-stranded polynucleotide with the membrane. This linkage method facilitates the accurate guidance and processing of double-stranded polynucleotides in nanopore sequencing or other related applications.

[0057] According to an embodiment of the present invention, the first chain sequentially includes a guide sequence, a helicase-binding domain, and a first complementary sequence, wherein the helicase variant binds to the helicase-binding domain; the second chain includes a second complementary sequence and a linker sequence, wherein the first complementary sequence is at least partially complementary to the second complementary sequence; the 3' free end of the first complementary sequence contains a free thymine deoxynucleotide; and the linker sequence is used to link the sequencing adapter complex to a membrane.

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

[0059] According to embodiments of the present invention, the transmembrane pores include protein nanopores or solid nanopores.

[0060] It should be noted that, in this invention, "nanopore" refers to a tiny pore or channel with a diameter only on the nanometer scale. This channel passes through a thin film and is typically composed of synthetic materials (such as silica gel or silicon nitride) or natural proteins (such as protein nanopores). When a double-stranded DNA molecule passes through a transmembrane nanopore, a voltage is applied across the nanopore, creating an ionic current. Nucleotides of the DNA molecule pass through the nanopore one by one, causing minute changes in current during their passage. These changes in current can be detected and recorded, and used to infer the DNA sequence. The diameter of the transmembrane nanopore is typically comparable to the size of the DNA molecule to allow it to pass through. Furthermore, the surface of the transmembrane nanopore usually possesses charge properties to attract the passage of DNA molecules.

[0061] According to embodiments of the present invention, the membrane is a biological membrane or a biomimetic membrane. In this invention, a "biological membrane" refers to a thin-film structure existing within a living organism or on its tissues, composed of biological macromolecules (such as lipids and proteins). These membranes play important biological functions inside and outside cells, such as cell membranes and organelle membranes. In this invention, a "biomimetic membrane" refers to an artificially synthesized thin film with a structure and properties similar to a biological membrane. These membranes can be prepared artificially, for example, 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 embodiments of the present invention, the membrane can be a biological membrane, i.e., a thin-film structure of biological origin; or it can be a biomimetic membrane, having a structure and properties similar to a biological membrane.

[0062] According to embodiments of the present invention, the membrane is an amphiphilic bilayer. Here, "amphiphilic bilayer" in the present invention refers to a bilayer structure composed of two substances with different affinities. In biological and biomimetic membranes, the most common amphiphilic bilayer is the 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, serving as a supporting structure for transmembrane pores and providing a suitable environment for guiding DNA molecules through.

[0063] According to an embodiment of the present invention, the membrane is a phospholipid membrane.

[0064] According to embodiments of the present invention, the helicase variant is obtained by mutating at least one amino acid in a region of the helicase associated with helicase activity. Helicases are enzymes that unwind the double helix structure of DNA or RNA, converting it into a single strand. The helicase activity-related region is crucial for the helicase's helicase activity, containing key amino acid residues that influence this activity. In this invention, by mutating at least one amino acid in the helicase activity-related region, these mutations cause the helicase to lose its active helicase activity or translocation activity. According to the method of embodiments of the present invention, the helicase variant transforms the active helicase movement of polynucleotides into passive helicase movement under the control of an electric field, eliminating the need for spacers to block the helicase, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0065] According to embodiments of the present invention, the amino acid sequence of the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology with the amino acid sequence shown in SEQ ID NO:2. According to the method of the present invention, the helicase variant changes from actively unwinding and moving polynucleotides to passively unwinding polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0066] According to embodiments of the present invention, the amino acid sequence of the helicase, compared with the amino acid sequence shown in SEQ ID NO:2, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology in the amino acid sequences other than V119, C133, C164, K199, C292, C323, C347, and L369. According to the method of embodiments of the present invention, the helicase variant changes from actively unwinding and moving polynucleotides to passively unwinding polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0067] According to embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the helicase variant includes a mutation site of at least one of the following first-position groups:

[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 embodiments of the present invention, the helicase variant changes from actively unwinding and moving polynucleotides to passively unwinding polynucleotides under the control of an electric field. This eliminates the need for a spacer to block the helicase, reduces costs, and ensures that the movement speed of polynucleotides is no longer limited by the helicase.

