Helicase and use thereof

Abstract

Provided is a helicase, in which an ectopic bond is formed, wherein the helicase retains the ability to control the movement of a target analyte, and the ectopic bond does not include an ectopic bond where the formation thereof involves cysteine. Further provided are a polypeptide, a construct, a polynucleotide, a vector and a host cell; a method for preparing the helicase, polypeptide or construct; a method for controlling the movement of a target analyte; a method for characterizing a target analyte; a method for forming a sensor for characterizing a target analyte; a sensor for characterizing a target analyte; the use of the helicase or construct in controlling the movement of a target analyte through a pore; a kit or device for characterizing a target analyte; and a set of two or more helicases linking to a target analyte.

Description

Helicase and its applications Technical Field

[0001] The present invention belongs to the technical field of sequencing, and particularly relates to a modified helicase and an application thereof in characterizing a target analyte. Background Art

[0002] Nanopore sequencing technology, also known as the fourth-generation sequencing technology, uses a nanopore that can provide an ion current channel, allowing analytes such as single-stranded nucleic acid molecules to pass through the nanopore under electrophoresis. When analytes such as nucleic acids pass through the nanopore, the current of the nanopore will be reduced, and the gene sequencing technology reads the sequence information of the different signals generated in real time.

[0003] Helicases are a useful tool for controlling the movement of nucleic acid molecules during nanopore sequencing, but current helicases present several challenges that need to be addressed. For example, the helicase can become detached from the nucleic acid molecule during sequencing, causing it to be pulled through the pore at an uncontrolled speed and pattern, resulting in current signals from analytes such as individual nucleotides being too brief to be resolved.

[0004] To address this issue, the prior art CN105899678A discloses a modified helicase. Specifically, at least one cysteine / unnatural amino acid is introduced into the tower / pin / 1A domain of the helicase, thereby improving the helicase's ability to continuously control rate in nanopore sequencing.

[0005] Summary of the Invention

[0006] This application provides a new helicase modification method, which can also improve the continuous rate control ability of helicase in nanopore sequencing, and has better effects in terms of sequencing stability and accuracy.

[0007] In a first aspect, the present application provides a helicase,

[0008] (a) comprising a polynucleotide binding domain and two RecA-like domains, wherein two or more amino acid residues located on the surface of the helicase surrounding the polynucleotide binding domain are connected by an ectopic bond, so that the polynucleotide bound to the polynucleotide binding domain is surrounded by the structure formed after the connection; or

[0009] (b) comprising a polynucleotide binding domain and two RecA-like domains, wherein two or more amino acid residues located on the surface of the helicase surrounding the polynucleotide binding domain can be connected by an ectopic bond, so that the polynucleotide bound to the polynucleotide binding domain is surrounded by the connected structure, and at least one of the two or more amino acid residues is a mutant amino acid residue; or

[0010] (c) comprising a polynucleotide binding domain, wherein the polynucleotide binding domain comprises an opening in at least one conformational state, through which a polynucleotide can be debound by the helicase, wherein the helicase forms an ectopic bond between two or more amino acids at the opening (forming the ectopic bond can prevent the polynucleotide from being debound by the helicase through the opening), and wherein the helicase retains its ability to control the movement of the target polynucleotide; or

[0011] (d) comprising a polynucleotide binding domain, wherein the polynucleotide binding domain comprises an opening in at least one conformational state, through which a polynucleotide can be debound by the helicase, wherein the helicase is modified so that it can form an ectopic bond between two or more amino acids at the opening (formation of the ectopic bond can prevent the polynucleotide from being debound by the helicase through the opening), and wherein the helicase retains its ability to control the movement of the target polynucleotide.

[0012] In one embodiment, the ectopic bond does not include an ectopic bond formed with the participation of cysteine, and the ectopic bond includes an ectopic bond formed with the participation of any natural amino acid except cysteine.

[0013] The arbitrary natural amino acid except cysteine ​​is selected from the group consisting of Ile, Val, Leu, Phe, Met, Ala, Gly, Thr, Ser, Trp, Tyr, Pro, His, Glu, Gln, Asp, Asn, Lys, Arg,

[0014] Preferably, the ectopic bond comprises Lys-His, Lys-Ser, Lys-Thr, Lys-Tyr, Lys-Lys, Lys-Glu, Lys-Asp, Lys-Gln, Lys-Arg, Arg-Glu, Lys-Met, Arg-Asp, Arg-Arg, Tyr-Tyr, Tyr-Trp, Met-Met ectopic bond or a combination thereof;

[0015] and / or, the helicase consists of one or more monomers,

[0016] And / or, the two amino acids forming the isopeptide bond are located in the same domain or different domains.

[0017] In one embodiment, the helicase is derived from a member of the natural or modified helicase family of the following group: Dda helicase, Pifl-like helicase, Upfl-like helicase, UvrD / Rep helicase, Ski-like helicase, Rad3 / XPD helicase, NS3 / NPH-II helicase, DEAD helicase, DEAHi RHA helicase, RecG-like helicase, REcQ-like helicase, T1R-like helicase, Swi / Snf-like helicase, and Rig-I-like helicase, preferably Dda helicase, more preferably T4-Dda helicase;

[0018] And / or, the helicase is derived from a natural or modified member of the helicase family of the following groups: RecD helicase, Upfl helicase, PcrA helicase, Rep helicase, UvrD helicase, Hel308 helicase, Mtr4 helicase, XPD helicase, NS3 helicase, Mssl 16 helicase, Prp43 helicase, RecG helicase, RecQ helicase, T1R helicase, RapA helicase and Hef helicase.

[0019] In one embodiment, the use of modified helicases, such as those with amino acid mutations, is complex and expensive to produce. Using an unmodified natural helicase for covalent linkage, such as an ectopic linkage, is simpler and easier for those skilled in the art and can also improve its stability in controlling the movement of nucleic acids through a nanopore. In one embodiment, the unmodified natural helicase comprises a polynucleotide-binding domain and two RecA-like domains. Links exist between natural amino acid residues located in different RecA-like domains, which have the ability to control polynucleotide movement; and the presence of these linkages enhances the ability of the modified helicase to control polynucleotide movement compared to the absence of these linkages. In one embodiment, the linked natural amino acid residues interact with each other and / or are spaced less than 50 angstroms apart. The interactions include electrostatic interactions, preferably generated after the helicase binds to the target polynucleotide. In one embodiment, the linked natural amino acid residues are naturally occurring in the helicase, preferably including glutamic acid, lysine, tyrosine, arginine, aspartic acid, cysteine, methionine, or tryptophan. In one embodiment, the natural amino acid residues present in the connection are located around the polynucleotide binding domain. In one embodiment, the connection is a covalent connection, preferably, the connection is an ectopic bond connection. In one embodiment, the covalent connection includes a connection formed by a chemical cross-linking agent, or a connection formed by an enzyme. In one embodiment, the chemical cross-linking agent includes EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), EDC / NHS (N-hydroxysuccinimide), EDC / s-NHS (N-hydroxysuccinimide sulfonic acid sodium salt), chlorpyrifos oxon, HRP (horseradish peroxidase), laccase, or tyrosinase. In one embodiment, the helicase belongs to the SF1B (Superfamily 1B) subfamily helicase (The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation, Structure. 2012 Jul 3; 20 (7): 1189-200). In one embodiment, the helicase comprises a Dda helicase, a RecD2 helicase, a Pif1 helicase, or a DNA helicase B. In one embodiment, the helicase is a Dda helicase from T4 phage. In one embodiment, the naturally occurring amino acid residues present in the linkage comprise E93, E94, K364, Y92, or Y363.In one embodiment, the linked natural amino acid residues are selected from at least one of the following groups: E93 and K364; E94 and K364; or Y92 and Y363.

[0020] In one embodiment, the natural or modified Dda helicase is from the Dda helicases disclosed in Table 1 and Table 2 in CN105899678A.

[0021] In one embodiment, the natural or modified Pifl-like helicase is from the Pifl-like helicases in Table 1 and Table 2 disclosed in CN113930406A.

[0022] In one embodiment, an ectopic bond is introduced between the natural amino acid residues in the 1A domain and the 2A domain of the helicase. In one embodiment, the ectopic bond comprises Lys-His, Lys-Ser, Lys-Thr, Lys-Tyr, Lys-Lys, Lys-Glu, Lys-Asp, Lys-Gln, Lys-Arg, Arg-Glu, Lys-Met, Arg-Asp, Arg-Arg, Tyr-Tyr, Tyr-Trp, Met-Met ectopic bond or a combination thereof. In one embodiment, the amino acid side chains are directly connected to form a covalent bond or are connected to form an ectopic bond by a linker molecule. In one embodiment, the helicase is derived from a Dda helicase, preferably a T4-Dda helicase.