[0069] According to embodiments of the present invention, 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 present invention, the helicase variant changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and further ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0070] According to embodiments of the present invention, the helicase variant further comprises at least one of the following mutations: F110A, L125M, V179W, N314A, L106A, and M147I. According to the method of the present invention, the helicase variant changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase and further ensuring that the movement speed of the polynucleotides is no longer limited by the helicase.

[0071] According to embodiments of the present invention, the helicase variant further includes the following mutation: F110A; or, the helicase variant includes the following mutation: L125M; or, the helicase variant includes the following mutation: V179W; or, the helicase variant includes the following mutation: N314A; or, the helicase variant includes the following mutations: L106A, L125M; or, the helicase variant includes the following mutations: M147I, V179W. According to the method of the present invention, the helicase variant changes from actively unwinding and moving polynucleotides to passively unwinding polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and further ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0072] According to embodiments of the present invention, the amino acid sequence of the helicase mutant, compared with the amino acid sequence shown in SEQ ID NO:2, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology in the amino acid sequences other than at least one of the sites in the first dot group (V119, C133, C164, K199, C292, C323, C347, L369, and the first dot group); or the helicase variant has the amino acid sequence 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. According to the method of the present invention, the helicase variant changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and further ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

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

[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, P 355, 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 present invention, the helicase variant changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase, reducing costs, and further ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0075] According to embodiments of the present invention, the non-natural 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-tyrosine, 3-(phenylselenoyl)-L-alanine, O-propynyl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, and 4-[(ethylthioalkyl)carbonyl]-L-phenylalanine.

[0076] According to an embodiment of the present invention, the pin structure domain is connected to the tower structure domain.

[0077] According to an embodiment of the present invention, the connection is formed by introducing a crosslinking agent, the crosslinking agent comprising one of maleimide, active ester, succinimide, azide, alkyne, phosgene, sulfonyl chloride, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine, and photosensitizer.

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

[0079] According to an embodiment of the present invention, the covalent linking is carried out by at least one of a cross-linking agent, protein fusion, polypeptide molecule, or small molecule.

[0080] According to embodiments of the present invention, the method further includes introducing a crosslinking agent, said crosslinking agent comprising at least one of maleimide, active ester, succinimide, azide, alkyne, difluorocycloalkyne, phosphine, haloacetyl, phosgene, sulfonyl chloride, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine, and photosensitizing agent.

[0081] According to embodiments of the present invention, 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, and L134 are derived from the pin domain of the helicase variant.

[0082] According to embodiments of the present invention, 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 are derived from the tower domain of the helicase variant.

[0083] Nanopore sequencing methods

[0084] In a second aspect, the present invention provides a method for nanopore sequencing. According to embodiments of the invention, the method includes using the method of the first aspect to move a target polynucleotide linked to a helicase variant through a nanopore under the control of an electric field; and determining the sequence of the target polynucleotide based on the current signal generated when the target polynucleotide passes through the nanopore. In the method of the embodiments of the invention, the helicase variant changes from actively unwinding the polynucleotide to passively unwinding it under the control of an electric field. In this process, there is no need to use a spacer to block the helicase, reducing the cost of nanopore sequencing; simultaneously, the method changes to using an electric field to control the speed of the helicase's movement of the polynucleotide, so that the movement speed of the polynucleotide is no longer limited by the helicase.

[0085] helicase variants

[0086] In a third aspect, the present invention provides a helicase variant. According to embodiments of the invention, the helicase variant, compared to the amino acid sequence shown in SEQ ID NO:2, has at least one amino acid mutation in the region related to helicase activity, and the helicase mutant does not possess active helicase activity or translocation activity. The helicase variant according to embodiments of the present invention does not possess active helicase activity or translocation activity.

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

[0088] According to embodiments of the present invention, the helicase variant includes a mutation site of at least one of the following first point 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 invention, the helicase variants change from active unwinding of polynucleotides to passive unwinding under the control of an electric field. This eliminates the need for spacers to block the helicase variants, reduces costs, and ensures that the movement speed of polynucleotides is no longer limited by the helicase.

[0090] According to embodiments of the present invention, the helicase variant includes at least one of the following mutation sites: F110, L125, V179, N314, L106, and M147. The helicase variant according to embodiments of the present invention changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for spacers to block the helicase variant, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0091] According to embodiments of the present invention, the helicase variant further includes at least one of the following mutations: F110A, L125M, V179W, N314A, L106A, and M147I. The helicase variant according to embodiments of the present invention changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for spacers to block the helicase variant, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0092] According to embodiments of the present invention, the helicase variant further includes the following mutation: F110A; or, the helicase variant includes the following mutation: L125M; or, the helicase variant includes the following mutation: V179W; or, the helicase variant includes the following mutation: N314A; or, the helicase variant includes the following mutations: L106A, L125M; or, the helicase variant includes the following mutations: M147I, V179W. The helicase variant according to embodiments of the present invention changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase variant, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0093] According to embodiments of the present invention, the amino acid sequence of the helicase mutant, compared with the amino acid sequence shown in SEQ ID NO:2, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology in the sequences of amino acids other than at least one site in the first dot group and V119, C133, C164, K199, C292, C323, C347, and L369; or the helicase variant has the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14. According to embodiments of the present invention, the helicase variant changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase variant, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

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

[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, P3 55, 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 embodiments of the present invention, the helicase variant changes the active unwinding and movement of polynucleotides to passive unwinding of polynucleotides under the control of an electric field, eliminating the need for a spacer to block the helicase variant, reducing costs, and ensuring that the movement speed of polynucleotides is no longer limited by the helicase.

[0096] According to embodiments of the present invention, the non-natural amino acid includes one of the following: 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-tyrosine, 3-(phenylselenoyl)-L-alanine, O-propynyl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, and 4-[(ethylthioalkyl)carbonyl]-L-phenylalanine.

[0097] According to an embodiment of the present invention, the pin structure domain is connected to the tower structure domain.

[0098] According to an embodiment of the present invention, the connection is formed by introducing a crosslinking agent, the crosslinking agent comprising one of maleimide, active ester, succinimide, azide, alkyne, phosgene, sulfonyl chloride, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine, and photosensitizer.

[0099] According to an embodiment of the present invention, the connection includes at least one of a covalent connection, a covalent and non-covalent combination connection, and a non-covalent connection.

[0100] According to an embodiment of the present invention, the covalent linking is carried out by at least one of a cross-linking agent, protein fusion, polypeptide molecule, or small molecule.

[0101] According to embodiments of the present invention, the method further includes introducing a crosslinking agent, said crosslinking agent comprising at least one of maleimide, active ester, succinimide, azide, alkyne, difluorocycloalkyne, phosphine, haloacetyl, phosgene, sulfonyl chloride, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine, and photosensitizing agent.

[0102] According to embodiments of the present invention, 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, and L134 are derived from the pin domain of the helicase variant.

[0103] According to embodiments of the present invention, 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 are derived from the tower domain of the helicase variant.

[0104] Sequencing adapter complex

[0105] In a fourth aspect, the present invention provides a sequencing adapter complex. According to an embodiment of the invention, the sequencing adapter complex comprises: a first strand and a second strand, and a helicase variant as described in the third aspect of the invention; wherein the first strand sequentially comprises 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 linker sequence, the first complementary sequence being at least partially complementary to the second complementary sequence; the 3' free end of the first complementary sequence contains a free thymine deoxynucleotide; the linker sequence is used to link the sequencing adapter complex to a membrane.

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

[0107] In this invention, "sequencing adapter complex" and "adapter complex" have the same meaning.

[0108] It should be noted that the 3' end of the first complementary sequence contains a free thymine deoxynucleotide (dT) linked to a polynucleotide via a TA linker, which is then used for subsequent nanopore sequencing.

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

[0110] According to an embodiment of the present invention, the oligonucleotide chain is further included, wherein the linker sequence is complementary to at least a portion of the sequence of the oligonucleotide chain, and the 5' end of the oligonucleotide chain is linked to a cholesterol molecule.

[0111] According to an embodiment of the present invention, the second chain is linked to a cholesterol molecule.