[0023] In one embodiment, the helicase is derived from T4-Dda helicase, and the helicase comprises:

[0024] (1) Tyr-Tyr (YY) heterotopic bond formed by x1 and x2;

[0025] (2) Lys-Gln(KQ) heterotopic bond formed by x3 and x4;

[0026] (3) Tyr-Lys (YK) heterotopic bond formed by x1 and x3;

[0027] (4) Tyr-Gln(YQ) heterotopic bond formed by x1 and x4;

[0028] (5) Tyr-Lys (YK) heterotopic bond formed by x2 and x3;

[0029] (6) Tyr-Gln(YQ) heterotopic bond formed by x2 and x4;

[0030] (7) Lys-Glu (KE) heterotopic bond formed between x3 and x5; or

[0031] (8) Any combination of the above.

[0032] In one embodiment, the helicase comprises any mutant of the 1A domain and / or any mutant of the 2A domain, so long as the helicase retains its ability to control the movement of the target analyte, preferably the target polynucleotide. In one embodiment, the target analyte is modified with one or more proteins or one or more markers, tags or spacers by methylation, oxidation, damage. In one embodiment, the target analyte is a target polynucleotide.

[0033] In a second aspect, the present application provides a polypeptide comprising the 1A domain and the 2A domain that form an ectopic bond from the aforementioned helicase, and a polynucleotide binding domain, and excluding other domains of the helicase.

[0034] In a third aspect, the present application provides a helicase comprising the polypeptide, wherein the helicase has the ability to control the movement of a target analyte, preferably a target polynucleotide.

[0035] In a fourth aspect, the present application provides a construct comprising a helicase or polypeptide, and a target polynucleotide bound to its polynucleotide binding domain, wherein two or more amino acid residues located on the surface of the helicase surrounding the polynucleotide binding domain are connected by an isopeptide bond, and the bound target polynucleotide is surrounded by the structure after the isopeptide bond connection.

[0036] In one embodiment, the construct comprises an adapter, the target polynucleotide is connected to the adapter, and the construct has the ability to control the movement of the target polynucleotide.

[0037] In one embodiment, the construct comprises two or more helicases. In one embodiment, the helicase can be any one of the helicases described above, or a combination thereof.

[0038] In a fifth aspect, the present application provides a polynucleotide comprising a sequence encoding the helicase, the polypeptide, the construct, or consisting of a sequence thereof.

[0039] In a sixth aspect, the present application provides a vector comprising the polynucleotide operably linked to a promoter.

[0040] In a seventh aspect, the present application provides a host cell comprising the vector.

[0041] In an eighth aspect, the present application provides a method for preparing the helicase, the polypeptide, or the construct, comprising expressing the polynucleotide, transfecting cells with the vector, or culturing the host cells.

[0042] In a ninth aspect, the present application provides a method for controlling the movement of a target analyte, preferably a target polynucleotide, comprising contacting the target polynucleotide with the helicase or polypeptide, or the construct, and thereby controlling the movement of the target analyte, preferably the target polynucleotide.

[0043] In one embodiment, the method is used to control the movement of a target analyte, preferably a target polynucleotide, through a pore.

[0044] In a tenth aspect, the present application provides a method for characterizing a target analyte, preferably a target polynucleotide, comprising:

[0045] (a) contacting a target analyte, preferably a target polynucleotide, with a pore and said helicase or polypeptide, or said construct, such that said helicase controls movement of said target analyte, preferably a target polynucleotide, through said pore; and

[0046] (b) obtaining one or more measurements as the target analyte, preferably target polynucleotide, moves relative to the pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target analyte, preferably target polynucleotide.

[0047] In one embodiment, the target analyte is selected from one or more of polynucleotides, polypeptides, polysaccharides and lipids, preferably polynucleotides or polypeptides, polysaccharides and lipids linked to polynucleotides, more preferably single-stranded polynucleotides, double-stranded polynucleotides or partially double-stranded polynucleotides; or

[0048] The target analyte is a target polynucleotide, and the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide; and (v) whether the target polynucleotide is modified.

[0049] In one embodiment, the target polynucleotide is modified with one or more proteins or one or more markers, tags or spacers by methylation, oxidation or damage. In one embodiment, one or more characteristics of the target analyte, preferably the target polynucleotide, are measured by electrical and / or optical measurements. In one embodiment, the electrical measurements are current measurements, voltage measurements, resistance measurements, capacitance measurements, inductance measurements, impedance measurements, tunneling measurements or field effect transistor measurements.

[0050] In one embodiment, the method includes:

[0051] (a) contacting the target analyte, preferably a target polynucleotide, with a pore and the helicase or the construct, such that the helicase or construct controls the movement of the target analyte, preferably a target polynucleotide, through the pore; and

[0052] (b) acquiring a current across the pore as the target analyte, preferably target polynucleotide, moves relative to the pore, wherein the current represents one or more characteristics of the target analyte, preferably target polynucleotide, and thereby characterizes the target analyte, preferably target polynucleotide.

[0053] In one embodiment, the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the helicase or construct. In one embodiment, the target analyte is a target polynucleotide, and at least a portion of the target polynucleotide is double-stranded. In one embodiment, the pore is a nanopore or a transmembrane pore, or the pore is selected from a biological pore, a solid-state pore, or a biological and solid-state hybrid pore. In one embodiment, the biological pore is derived from hemolysin, leukocidin, CsGG, Mycobacterium smegmatis porin A (MspA), porin B, porin C, porin D, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria autotransporter lipoprotein, and WZA; and the solid-state pore is derived from a graphene nanopore, a MoS2 nanopore, a BN nanopore, or a PA63 nanopore.

[0054] In the eleventh aspect, the present application provides a method for forming a sensor for characterizing a target analyte, preferably a target polynucleotide, comprising forming a complex between (a) a hole and (b) the helicase or polypeptide, or the construct, and thereby forming a sensor for characterizing the target analyte, preferably a target polynucleotide.

[0055] In one embodiment, the complex is formed by (a) contacting the pore and the helicase or construct in the presence of the target analyte, preferably a target polynucleotide, and (b) applying an electric potential across the pore. In one embodiment, the electric potential is a voltage potential or a chemical potential. In one embodiment, the complex is formed by covalently linking the pore to the helicase or construct.

[0056] In a twelfth aspect, the present application provides a sensor for characterizing a target analyte, preferably a target polynucleotide, comprising (a) a pore and (b) the helicase or polypeptide, or a complex between the construct.

[0057] In a thirteenth aspect, the present application provides the use of the helicase or polypeptide, or the construct in controlling the movement of a target analyte, preferably a target polynucleotide, through a pore.

[0058] In a fourteenth aspect, the present application provides a kit for characterizing a target analyte, preferably a target polynucleotide, comprising (a) a pore and (b) the helicase or polypeptide, or the construct.

[0059] In one embodiment, the kit further comprises a chip comprising an amphiphilic membrane.

[0060] In a fifteenth aspect, the present application provides a device for characterizing a target analyte, preferably a target polynucleotide, comprising (a) a plurality of pores and (b) a plurality of the helicases or polypeptides, or the constructs.

[0061] In one embodiment, the apparatus comprises: a sensor device capable of supporting a plurality of pores and operable to use the pores and a helicase or a construct to characterize a target analyte, preferably a target polynucleotide; and at least one port for delivering a material for characterization. In one embodiment, the apparatus comprises: a sensor device capable of supporting a plurality of pores and operable to use the pores and a helicase or a construct to characterize a target analyte, preferably a target polynucleotide; and at least one reservoir for storing a material for characterization; a fluidics system configured to controllably provide the material from the at least one reservoir to the sensor device; and one or more containers for receiving corresponding samples, the fluidics system being configured to selectively provide the sample from the one or more containers to the sensor device.

[0062] In a sixteenth aspect, the present application provides a method for preparing the helicase, comprising:

[0063] (a) providing a helicase, preferably a Dda helicase, more preferably a T4-Dda helicase; and

[0064] (b) forming an ectopic bond within the helicase protein to prepare the helicase;

[0065] Preferably, in step (b), the step of contacting the helicase with the polynucleotide is further included.

[0066] Preferably, the ectopic bond is formed by catalysis using H2O2, Hemin and H2O2, HRP enzyme and H2O2, transglutaminase, EDC, or Ru. More preferably, HRP enzyme and H2O2 2、 or EDC.