[0112] Nucleic acid molecules

[0113] In a fifth aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes a helicase variant described in the third aspect of the invention.

[0114] expression carrier

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

[0116] Recombinant cells

[0117] In a seventh aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the fifth aspect of the present invention or the expression vector described in the sixth aspect of the present invention. According to embodiments of the present invention, the recombinant cell includes, but is not limited to, recombinant bacterial strains, recombinant single-celled organisms, and recombinant multicellular organisms.

[0118] According to embodiments of the present invention, the recombinant strain includes, but is not limited to, Escherichia coli.

[0119] According to embodiments of the present invention, the recombinant single-celled organism includes, but is not limited to, yeast.

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

[0121] Methods for preparing helicase variants

[0122] In an eighth aspect, the present invention provides a method for preparing a helicase variant. According to an embodiment of the invention, the method includes: culturing recombinant cells according to the seventh aspect of the invention under conditions suitable for protein expression to obtain the helicase variant.

[0123] The sequence list of the present invention is shown below:

[0124] sequence list

[0125] It should be noted that the X in SEQ ID NO:17 refers to iSPC3.

[0126] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0127] Example 1: Expression of BCH105 helicase variant

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

[0129] (1) Construction: The sequences BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 shown in Table 1 were ligated into the PET.28a(+) plasmid using the double restriction sites Nde1 and Xho1. The BCH105-Mut1 helicase expressed by this plasmid has a 6×His tag and a thrombin restriction site at its N-terminus.

[0130] Table 1

[0131] (2) Purification: Recombinant plasmids containing BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 were transformed into *E. coli* expression bacteria ArcticExpress (DE3) or its derivatives. Single colonies were picked and inoculated into 20 mL of LB medium containing kanamycin resistance, and cultured overnight at 37°C with shaking. Then, they were transferred into 2 L of LB medium containing kanamycin resistance, cultured at 37°C with shaking until OD600 = 0.6-0.8, cooled to 10°C, and IPTG was added to a final concentration of 500 μM to induce expression for 40-46 hours, and the bacterial cells were collected.

[0132] At 4°C, bacterial cells expressing helicase variants BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 were collected by centrifuging at 5000 rpm. The bacterial cells were then resuspended in Buffer A, disrupted using a cell disruptor, and the supernatant was collected after high-speed centrifugation. The supernatant was mixed with Ni-NTA packing material pre-equilibrated with Buffer A (formulation: 20mM Tris-HCl pH 8.0, 200mM NaCl, 20mM Imidazole) and incubated at 4°C for 1 hour. The packing material was then washed with 5-10 column volumes of Buffer B (formulation: 20mM Tris-HCl pH 8.0, 100mM NaCl, 20mM Imidazole) until no extraneous proteins were eluted. Next, 10 mL of Buffer B and 50 μL of thrombin were added to the packing material and incubated overnight at 4°C for 14-16 hours. Afterward, the flow-through of the overnight digested sample was collected, and 10 mL of Buffer B was added again for washing. The flow-through was then collected. The eluted target protein was diluted to a salt concentration of 50 mM using Buffer C (formulation: 20 mM Tris-HCl pH 8.0) and loaded onto Hitrap Q HP (Cytiva). Then, a gradient elution was performed using Buffer D (formulation: 20 mM Tris-HCl pH 8.0, 50 mM NaCl) and Buffer E (formulation: 20 mM Tris-HCl pH 8.0, 1000 mM NaCl). The eluted target protein was then concentrated to 1 mL and further purified using a Superdex 200 increase 10 / 300 GL molecular sieve (Cytiva) with Buffer F (formulation: 20 mM Tris-HCl pH 8.0, 80 mM NaCl). After identifying the elution peaks of the target proteins (BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 helicase variants) based on the elution curves of the molecular sieve and SDS-PAGE, the peaks were collected and concentrated. The final target proteins were then stored at -80°C.

[0133] This embodiment exemplifies the purification results of the BCH105-Mut1 helicase variant, as shown in Figure 1. The final obtained BCH105-Mut1 helicase variant exhibits good protein purity and high expression levels. Other helicase variants not shown, such as BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6, showed similar purification results to the BCH105-Mut1 helicase variant, exhibiting good protein purity and high expression levels.