[0067] In one embodiment, the method further comprises (c) determining whether the obtained helicase is capable of controlling the movement of a target analyte, preferably a target polynucleotide.

[0068] In the seventeenth aspect, the present application provides a method for preparing the construct, comprising connecting the helicase or polypeptide to a linker and thereby preparing the construct.

[0069] In one embodiment, the method further comprises determining whether the obtained construct can control the movement of the target analyte, preferably the target polynucleotide.

[0070] In a seventeenth aspect, the present application provides a group of two or more helicases linked to a target analyte, preferably a target polynucleotide, wherein at least one of the two or more helicases is the aforementioned helicase. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The drawings described are only schematic and are non-limiting.

[0072] Figures 1 to 4 show the effects of enzyme cross-linking under the addition of different catalysts.

[0073] FIG5 shows the locked enzyme cross-linking effect of different enzymes under HRP / H2O2 catalysis according to one embodiment.

[0074] FIG6 shows the column purification elution process of a locked enzyme mutant based on Tyr-Tyr cross-linking according to one embodiment.

[0075] FIG. 7 shows a PAGE gel image of column purification elution of a nucleic acid-protein complex based on Tyr-Tyr cross-linking according to one embodiment.

[0076] FIG8 shows a graph of on-machine test signals of a nucleic acid-protein complex based on Tyr-Tyr cross-linking according to one embodiment.

[0077] FIG9 shows the gel electrophoresis diagram of the nucleic acid-protein complex based on Tyr-Tyr cross-linking prepared in Example 2.1.

[0078] FIG10 shows the gel electrophoresis diagram of the Lys-Glu cross-linked nucleic acid-protein complex prepared in Example 2.2.

[0079] FIG11 shows the column purification elution profile of the nucleic acid-protein complex purified in Example 2.3.

[0080] FIG12 shows the gel electrophoresis diagram of the nucleic acid-protein complex after elution in Example 2.3, wherein FT, E1, E2, and E3 represent the nucleic acid-protein complexes corresponding to the different main peaks in the elution process shown in FIG11 .

[0081] FIG13 shows gel electrophoresis images of nucleic acid-protein complexes after treatment with different reagents in Example 2.3.

[0082] FIG14 shows the results of ATP consumption detection of the nucleic acid-protein complex in Example 2.4.

[0083] FIG15 shows the sequencing signal diagram of the nucleic acid-protein complex in Example 2.5, wherein FIGA is a complete sequencing signal diagram of 10 kb, and FIGB is a local amplified signal step diagram. DETAILED DESCRIPTION

[0084] It should be understood that different applications of the disclosed products and methods can be adapted according to the specific needs of the field. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments of the present application only and is not intended to be limiting.

[0085] Additionally, as used in this specification and claims, the singular forms "a," "an," and "the" include pluralities unless the context clearly dictates otherwise. For example, reference to "a nucleotide" includes two or more nucleotides, and reference to "a helicase" includes two or more helicases.

[0086] As used herein, the term "comprising" means that any listed elements must be included and other elements may also be optionally included. "Consisting of" means that all unlisted elements are not included. Embodiments defined by each of these terms are within the scope of the embodiments of the present application.

[0087] As used herein, "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. The term refers only to the primary structure of the molecule. Thus, the term includes double-stranded and single-stranded DNA and RNA.

[0088] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded covalently linked sequence of nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Nucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can include DNA and RNA and can be synthesized in vitro or isolated from natural resources. Nucleic acids can further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modifications, such as 5'-capping with 7-methylguanosine, 3'-end processing, such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA), and peptide nucleic acids (PNA). The size of a nucleic acid (or polynucleotide) is typically expressed in terms of the number of base pairs (bp) for double-stranded polynucleotides, or in terms of the number of nucleotides (nt) for single-stranded polynucleotides. One thousand bp or nt is equal to one kilobase pair (kb). Polynucleotides of less than about 40 nucleotides in length are often referred to as "oligonucleotides," and may comprise primers used in DNA manipulations, such as by the polymerase chain reaction (PCR).

[0089] A polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides may be naturally occurring or artificially synthesized. One or more nucleotides in the polynucleotide may be oxidized or methylated. One or more nucleotides in the polynucleotide may be damaged. For example, the polynucleotide may comprise a pyrimidine dimer. Such dimers are generally associated with damage caused by ultraviolet light and are a major cause of melanoma of the skin. One or more nucleotides in the polynucleotide may be modified, for example with a conventional marker or label. The polynucleotide may comprise one or more nucleotides that are abasic (i.e., lack a nucleobase) or lack a nucleobase and a sugar (i.e., C3).

[0090] The nucleotides in the polynucleotide can be linked to each other in any manner. The nucleotides are typically linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked by their nucleobases, as in pyrimidine dimers.

[0091] A polynucleotide may be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded. A polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide may comprise an RNA strand hybridized to a DNA strand. A polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers having nucleotide side chains. The PNA backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone is composed of repeating ethylene glycol units linked by phosphodiester bonds. The TNA backbone is composed of repeating threose groups linked together by phosphodiester bonds. LNA is formed from the above-mentioned ribonucleic acids and has an additional bridge structure connecting the 2' oxygen and 4' carbon of the ribose moiety. Bridged nucleic acids (BNA) are modified RNA nucleotides. They can also be referred to as constrained or inaccessible RNA. BNA monomers can contain 5-, 6-, or even 7-membered bridges with a "fixed" C3'-endo sugar puckering. The bridges are synthetically introduced into the 2', 4'-positions of the ribose to produce 2', 4'-BNA monomers.

[0092] The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The polynucleotide can be of any length. For example, the length of the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotides or nucleotide pairs. The length of the polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs or 100,000 or more nucleotides or nucleotide pairs.

[0093] Any number of polynucleotides can be studied. For example, the methods of the embodiments can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they can be different polynucleotides or the same polynucleotide.

[0094] The polynucleotide can be naturally occurring or artificially synthesized. For example, the method can be used to verify the sequence of the prepared oligonucleotide. The method is usually performed in vitro.

[0095] The terms "protein," "polypeptide," and "peptide" are further used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs of amino acid residues. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like.

[0096] Conservative substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties or similar side chain volumes. The amino acids introduced can have polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge similar to the amino acids they replace. Alternatively, conservative substitutions can introduce another aromatic or aliphatic amino acid to replace pre-existing aromatic or aliphatic amino acids. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 main amino acids defined in Table 1 below. When amino acids have similar polarity, this can also be determined with reference to the hydrophilicity scale of the amino acid side chains in Table 2.

[0097] Table 1 - Chemical properties of amino acids

[0098] Table 2 - Hydrophilicity Scale

[0099] It is well known that conservative substitutions between amino acids with similar properties usually do not affect the activity of the peptide sequence. Conservative substitutions are shown in Table 3.

[0100] Table 3 Conservative amino acid substitutions

[0101] Polynucleotide sequences can be derived and replicated using standard methods in the art. Suitable methods for site-directed mutagenesis are known in the art and include, for example, combinatorial chain reactions. The polynucleotides encoding the constructs of the embodiments can be prepared using techniques known in the art, such as those described in Sambrook, J. and Russell, D. (2001). Molecular Cloning A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0102] The resulting polynucleotide sequence can then be incorporated into a recombinant replicable vector, such as a cloning vector. The vector can be used to replicate the polynucleotide in a compatible host cell. Thus, a polynucleotide sequence can be prepared by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that cause the vector to replicate. The vector can be recovered from the host cell.

[0103] Because the four bases that make up DNA, adenine (A), guanine (G), cytosine (C), and thymine (T), have different molecular structures and sizes, when single-stranded DNA (ssDNA) is driven by a rate-controlling enzyme (such as the helicase in the embodiment of the present application) and an electric field through a nanoscale pore, the differences in the chemical properties of the different bases result in different changes in the current caused by passing through the nanopore, protein pore, or transmembrane pore, thereby obtaining sequence information of the measured nucleic acid, such as DNA.

[0104] In an embodiment of a nanopore / protein pore / transmembrane pore sequencing experiment, the nanopore is the only channel for ions to pass through on both sides of the phospholipid membrane. Speed-controlling proteins such as polynucleotide binding proteins (for example, the helicase of the present application embodiment) act as motor proteins for nucleic acid molecules such as DNA, pulling the DNA chain so that it passes through the nanopore / protein pore / transmembrane pore in sequence with a step length of a single nucleotide. Whenever a nucleotide passes through the nanopore / protein pore / transmembrane pore, the corresponding pore-blocking signal will be recorded. By analyzing these sequence-related current signals through the corresponding algorithm, the sequence information of nucleic acid molecules such as DNA can be inferred.