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

[0135] This embodiment detected the ATPase activity of helicase variants BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6, respectively. The specific experimental methods are as follows:

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

[0137] The sequences SEQ ID NO:16 and SEQ ID NO:17 (from Liuhe Huada) were synthesized. SEQ ID NO:16 and SEQ ID NO:17 were annealed to form 5' suspended 20 T ovDNA-1. The annealing process involved incubation at 95°C for 5 minutes, followed by cooling to 25°C at a rate of 0.1°C / s, and then incubation for another 30 minutes. The annealing formulation is shown in Table 2. 100 μM of SEQ ID NO:17 was diluted to 10 μM with TE buffer (pH = 8) to obtain ssDNA.

[0138] Table 2: OvDNA-1 Annealing Formulation

[0139] (2) Perform ATP hydrolysis reactions separately: Add the corresponding reagents according to the reaction system in Table 2, incubate at 30℃ for 30 min to test the ATP hydrolysis reaction, and inactivate at 80℃ for 5 min. Among them, ①② are experimental groups, and ③④⑤⑥ are corresponding control groups, with 3 replicates for each group. The formula of the high-salt reaction buffer (2×) in Table 3 is as follows: 20mM HEPES (pH 8.0), 4mM ATP, 4mM MgCl2, 300mM KCl. Dilute the protein: Dilute the BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 helicases to 10μM with 1×PBS.

[0140] Table 3: ATP hydrolysis reaction system

[0141] (3) Detection of residual ATP in the reaction: Using the ATP detection kit (Beyotime, S0026B), the concentration of residual ATP in the reaction was determined according to the manufacturer's instructions.

[0142] This embodiment exemplifies the results of BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, and BCH105-Mut1_4, as shown in Figure 2. BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, and BCH105-Mut1_4 exhibit equivalent ATP hydrolysis activity.

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

[0144] This embodiment tested the dsDNA unwinding activity of helicase variants BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6, respectively. The specific experimental methods are as follows:

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

[0146] The sequences SEQ ID NO:18 and SEQ ID NO:19 (BGI Genomics) were synthesized. The sequences SEQ ID NO:18 and SEQ ID NO:19 were annealed to form 5' suspended 20 T ovDNA-2. The annealing process was as follows: incubation at 95°C for 5 minutes, cooling to 25°C at a rate of 0.1°C / s, and incubation for 30 minutes. The annealing formula for forming ovDNA-2 is shown in Table 4.

[0147] Table 4: OvDNA-2 Annealing Formulation

[0148] (2) Preparation of the chain-breaking reaction:

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

[0150] Take 1 μL of 10 μM SEQ ID NO:19, 2 μL of 100 μM SEQ ID NO:20 (20-fold competitive DNA), and 2 μL of 100 mM ATP and add them to 195 μL of reaction buffer (formulation: 100 mM HEPES (pH=8.0), 1 mg / mL BSA, 10 mM MgCl2, 150 mM KCl) to prepare a positive control solution.

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

[0152] Subsequently, the corresponding reagents prepared above were added according to Table 5, with a representing the experimental group, b representing the negative control group, and c representing the positive control group. The kinetic changes in fluorescence intensity within 30 minutes of reaction were detected using an ELISA reader at 30℃, with three replicates per group. Within the acceptable error range and instrument fluctuation limits, the experimental results were statistically analyzed by calculating the ratio of fluorescence values ​​in the experimental group to those in the positive control group, and the ratio of fluorescence values ​​in the negative control group to those in the positive control group.

[0153] Table 5: Chain Desolution Reactions

[0154] This embodiment exemplifies the results of BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, and BCH105-Mut1_4, as shown in Figure 3. The negative control group in each experiment remained unchanged throughout the measurement process, while the fluorescence value of the experimental groups gradually increased with the increase of reaction time. Among them, BCH105-Mut1 had the highest activity, while the enzyme activities of BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, and BCH105-Mut1_4 helicase variants decreased to varying degrees.

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

[0156] In this embodiment, BCH105-Mut1, BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 helicase variant-linker complexes were prepared respectively. The specific experimental methods are as follows:

[0157] (1) Preparation of adapters: 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 adapters. A schematic diagram of the adapters is shown in Figure 5. The adapters do not have spacers that block the advance of helicase variants. The addition of single-stranded complementary DNA with cholesterol is to prevent helicase variants from binding to the second strand and affecting sequencing.

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

[0159] Table 6: Preparation of helicase variants and adaptor reaction systems

[0160] Add 11 μL of 400 μM bismaleimide ethane (BMOE) to the incubated helicase variant-adaptor complex, and mix thoroughly by pipetting 6-8 times. Incubate the 1.5 mL DNA LoBind Tube Microcentrifuge Tube in a metal bath at 28°C for 30 minutes. Add 0.22 μL of 1 M dithiothreitol (DTT) solution to the cross-linked helicase variant-adaptor complex, and mix thoroughly by pipetting 6-8 times. Incubate at room temperature for 15 minutes.

[0161] The cross-linked helicase variant-linker complex was mixed with Buffer H (formulation: 50mM Hepes-Na, pH 7.5, 1M NaCl, 4mM ATP, 20mM MgCl2) at a volume ratio of 1:1. The mixture was pipetted 6-8 times to ensure homogeneity. The homogeneous sample was then incubated in a preheated metal bath at 30°C for 1 hour.

[0162] Beforehand, remove the magnetic DNA Clean Beads into a DNA LoBind Tube Microcentrifuge Tube and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette. Add Buffer I (formulation: 50mM Hepes-Na, pH 7.5, 2.5M NaCl) to the magnetic beads and wash them twice, each time using the "magnetic bead swimming method" to ensure the magnetic beads move through the solution at least 5 times to thoroughly mix them. Each time you move the test tube, allow the magnetic beads to stand for 2-5 minutes until the liquid is completely clear. Finally, carefully aspirate and discard the supernatant using a pipette. Add Buffer J (formulation: 50mM Hepes-Na, pH 7.5, 2.5M NaCl, 24% PEG8000, 0.05% TWEEN-20) to the cleaned magnetic beads. Use the "magnetic bead swimming method" to allow the magnetic beads to swim in the solution more than 5 times to ensure thorough mixing. After standing, carefully aspirate and discard the supernatant using a pipette. Then, repeat the equilibration of the magnetic beads with Buffer J once more. Finally, add an appropriate volume of Buffer J to resuspend the magnetic beads.

[0163] The ATP-treated helicase variant-adaptor complex system was mixed separately with equilibrated magnetic DNA Clean Beads at a volume ratio of 1:2 and incubated at room temperature for 30 minutes using a rotary mixer. After incubation, the helicase variant-adaptor complexes were placed on a magnetic rack and allowed to stand until the liquid was completely clear (approximately 5-10 minutes). The supernatant was carefully aspirated and discarded using a pipette. The helicase variant-adaptor complexes were then removed from the magnetic rack and washed with Buffer K (formulation: 50mM Hepes-Na, pH 7.5, 2.5M NaCl, 20% PEG8000). The magnetic beads were swirled in the solution at least 5 times using the "magnetic bead swimming method" to ensure thorough mixing. Each time the test tube was moved, the magnetic beads were allowed to stand for 2-5 minutes until the liquid was completely clear. Finally, the supernatant was carefully aspirated and discarded. The washing process was repeated once with Buffer K, and the liquid in the tube was aspirated as dry as possible to remove any Buffer K adhering to the surface of the magnetic beads. Then, 40 μL of Buffer L (formulation: 50 mM Hepes-Na, pH 7.5, 20 mM NaCl) was added for elution, and the mixture was incubated at room temperature for 5 min. The incubated helicase variant-adaptor complexes were then placed on a magnetic rack and allowed to stand for 2-5 min until the liquid became clear. The supernatant was then transferred to a new 1.5 mL DNA LoBind Tube Microcentrifuge Tube tube, and the helicase variant-adaptor complexes were 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-adaptor complexes, which can be used for library construction for nanopore sequencing.