[0105] Helicase

[0106] The present application embodiment provides the helicase of modification, the helicase of DNA dependent ATPase of modification (Dda helicase), more preferably T4-Dda helicase.The modification allows the helicase of modification to keep the combination with analyte preferably polynucleotide for a longer time.The helicase of modification retains the ability of its control analyte preferably polynucleotide to move.In other words, the helicase of modification can still control the movement of analyte preferably polynucleotide.The degree of movement of the helicase control analyte preferably polynucleotide is usually changed by the modification, as described in detail below.

[0107] In one embodiment, the helicase, preferably the Dda helicase, more preferably the T4-Dda helicase is modified. The modified helicase is typically modified compared to the corresponding wild-type helicase or native helicase. The helicase of the present application embodiment is artificial or non-natural.

[0108] The ability of a helicase to bind or not bind to a polynucleotide can be determined using any method known in the art. Suitable binding / not binding assays include, but are not limited to, native polyacrylamide gel electrophoresis (PAGE), fluorescence anisotropy, calorimetry, and surface plasmon resonance (SPR, such as Biacore). TM Of course, the ability of a helicase to unwind a polynucleotide can be determined by measuring the time it takes for the helicase to control the movement of the polynucleotide. This can also be determined using any method known in the art. The ability of a helicase to control the movement of a polynucleotide is typically analyzed in a nanopore system.

[0109] Modified helicases are a useful tool for controlling the movement of polynucleotides during strand sequencing. In one embodiment, the Dda helicase can operate in at least two active modes (when all the necessary components to promote movement, such as ATP and Mg, are provided to the helicase). 2+The Dda helicase controls the movement of DNA in two modes: when the DNA is trapped 5'-down and an inactive mode of operation (when the helicase is not provided with the necessary components to promote movement). When all the necessary components to promote movement are provided, the Dda helicase moves along the DNA in a 5'-3' direction, but the orientation of the DNA in the nanopore (depending on which end of the DNA is captured) means that the enzyme can be used to move the DNA out of the nanopore against the direction of the applied field, or to move the DNA into the nanopore in the direction of the applied field. When the 3' end of the DNA is captured, the helicase works against the direction of the field applied by the potential, pulling the helical DNA out of the nanopore and into the cis compartment. However, when the DNA is captured in the nanopore 5'-down, the helicase works in the direction of the field applied by the potential, pushing the helical DNA into the nanopore and into the trans compartment. When the Dda helicase is not provided with the necessary components to promote movement, the Dda helicase can bind to the DNA and act as a brake to slow the DNA's movement when the DNA is pulled into the pore by the applied field. In the inactive mode, it doesn't matter whether the DNA is trapped with the 3' or 5' end facing down; the applied field pulls the DNA into the nanopore toward the trans side, and the enzyme acts as a brake. The helicase's control of DNA movement when in the inactive mode can be described in a variety of ways, including ratcheting, sliding, and braking.

[0110] A problem that exists in the sequencing process of polynucleotides, especially 500 nucleotides or more, is that the molecular motor that controls the movement of the polynucleotide may be uncoupled from the polynucleotide. This allows the polynucleotide to be rapidly pulled through the pore in an uncontrolled manner in the direction of the applied field. The modified helicase of the present application embodiment does not unwind or uncouple from the polynucleotide being sequenced. When the modified helicase controls the movement of the polynucleotide through the nanopore, the modified helicase can provide an increased polynucleotide read length. The ability to move the entire polynucleotide through the nanopore under the control of the modified helicase of the present application embodiment allows the polynucleotide to be evaluated to be characterized, such as its sequence, with accuracy and speed improved over known methods. This becomes even more important when chain length increases and a molecular motor with improved processivity is required. The modified helicase of the present application embodiment is particularly effective for controlling the movement of 500 nucleotides or more nucleotides, such as 1000 nucleotides, 5000, 10000, 20000, 50000, 100000 or more nucleotides.

[0111] In addition, the use of the modified helicase according to the examples of the present application means that lower helicase concentrations can be used.

[0112] The helicase of the modification of the present application embodiment is also a useful tool for isothermal polymerase chain reaction (PCR). In this method, the chain of double-stranded DNA is first separated by helicase and covered by single-stranded DNA (ssDNA)-binding protein. In a second step, two sequence-specific primers are usually hybridized to each edge of the DNA template. The primer annealed to the template can then be used to prepare double-stranded DNA using an archaeal dna polymerase extension, and then two newly synthesized DNA products can be used as substrates by the helicase of the present application embodiment, enter the next round of reaction. Therefore, synchronous chain reaction occurs, causing the exponential amplification of the target sequence selected.

[0113] The modified helicase has the ability to control the movement of a polynucleotide. The ability of the helicase to control the movement of a polynucleotide can be analyzed by any method known in the art. For example, the helicase can be contacted with a polynucleotide and the position of the polynucleotide can be determined using standard methods. The ability of the modified helicase to control the movement of a polynucleotide is analyzed in a nanopore system.

[0114] The modified helicases of the present application's embodiments can be separated, substantially separated, purified or substantially purified. If the helicase does not contain any other components such as lipids, polynucleotides, hole monomers or other proteins at all, the helicase is separated or purified. If the helicase is mixed with a carrier or diluent that does not interfere with its intended use, the helicase is substantially separated. For example, if the helicase exists in a form containing less than 10%, less than 5%, less than 2% or less than 1% other components such as lipids, polynucleotides, hole monomers or other proteins, the helicase is substantially separated or substantially purified.

[0115] Any helicase, preferably Dda helicase, more preferably T4-Dda helicase can be modified according to the embodiments of the present application. In one embodiment, the Dda helicase is T4-Dda helicase.

[0116] Dda helicases typically contain the following five domains: 1A (RecA-type motor) domain, 2A (RecA-type motor) domain, tower domain, pin domain, and hook domain (Xiaoping He et al., 2012, Structure; 20: 1189-1200). These domains can be identified using the following methods: protein modeling, X-ray diffraction measurements of proteins in crystal form (Rupp B (2009). Biomolecular Crystallograph: Principles, Practice and Application to Structural Biology. New York: Gar 1 and Science.), and nuclear magnetic resonance (NMR) spectroscopy of protein solutions (Mark Rance; Cavanagh, John; Wayne J. Fairbrother; Arthur W. Hunt III; Skelton, N. Nicholas J. (2007). Protein NMR spectroscopy: principles and practice (2 nd ed.).Boston:Academic Press.) or cryo-electron microscopy of cold-hydrated proteins (van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, Pape T, Cohen D, Stark H, Schmidt R, Schatz M, Patwardhan A (2000). "Single-particle electron cryo-microscopy: toward atomic resolution." Q Rev Biophys. 33:307-69). The structural information of proteins determined by the above methods is publicly available in the Protein Data Bank (PDB).

[0117] In one embodiment, the modified helicase is further modified to reduce its surface negative charge. Surface residues can be identified in the same manner as described above for the Dda domain. Surface negative charge is typically a surface negatively charged amino acid, such as aspartic acid (D) and glutamic acid (E).

[0118] The helicase is preferably modified to neutralize one or more surface negative charges by: replacing one or more negatively charged amino acids with one or more positively charged amino acids, uncharged amino acids, non-polar amino acids and / or aromatic amino acids, or by introducing one or more positively charged amino acids, preferably adjacent to one or more negatively charged amino acids. Suitable positively charged amino acids include, but are not limited to, histidine (H), lysine (K) and arginine (R). Uncharged amino acids have no net charge. Suitable uncharged amino acids include, but are not limited to, cysteine ​​(C), serine (S), threonine (T), methionine (M), asparagine (N) and glutamine (Q). Non-polar amino acids have non-polar side chains. Suitable non-polar amino acids include, but are not limited to, glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L) and valine (V). Aromatic amino acids have aromatic side chains. Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0119] Preferred substitutions include, but are not limited to, R for E, K for E, N for E, K for D, and R for D.

[0120] Modification of 1A and 2A domains

[0121] The present invention provides a modified helicase that forms an ectopic bond between the 1A and 2A domains of the helicase. The introduction of unnatural amino acids is cumbersome and costly, while the introduction of disulfide bonds is less stable and easily broken by reducing agents. In contrast, the introduction of natural side chains of ectopic bond-forming amino acid residues into the 1A and 2A domains mediates the formation of stable covalent ectopic bonds, enhancing the enzyme's ability to sustain rate control during sequencing.