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

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

[0167] First, a patch-clamp amplifier was used to acquire the current signal. The Ag / AgCl electrode was immersed in sequencing buffer (formulation: 0.47M KCl, 25mM HEPES, 1mM EDTA, 30mM ATP, 25mM MgCl2, pH 8) with the electrode located in the cis and trans regions of the electrolytic cell, respectively. After a bilayer phospholipid membrane was formed at the micropores of the two chambers, nanoporous proteins were added. After a single nanoporous protein was inserted into the phospholipid membrane, the electrical measurement was obtained.

[0168] Subsequently, sequencing libraries containing helicase variant-adaptor complexes prepared above were added, 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 retardation current amplitude value.

[0169] As shown in Figure 14, the helicase variant BCH105-Mut1 showed a significant number of current changes, indicating that when the adapter does not contain a spacer, the helicase variant BCH105-Mut1 will untie the double-stranded DNA in solution, resulting in signals of single-stranded nucleic acid directly piercing or blocking the pore.

[0170] The results are shown in Figures 15-20. The helicase variants BCH105-Mut1_1, BCH105-Mut1_2, BCH105-Mut1_3, BCH105-Mut1_4, BCH105-Mut1_5, and BCH105-Mut1_6 showed fewer current changes and were all able to be sequenced normally. This indicates that weakening the enzyme activity can inhibit the helicase from unwinding double-stranded DNA in sequencing solutions containing ATP and, under the action of an electric field, control the DNA to pass through the nanopore at a uniform speed.

[0171] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method of controlling movement of a polynucleotide, comprising, include: Polynucleotides linked to helicase variants move through transmembrane pores under the control of an electric field. The helicase variant does not possess active unwinding or translocation activity.

2. The method of claim 1, wherein, The helicase variant is attached to a polynucleotide by binding to a linker, which may or may not have a spacer.

3. The method of claim 2, wherein, The polynucleotide is a double-stranded polynucleotide, and the linker includes a first strand and a second strand, wherein at least a portion of the sequences of the first strand and the second strand are complementary. The double-stranded polynucleotide is attached to the membrane via the adapter, and the transmembrane pore is located on the membrane.

4. The method of claim 3, wherein, The double-stranded polynucleotide is attached to the membrane via the linker, comprising: the second strand of the linker being directly attached to the membrane; or, the membrane being attached with an oligonucleotide chain that is at least partially complementary to the second strand, wherein the double-stranded polynucleotide is attached to the membrane through the complementarity of the second strand with the oligonucleotide chain. Preferably, the second chain is connected to the membrane via cholesterol modification; or, the oligonucleotide chain is connected to the membrane via cholesterol modification.

5. The method of claim 4, wherein, The first chain comprises, in sequence, a guide sequence, a helicase binding domain, and a first complementary sequence, wherein the helicase variant binds to the helicase binding domain; The second strand includes a second complementary sequence and a linker sequence, wherein the first complementary sequence is at least partially complementary to the second complementary sequence; the 3' free end of the first complementary sequence contains a free thymine deoxynucleotide; and the linker sequence is used to link the sequencing adapter complex to the membrane.

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

23.

7. The method of claim 3, wherein, The transmembrane pores include protein nanopores or solid nanopores; Optionally, the membrane is a biological membrane or a biomimetic membrane.

8. The method according to any one of claims 1 to 7, characterized in that, The helicase variant is obtained by mutating at least one amino acid in a region of the helicase that is associated with unwinding activity.

9. The method of claim 8, wherein, The amino acid sequence of the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology with the amino acid sequence shown in SEQ ID NO:

2.

10. The method of claim 9, wherein, The amino acid sequence of the helicase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology with the amino acid sequence shown in SEQ ID NO:2, except for V119, C133, C164, K199, C292, C323, C347, and L369.

11. The method of claim 10, wherein, Compared to the amino acid sequence shown in SEQ ID NO:2, the helicase variant includes a mutation site in at least one of the following first-position groups: 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 includes at least one of the following mutation sites: F110, L125, V179, N314, L106, and M147; Preferably, the helicase variant includes at least one of the following mutations: F110A, L125M, V179W, N314A, L106A, and M147I.