[0122] Eccentric bond

[0123] Heteropeptide bonds are a type of covalent bond formed within proteins by the reaction of amino acid R chains, in addition to backbone peptide bonds. Heteropeptide bonds can form naturally within or between protein molecules. Intramolecular isopeptide bonds generally help proteins respond to environmental stresses and enhance their stability (DOI:10.1016 / j.tibs.2010.09.007), while intermolecular isopeptide bonds can be used in the food industry (doi:10.1007 / s12223-013-0287-x) or in protein-protein interaction studies. Heteropeptide bond formation can be categorized into three pathways: autocatalytic formation within proteins, enzymatic catalysis, and chemical catalysis. Specific examples of these three formation methods are listed below.

[0124] Spontaneous covalent bond formation within proteins: For example, studies have found that Lys-Asp ectopic bonds (also known as isopeptide bonds) can spontaneously form within proteins in a hydrophobic environment and in the presence of carboxyl groups as catalysts (DOI: 10.1002 / anie.201004340), with CnaA / CnaB proteins being a common example. Therefore, by creating a hydrophobic environment and introducing specific reactive and catalytic amino acids, spontaneous ectopic bonds can be formed within proteins. This type of ectopic bond can also be modified into a polypeptide ligase / bisubstrate form, that is, the protein that can spontaneously form an isopeptide bond is divided into a catalytic protein and two polypeptides with side chains that can react to form an ectopic bond. The polypeptides with side chains that can react to form an ectopic bond can be integrated into the site where a covalent bond needs to be formed in the form of a recombinant protein. Examples include the SpyTag / KTag ectopic bond connection catalyzed by SpyLiagse based on CnaB modification (doi / 10.1073 / pnas.ss11113) and the SnoopTagJr / DogTag connection catalyzed by SnoopLigase based on RrgA C-terminal domain modification.

[0125] Enzymatic catalysis: This type of method involves a variety of reactions, such as the covalent formation of dityrosine or trityrosine bonds catalyzed by peroxidases (HRP, myeloperoxidase, laccase, etc.) (DOI:10.1021 / acsbiomaterials.6b00454). This cross-linking reaction can also be catalyzed by G4-hemin nuclease (DOI:10.1002 / chem.201101941); the Lys-Gln heterotopic bond (also known as an isopeptide bond) catalyzed by transglutaminase (https: / / doi.org / 10.1016 / j.ijbiomac.2017.10.115); and Lys-Lys cross-linking catalyzed by lysyloxidases (doi:10.1021 / Bm1010195).

[0126] Chemical cross-linking: Chemical cross-linking produces many ectopic bonds, such as the formation of isopeptide bonds between the side chains of D / E acidic amino acids and K / R basic amino acids catalyzed by EDC / NHS / S-NHS, Tyr-Tyr covalent cross-linking catalyzed by H2O2 / Hemin, ruthenium, and cross-linking between D / E and D / E mediated by diazo-containing cross-linkers (doi.org / 10.1021 / acs.analchem.7b03789).

[0127] The types of ectopic bonds in which non-Cys groups participate include, but are not limited to, Lys-His, Lys-Ser, Lys-Thr, Lys-Tyr, Lys-Lys, Lys-Glu, Lys-Asp, Lys-Gln, Lys-Arg, Arg-Glu, Lys-Met, Arg-Asp, Arg-Arg, Tyr-Tyr, Tyr-Trp, and Met-Met. These amino acid side chains can directly form covalent bonds or can be connected through other linker molecules. In one embodiment, the ectopic bond can be Tyr-Tyr, Lys-Lys, Gln-Lys, Glu-Lys, etc.

[0128] In one embodiment, an ectopic bond is formed between the 1A domain and the 2A domain of the helicase, preferably the Dda helicase, more preferably the T4-Dda helicase. In another embodiment, an ectopic bond is introduced between the native amino acid residues of the 1A domain and the 2A domain of the helicase.

[0129] In one embodiment, the ectopic bond comprises a Tyr-Tyr ectopic bond, a Lys-Lys ectopic bond, a Gln-Lys ectopic bond, a Glu-Lys ectopic bond, or a combination thereof. In one embodiment, the helicase is derived from a T4-Dda helicase, and the helicase comprises: (1) a Tyr-Tyr ectopic bond formed between x1 and x2; (2) a Lys-Gln ectopic bond formed between x3 and x4; (3) a Tyr-Lys ectopic bond formed between x1 and x3; (4) a Tyr-Gln ectopic bond formed between x1 and x4; (5) a Tyr-Lys ectopic bond formed between x2 and x3; (6) a Tyr-Gln ectopic bond formed between x2 and x4; (7) a Lys-Glu ectopic bond formed between x3 and x5; or (8) any combination thereof.

[0130] In one embodiment, the helicase is further modified to reduce its surface negative charge. In one embodiment, one or more surface negative charges are neutralized by replacing one or more negatively charged amino acids with one or more positively charged amino acids, uncharged amino acids, non-polar amino acids and / or aromatic amino acids, or by introducing one or more positively charged amino acids near one or more negatively charged amino acids.

[0131] In one embodiment, the helicase comprises any mutant of the 1A domain or any mutant of the 2A domain, as long as the helicase retains its ability to control the movement of a target analyte, preferably a target polynucleotide.

[0132] In one embodiment, the target analyte comprises a polynucleotide, a polypeptide, a polysaccharide, or a lipid, preferably a polynucleotide. In one embodiment, the polynucleotide comprises DNA and / or RNA.

[0133] Variants

[0134] Helicase variants, such as Dda helicase variants, are enzymes having amino acid sequences that vary from the amino acid sequence of the wild-type helicase and retain polynucleotide binding activity. Polynucleotide binding activity can be determined using methods known in the art. Suitable methods include, but are not limited to, fluorescence anisotropy, tryptophan fluorescence, and electrophoretic mobility shift assay (EMSA).

[0135] The variant retains helicase activity. This can be determined using a variety of methods. For example, the ability of the variant to translocate along a polynucleotide can be determined using electrophysiological methods, fluorescence analysis, or ATP hydrolysis.

[0136] The variants may include modifications that facilitate processing of the helicase encoding polynucleotide or facilitate activity of the polynucleotide at high salt concentrations and / or room temperature.

[0137] Helicase fragment

[0138] The embodiment provides the fragment of helicase, for example the fragment of Dda helicase (more preferably T4-Dda helicase), which can be used to prepare the helicase of the present application embodiment. In one embodiment, the polypeptide includes the 1A domain and the 2A domain from the Dda helicase (preferably T4-Dda helicase) and does not include any other domain from the Dda helicase, wherein an ectopic bond is formed between the 1A domain and the 2A domain. The polypeptide may include variants of any of the 1A domains and 2A domains defined above.

[0139] Construct

[0140] The present invention also provides a construct comprising a Dda helicase or a modified Dda helicase (preferably a modified T4-Dda helicase) of the present invention and an adapter, wherein the helicase is connected to the adapter and the construct has the ability to control the movement of a target analyte, preferably a target polynucleotide. The construct is artificial or non-natural.

[0141] In one embodiment, the construct is a useful tool for controlling the movement of the target analyte, preferably the target polynucleotide, during strand sequencing. The construct of the embodiment does not free the modified helicase of the embodiment from the target analyte, preferably the target polynucleotide being sequenced. As the construct controls the displacement of the target analyte, preferably the target polynucleotide through the nanopore, the construct can even provide a longer read length of the target analyte, preferably the target polynucleotide.

[0142] Targeting constructs can also be designed to bind to specific polynucleotide sequences. As discussed in detail below, the adaptor can bind to a specific polynucleotide sequence and thereby target the helicase portion of the construct to that specific sequence.

[0143] The construct has the ability to control the movement of a target analyte, preferably a target polynucleotide. This can be determined as described above.

[0144] The constructs of the present application embodiments can be separated, substantially separated, purified or substantially purified. If the construct is completely free of any other components such as lipids, polynucleotides or pore monomers, the construct is separated or purified. If the construct is mixed with a carrier or diluent that does not interfere with its intended use, the construct is substantially separated. For example, if the construct is present in a form containing less than 10%, less than 5%, less than 2% or less than 1% of other components such as lipids, polynucleotides or pore monomers, the construct is substantially separated or substantially purified.

[0145] The Dda helicase can be any Dda helicase. Preferred Dda helicases include, but are not limited to, T4-Dda helicase and variants thereof. The variants are as described above.

[0146] The helicase is preferably a modified Dda helicase, more preferably a T-Dda helicase. The helicase of any embodiment can be present in the construct of the embodiment of the present application.

[0147] The helicase is preferably covalently linked to the adapter. The helicase can be linked to the adapter at more than one site, such as two or a single site. The helicase can be linked to the adapter using any method known in the art.

[0148] The helicase and the adapter can be prepared separately and then connected together. The two components can be connected in any configuration. For example, they can be connected by their terminal (i.e., amino terminal or carboxyl terminal) amino acid. Suitable configurations include but are not limited to, the amino terminal of the adapter is connected to the carboxyl terminal of the helicase and vice versa. Alternatively, the two components can be connected by the amino acid in its sequence. For example, the polynucleotide binding portion can be connected to one or more amino acids in the helicase ring region. In a preferred embodiment, the terminal amino acid of the polynucleotide binding portion is connected to one or more amino acids in the ring region of the helicase.

[0149] In a preferred embodiment, the helicase is chemically linked to the adapter, for example, by one or more connector molecules. Each connector can have two or more functional ends, such as two, three or four functional ends. Suitable terminal configurations in connectors are known in the art. In another preferred embodiment, the helicase is genetically fused to the adapter. If the entire construct is expressed from a single polynucleotide sequence, the helicase is genetically fused to the adapter. The coding sequences of the helicase and the polyadapter can be combined in any manner to form a single polynucleotide sequence encoding the construct.

[0150] The helicase and the adapter can be genetically fused in any configuration. The helicase and the polynucleotide binding portion can be fused through their terminal amino acids. For example, the amino terminus of the polynucleotide binding portion can be fused to the carboxyl terminus of the helicase and vice versa. The amino acid sequence of the polynucleotide binding portion is preferably added to the amino acid sequence of the helicase within the framework. In other words, the polynucleotide binding portion is preferably inserted into the sequence of the helicase. In this embodiment, the helicase and the distribution are usually connected at two sites, i.e., by the amino terminus and carboxyl terminus of the adapter. If the adapter is inserted into the sequence of the helicase, the amino terminus and carboxyl terminus of the preferred part are very close and are each connected to adjacent amino acids in the sequence of the helicase or its variant. In a preferred embodiment, the adapter is inserted into the annular region of the helicase.

[0151] The helicase can be directly connected to the adapter. The helicase is preferably connected to the portion using one or more, such as two or three, connectors as described above. One or more connectors can be designed to limit the mobility of the portion. The helicase and / or the adapter can be modified to facilitate the connection of one or more connectors as described above.

[0152] In another embodiment, the invention relates to a cleavable connector that can be used to help separate the construct from non-connected components and can be used to further control the synthesis reaction. For example, a heterogeneous-bifunctional connector can react with a helicase, but not with the partial reaction. If the free end of the connector is used to connect the helicase protein to a surface, the unreacted helicase from the first reaction can be removed from the mixture. Subsequently, the connector can be sheared to expose the group that reacts with the adapter. In addition, according to the order of this ligation reaction, the reaction conditions with the helicase can be first optimized, and then the reaction conditions with the adapter can be optimized after shearing off the connector. The second reaction can be carried out more directly towards the correct site that reacts with the adapter because the connector is confined to the region where it has been connected.

[0153] The helicase can be covalently linked to the bifunctional crosslinker before the helicase / crosslinker complex is covalently linked to the adaptor. Alternatively, the moiety can be covalently linked to the bifunctional crosslinker before the bifunctional crosslinker / moiety complex is linked to the helicase. The helicase and the adaptor can be covalently linked to the chemical crosslinker simultaneously.

[0154] Cross-linking of the helicase or moiety itself can be prevented by maintaining a significant excess of the connector concentration relative to the helicase and / or moiety. Alternatively, a "lock and key" setup can be used where two connectors are used. Only one end of each connector can react together to form a longer connector, and the other ends of the connectors react separately with different parts of the construct (i.e., the helicase or moiety).

[0155] The attachment site is selected such that, when the construct is contacted with the polynucleotide, both the helicase and the adaptor can bind to the polynucleotide and control its movement.

[0156] The helicase and the adapter can be used to promote connection. For example, complementary polynucleotides can be used to combine the helicase and the adapter when the helicase and the adapter are hybridized. The helicase can be bound to a polynucleotide and the adapter can be bound to a complementary polynucleotide. The two polynucleotides can then hybridize with each other. This allows the helicase to be in close contact with the adapter, allowing the ligation reaction to be more effectively carried out. This is especially conducive to connecting two or more helicases in the correct direction to control the movement of the target polynucleotide.

[0157] Tags can be added to the construct to facilitate purification of the construct. These tags can then be chemically or enzymatically cleaved if desired to remove them. Fluorophores or chromophores can also be included, which can also be cleaved.

[0158] A simple way to purify the construct is to include different purification tags on each protein (i.e. the helicase and the polynucleotide binding portion), such as a 6His tag and a Strep tag. This method is particularly useful if the two proteins are distinct from each other. Using two tags only allows substances that have both tags to be easily purified. If the two proteins do not have two distinct tags, other methods can be used.

[0159] The constructs can be purified from unreacted proteins based on their different DNA processing properties. In particular, the constructs can be purified from unreacted proteins based on: increased affinity for the target analyte, preferably the target polynucleotide, reduced likelihood of dissociation from the analyte, preferably the polynucleotide, after binding, and / or increased read length of the analyte, preferably the polynucleotide, when they control translocation of the analyte, preferably the polynucleotide through the nanopore.

[0160] Targeting constructs can also be designed to bind to specific polynucleotide sequences. The adaptor can bind to a specific polynucleotide sequence and thereby target the helicase portion of the construct to that specific sequence.

[0161] Connector

[0162] In one embodiment, the construct includes an adapter. In one embodiment, the adapter is a polypeptide that can bind to the analyte, preferably a polynucleotide. The adapter preferably can specifically bind to a defined polynucleotide sequence. In some embodiments, the adapter binds to a specific polynucleotide sequence, while binding to a different sequence cannot be detected. The portion that binds to a specific sequence can be used to design a construct that targets the sequence.

[0163] In one embodiment, the adapter has at least one property of interacting with or modifying a polynucleotide. The adapter can modify the polynucleotide by shearing it to form a single nucleotide or a shorter nucleotide chain, such as two or three nucleotides. The adapter can modify the polynucleotide by positioning it or moving it to a specific position, i.e., controlling its movement.

[0164] In one embodiment, the adapter is derived from a polynucleotide binding enzyme. A polynucleotide binding enzyme is a polypeptide that can bind to a polynucleotide and can interact with the polynucleotide and modify at least one of its properties. The enzyme can modify the polynucleotide by shearing the polynucleotide into a single nucleotide or a shorter nucleotide chain, such as a dinucleotide or trinucleotide. The enzyme can modify the polynucleotide by directing or moving it to a specific position. The polynucleotide binding portion does not need to show enzymatic activity, as long as it can bind to the polynucleotide and control its movement. For example, the portion can be derived from an enzyme that has been modified to remove its enzymatic activity or can be used under conditions that prevent it from acting as an enzyme.

[0165] In one embodiment, the adaptor is derived from a nucleolytic enzyme.In one embodiment, preferred enzymes are exonucleases, polymerases, helicases and topoisomerases, such as gyrase.

[0166] In one embodiment, the adapter is connected to the helicase and the target analyte (preferably the target polynucleotide). In one embodiment, the adapter is a nucleic acid. In one embodiment, the adapter is a polynucleotide. In another embodiment, the adapter includes a double-stranded nucleic acid region that can connect other double-stranded nucleic acids, such as the target polynucleotide. In one embodiment, the adapter is a Y-shaped adapter. In one embodiment, the adapter is composed of three DNA chains. In one embodiment, the adapter includes a spacer, preferably a spacer inserted in the adapter, or the spacer is located at the 5' end or 3' end of one chain. In one embodiment, the adapter is a polynucleotide comprising a spacer. In one embodiment, the adapter is a Y-shaped polynucleotide comprising a spacer. In one embodiment, the adapter is a polynucleotide, polypeptide, polysaccharide or lipid containing a spacer. The helicase can be stalled on the spacer of the adapter. By contacting the helicase and the polynucleotide (or polypeptide or polysaccharide or lipid) on the adapter with a transmembrane pore and applying an electric potential, the stalled helicase can be moved through the spacer on the adapter. Since the helicase cannot pass through the transmembrane pore, the polynucleotide (or polypeptide or polysaccharide or lipid) of the adapter moves along the electric potential through the transmembrane pore and the force generated moves the helicase through the spacer. In one embodiment, a plurality of adapters are included. In one embodiment, the adapter is part of the target analyte, or is not part of the target analyte. In one embodiment, other adapters may be interspersed in the target analyte (preferably target polynucleotide).