12. The method of claim 11, wherein, The helicase variants include the following mutations: F110A; or, The helicase variants include the following mutations: L125M; or, The helicase variants include the following mutations: V179W; or, The helicase variants include the following mutations: N314A; or, The helicase variants include the following mutations: L106A, L125M; or, The helicase variants include the following mutations: M147I and V179W.

13. The method of claim 11, wherein, The amino acid sequence of the helicase mutant, compared with the amino acid sequence shown in SEQ ID NO:2, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology in all other amino acid sequences except for at least one site in the first dot group 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.

14. The method of claim 11, wherein, Compared to the amino acid sequence shown in SEQ ID NO:2, the helicase variant further comprises mutating at least one site in the second site group to cysteine ​​or introducing a non-natural amino acid: 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, P 355, 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 non-natural 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-tyrosine, 3-(phenylselenoyl)-L-alanine, O-propynyl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, and 4-(ethylthioalkyl)carbonyl]-L-phenylalanine.

15. The method of claim 14, wherein, The pin structure domain is connected to the tower structure domain; Preferably, the connection is formed by introducing a crosslinking agent, which includes one of maleimide, active ester, succinimide, azide, alkyne, phosgene, sulfonyl chloride, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine, and photosensitizer.

16. A method of nanopore sequencing, characterized in that, include: Using the method described in any one of claims 1 to 15, a test polynucleotide linked to a helicase variant is moved through a nanopore under the control of an electric field. The sequence of the polynucleotide to be tested is determined based on the current signal generated when the polynucleotide passes through the nanopore.

17. A helicase variant, characterized in that, Compared with the amino acid sequence shown in SEQ ID NO:2, the helicase variant has at least one amino acid mutation in the region related to unwinding activity, and the helicase mutant does not have active unwinding activity or translocation activity.

18. The helicase variant of claim 17, wherein, The amino acid sequence of the helicase mutant, compared with the amino acid sequence shown in SEQ ID NO:2, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology in the amino acid sequences other than V119, C133, C164, K199, C292, C323, C347, and L369.

19. The helicase variant of claim 18, wherein, The helicase variant includes a mutation site of at least one of the following first point groups: 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 includes at least one of the following mutation sites: F110, L125, V179, N314, L106, and M147; Preferably, the helicase variant includes at least one of the following mutations: F110A, L125M, V179W, N314A, L106A, and M147I.

20. The helicase variant of claim 19, wherein, The helicase variants include the following mutations: F110A; or, The helicase variants include the following mutations: L125M; or, The helicase variants include the following mutations: V179W; or, The helicase variants include the following mutations: N314A; or, The helicase variants include the following mutations: L106A, L125M; or, The helicase variants include the following mutations: M147I and V179W.

21. The helicase variant of claim 19, wherein, The amino acid sequence of the helicase mutant, compared with the amino acid sequence shown in SEQ ID NO:2, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology in the sequence of amino acids other than at least one site in the first dot group 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, Compared to the amino acid sequence shown in SEQ ID NO:2, the helicase variant further comprises mutating at least one site in the second site group to cysteine ​​or introducing a non-natural amino acid: 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, P 355, 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 non-natural amino acid includes one of the following: 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-tyrosine, 3-(phenylselenoyl)-L-alanine, O-propynyl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, and 4-[(ethylthioalkyl)carbonyl]-L-phenylalanine.

23. A sequencing adaptor complex, characterized in that, Includes: the first strand, the second strand, and the helicase variant according to any one of claims 17-22; The first chain comprises, in sequence, a guide sequence, a helicase binding domain, and a first complementary sequence, wherein the helicase variant binds to the helicase binding domain; The second strand includes a second complementary sequence and a linker sequence, wherein the first complementary sequence is at least partially complementary to the second complementary sequence; the 3' free end of the first complementary sequence contains a free thymine deoxynucleotide; the linker sequence is used to link the sequencing adapter complex to a membrane; Preferably, the sequence of the first chain is as shown in SEQ ID NO:21, and the sequence of the second chain is as shown in SEQ ID NO:

22.

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

25. An expression vector comprising the nucleic acid of claim 24. It includes the nucleic acid molecule as described in 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, This includes: culturing the recombinant cells of claim 26 under conditions suitable for protein expression to obtain the helicase variant.

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