[0167] Polynucleotide sequence

[0168] The present invention provides polynucleotides containing sequences encoding the helicases, polypeptides, or constructs of the present invention. The polynucleotides may contain such sequences. The polynucleotides may be any of the polynucleotides discussed above.

[0169] Any of the proteins described herein can be expressed using methods known in the art. Polynucleotide sequences can be isolated and replicated using standard methods in the art. Chromosomal DNA can be extracted from organisms that produce helicases, such as Methanococcus burtonii and / or organisms that produce SSB, such as Escherichia coli. PCR with specific primers can be used to amplify the gene coding sequence of interest. The amplified sequence can then be inserted into a recombinant replicating vector, such as a cloning vector. The vector can be used to replicate the polynucleotide in a compatible host cell. Therefore, a polynucleotide sequence can be prepared by introducing a polynucleotide encoding a sequence of interest into a replicable vector, including introducing the vector into a compatible host cell and growing the host cell under conditions that induce vector replication. The vector can be recovered from the host cell. Suitable host cells for cloning polynucleotides are known in the art and are described in more detail below.

[0170] The polynucleotide sequence can be cloned into an appropriate expression vector. In an expression vector, the polynucleotide sequence is usually operably linked to a control sequence that provides for expression of the coding sequence by the host cell. The expression vector can be used to express the construct.

[0171] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated under conditions compatible with the control sequences in such a manner as to achieve expression of the coding sequence. Multiple copies of the same or different polynucleotides may be introduced into a vector.

[0172] The expression vector can then be introduced into a suitable host cell. Thus, a construct can be prepared by introducing a polynucleotide sequence encoding the construct into an expression vector, including introducing the vector into a compatible bacterial host cell and growing the host cell under conditions that cause expression of the polynucleotide sequence.

[0173] The vector can be, for example, a plasmid, virus or phage vector having an origin of replication, optionally a promoter for expressing the polynucleotide sequence, and optionally a regulatory factor for the promoter. The vector can contain one or more selectable marker genes, such as an ampicillin resistance gene. Promoters and other expression control signals compatible with the host cell for which the expression vector is designed can be selected. Typically, T7, trc, lac, ara or k promoters are used.

[0174] The host cell typically expresses the construct at high levels. A host cell compatible with the expression vector used to transform the host cell is selected for polynucleotide sequence transformation. The host cell is typically bacterial and preferably Escherichia coli. Any cell with a lambda DE3 lysogen, such as Rosetta2(DE3)pLys, C41(DE3), BL21(DE3), JM109(DE3), B834(DE3), TUNER, Origami, and Origami B, can express vectors containing a T7 promoter.

[0175] Helicase group

[0176] The present application embodiment also provides the group (series) of two or more helicases that connect (or combine) polynucleotide, wherein at least one in said two or more helicases is the helicase of the present application embodiment, preferably Dda helicase, more preferably T4-Dda helicase.Described group can comprise any number of helicases, such as 2,3,4,5,6,7,8,9,10 or more helicases.Any number of helicases can be the helicase of the present application embodiment, preferably Dda helicase, more preferably T4-Dda helicase.All of two or more helicases are preferably the helicase of the present application embodiment, preferably Dda helicase, more preferably T4-Dda helicase.One or more helicases of the present application embodiment, preferably Dda helicase, more preferably T4-Dda helicase can be the helicase discussed above arbitrarily, preferably Dda helicase, more preferably T4-Dda helicase.

[0177] The two or more helicases can be the same or different helicases. For example, if the group includes two or more helicases of the present application embodiments, preferably Dda helicases, more preferably T4-Dda helicases, the helicases of the present application embodiments, preferably Dda helicases, more preferably T4-Dda helicases can be the same or different.

[0178] The group can include the helicase of the present application embodiment of any number and combination, preferably Dda helicase, more preferably T4-Dda helicase. The group of two or more helicases preferably includes the helicase of at least two present application embodiments, preferably Dda helicase, more preferably T4-Dda helicase. The group can include two or more helicases, preferably Dda helicase, more preferably T4-Dda helicase. Each helicase, preferably Dda helicase, more preferably T4-Dda helicase includes modification as described above, including that the 1A domain and the 2A domain form an ectopic bond.

[0179] In addition to one or more helicases of the present application embodiments, preferably Dda helicase, more preferably T4-Dda helicase, the group may include one or more helicases that are not a part of the present application embodiments. The one or more helicases may be or be derived from Hel308 helicase, RecD helicase, such as Tral helicase or TrwC helicase, XPD helicase or Dda helicase. The one or more helicases may be any helicase, modified helicase or helicase construct disclosed in International Application Nos. PCT / GB2012 / 052579 (published as WO 2013 / 057495); PCT / GB2012 / 053274 (published as WO 2013 / 098562); PCT / GB2012 / 053273 (published as WO 2013 / 098561); PCT / GB2013 / 051925 (published as WO 2014 / 013260); PCT / GB2013 / 051924 (published as WO 2014 / 013259) and PCT / GB2013 / 051928 (published as WO 2014 / 013269). 2014 / 013262); and UK Application No. 1318464.3 filed on October 18, 2013. In particular, the one or more helicases are preferably modified to reduce the size of an opening in the polynucleotide binding domain, through which the polynucleotide can pass in at least one conformational state and be released from the helicase. This is disclosed in WO 2014 / 013260.

[0180] The two or more helicases in the group may be separated from each other. The two or more helicases in the group may be bound together by the transmembrane pore as the polynucleotide moves through the pore. The two or more helicases in the group may be in contact with each other.

[0181] Preferably, the two or more helicases are not linked to each other other than by the polynucleotide. Preferably, the two or more helicases are not covalently linked to each other.

[0182] The two or more helicases can be linked to each other or covalently linked to each other. The helicases can be linked in any order and using any method. A group of linked helicases can be referred to as a team.

[0183] Method for controlling the movement of a target analyte, preferably a target polynucleotide

[0184] The present application embodiment provides a method for controlling the movement of a target analyte, preferably a target polynucleotide. The method comprises contacting the target analyte, preferably a target polynucleotide, with a helicase of the embodiment, preferably a Dda helicase, more preferably a T4-Dda helicase, or a construct of the present application embodiment, and thereby controlling the movement of the target analyte, preferably a target polynucleotide. The method is preferably implemented under an electric potential applied across the hole. As described in detail below, the applied electric potential generally results in the formation of a complex between the hole and the helicase or construct. The applied electric potential can be a voltage potential. Alternatively, the applied electric potential can be a chemical potential. An example thereof is the use of a salt gradient across an amphiphilic layer. Salt gradients are disclosed in Holden et al., J Am Chem Soc. 2007 Jul 11; 129(27): 8650-5.

[0185] The present application embodiment also provides the method for characterizing target analyte preferably target polynucleotide.Described method comprises (a) by target analyte preferably target polynucleotide and hole and the helicase of embodiment, preferably Dda helicase, more preferably T4-Dda helicase or the construct of the present application embodiment contact, make described helicase or construct control target analyte preferably target polynucleotide pass the movement of described hole.Described method also comprises (b) along with described target analyte preferably polynucleotide moves relative to described hole, obtains one or more measured values, wherein said measured value represents one or more features of described target analyte preferably target nucleotide, and characterizes described target analyte preferably target polynucleotide thus.

[0186] In all methods of the embodiments of the present application, the helicase can be any helicase as described above for the constructs of the embodiments of the present application, including the modified helicases of the embodiments of the present application, preferably Dda helicases, more preferably T4-Dda helicases, and helicases not modified according to the embodiments of the present application, preferably Dda helicases, more preferably T4-Dda helicases.

[0187] In these methods, any number of helicases of the present application's embodiments can be used, preferably Dda helicase, more preferably T4-Dda helicase. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases can be used. If two or more helicases of the present application's embodiments are used, preferably Dda helicase, more preferably T4-Dda helicase, they can be identical or different. Applicable number and combination are as described above in the group for the present application's embodiments. They can be equally applied to the method for the present application's embodiments.

[0188] If two or more helicases are used, they may be linked to one another. The two or more helicases may be covalently linked to one another. The helicases may be linked in any order and using any method.

[0189] If two or more helicases are used, they are preferably not linked to each other except by the polynucleotide.The two or more helicases are more preferably not covalently linked to each other.

[0190] Steps (a) and (b) are preferably performed under conditions where a potential is applied across the pore as described above. In some cases, the sequence of the target polynucleotide is determined using the current passing through the pore as the polynucleotide moves relative to the pore. This is known as strand sequencing.

[0191] The method of the embodiment of the present application is used to characterize a target analyte, preferably a target polynucleotide. The polynucleotide is as defined above.

[0192] The entire or only a portion of the target polynucleotide can be characterized using this method. The target polynucleotide can be of any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotide pairs in length. The polynucleotide can be 1,000 or more nucleotide pairs in length, 5,000 or more nucleotide pairs in length, or 100,000 or more nucleotide pairs in length.

[0193] The target polynucleotide is typically present in any suitable sample. The present embodiments are typically implemented on samples known to contain or suspected of containing the target polynucleotide. Alternatively, the present embodiments can be implemented on samples to confirm the identity of one or more target polynucleotides known or expected to be present in the sample.

[0194] The sample can be a biological sample. The embodiments of the present application can be performed in vitro on samples obtained or extracted from any organism or microorganism. The organism or microorganism is typically archaeal, prokaryotic, or eukaryotic, and typically belongs to one of the following five kingdoms: Plantae, Animalia, Fungi, Ankaryotic Protozoa, and Protists. The embodiments of the present application can be performed in vitro on samples obtained or extracted from any virus. The sample is preferably a liquid sample. The sample typically includes a patient's body fluid. The sample can be urine, lymph, saliva, mucus, or amniotic fluid, but is preferably blood, plasma, or serum. Typically, the sample is of human origin, but can alternatively be from other mammals, such as commercially raised animals such as horses, cattle, sheep, or pigs, or pets such as cats or dogs. Alternatively, samples of plant origin are typically obtained from commercial crops, such as cereals, legumes, fruits, or vegetables, for example, wheat, barley, oats, rapeseed, corn, soybeans, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugarcane, cocoa, and cotton.

[0195] The sample may be a non-biological sample. The non-biological sample is preferably a liquid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

[0196] Samples are typically processed prior to use in the present embodiments, for example by centrifugation or membrane filtration to remove unwanted molecules or cells, such as red blood cells. Testing can be performed immediately after obtaining the sample. Samples can also typically be stored, preferably below -70°C, prior to analysis.

[0197] A pore is a structure that spans a membrane to some extent. It allows hydrated ions to flow across or within the membrane, driven by an applied electrical potential. Pores typically span the entire membrane, allowing hydrated ions to flow from one side of the membrane to the other. However, a pore does not necessarily span the membrane. It can be closed at one end. For example, a pore can be a well in the membrane through which hydrated ions can flow or flow.

[0198] Any pore may be used in the embodiments of the present application. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores.

[0199] Suitable membranes are preferably amphiphilic layers. An amphiphilic layer is a layer formed by amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or naturally occurring. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported. The membrane can be a lipid bilayer. The lipid bilayer is formed by two opposing layers of lipids. The two layers of lipids are arranged so that their hydrophobic tail groups face each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outward toward the aqueous environment on each side of the bilayer. The membrane includes a solid layer. The solid layer can be formed from organic and inorganic materials. If the membrane includes a solid layer, pores are typically present in the amphiphilic membrane or in a layer included within the solid layer, such as holes, wells, gaps, channels, grooves, or slits within the solid layer.

[0200] The polynucleotide can be attached to the membrane. This can be accomplished using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer, the polynucleotide is preferably attached to the membrane via a polypeptide present in the membrane or via a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube, or amino acid.

[0201] The polynucleotide can be directly connected to the membrane. The polynucleotide is preferably connected to the membrane through an adapter. Preferred adapters include, but are not limited to, polymers, such as polynucleotides, polyethylene glycol (PEG) and polypeptides. If the polynucleotide is directly connected to the membrane, due to the distance between the membrane and the helicase, the characterization cannot proceed to the end of the polynucleotide, and some data will be lost. If an adapter is used, the polynucleotide can be characterized completely. If an adapter is used, the adapter can be connected to any position of the polynucleotide. The adapter is usually connected to the tail polymer of the polynucleotide.

[0202] In another embodiment, the polynucleotide is connected to an amphiphilic layer.

[0203] Transmembrane pores or nanopores, preferably transmembrane protein pores, define a channel or pore that allows molecules and ions to translocate from one side of the membrane to the other. The translocation of ionic species through the pore can be driven by a potential difference applied to either side of the pore. A "nanopore" is a protein pore or transmembrane pore in which the minimum diameter of the channel through which a molecule or ion passes is on the order of nanometers (10 -9 m). In some embodiments, the protein pore can be a transmembrane protein pore or a transmembrane pore or a nanopore. The transmembrane protein structure of the transmembrane pore can be a monomer or an oligomer in nature. Typically, the pore comprises a plurality of polypeptide subunits arranged around a central axis, thereby forming a protein-lined channel extending substantially perpendicular to the membrane in which the pore resides. There is no limit to the number of polypeptide subunits. Typically, the number of subunits is 5 to 30, and suitably the number of subunits is 6 to 10. Alternatively, the number of subunits is not defined as in the case of perfringolysin or related large membrane pores. The protein subunit portion that forms the protein-lined channel in the pore typically comprises a secondary structural motif that may include one or more transmembrane β-barrels and / or α-helical portions.

[0204] The methods of the embodiments can measure two, three, four, or five or more characteristics of a polynucleotide. The one or more characteristics are preferably selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified. In one embodiment, any combination of (i) to (v) can be measured.

[0205] For (i), the length of the polynucleotide may be measured, for example, by determining the number of interactions between the polynucleotide and the pore or the duration of interactions between the polynucleotide and the pore.

[0206] Regarding (ii), the identity of a polynucleotide can be measured in a variety of ways. The identity of a polynucleotide can be measured in conjunction with or without measuring the polynucleotide sequence. The former is relatively simple; the polynucleotide is sequenced and identified. The latter can be accomplished in several different ways. For example, the presence of a specific motif in a polynucleotide can be measured (without measuring the rest of the polynucleotide sequence). Alternatively, the measurement of a specific electrical and / or optical signal in the described methods can identify the polynucleotide as originating from a specific source.

[0207] For (iii), the sequence of the polynucleotide can be determined as previously described.

[0208] Regarding (iv), secondary structure can be measured using a variety of methods. For example, if the method involves electrical measurement, changes in residence time or changes in the current flowing through the pore can be used to measure the secondary structure. This allows for differentiation between regions of single-stranded and double-stranded polynucleotides.

[0209] For (v), the presence or absence of any modifications can be measured. The method preferably comprises determining whether the polynucleotide is modified by methylation, oxidation, damage, modification with one or more proteins, or modification with one or more markers, tags, or modification without bases or lacking nucleobases and sugars. Specific modifications will result in specific interactions with the pore, which can be measured using the following methods. For example, methylcytosine can be distinguished from cytosine based on the current flowing through the pore during its interaction with each nucleotide.

[0210] Various different types of measurements can be performed. This includes, but is not limited to, electrical measurements and optical measurements. Electrical measurements include voltage measurements, capacitance measurements, current measurements, impedance measurements, tunneling measurements (Ivanov AP et al., Nano Lett. 2011 Jan 12; 11(1): 279-85) and FET measurements (International Application WO 2005 / 124888). Optical measurements can be combined with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan; 81(1)014301). The measurement can be a transmembrane current measurement, such as a measurement of the ionic current flowing through the pore. In one embodiment, the electrical measurement or optical measurement can be a conventional electrical or optical measurement.

[0211] Electrical measurements can be performed using standard single-channel recording equipment as described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19)7702-7, Lieberman KR et al, J Am Chem SOC. 2010; 132(50)17961-72 and international application WO 2000 / 28312. Alternatively, electrical measurements can be performed using a multi-channel system, such as described in international application WO 2009 / 077734 and international application WO 2011 / 067559.

[0212] The method is preferably performed using an applied potential across the membrane. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. One example is the use of a salt gradient across the membrane, such as across a layer of amphiphilic molecules. Salt gradients are disclosed in Holden et al., J Am Chem SOC. 2007 Jul 11;129(27):8650-5. In some cases, the current flowing through the pore as the polynucleotide moves relative to the pore is used to estimate or determine the sequence of the polynucleotide. This is known as strand sequencing.

[0213] The method may include measuring the current flowing through the pore as the polynucleotide moves relative to the pore. Thus, the apparatus used in the method may also include circuitry capable of applying an electric potential and measuring the electrical signal passing through the membrane and the pore. The method may be performed using a patch clamp or a voltage clamp.

[0214] The method can include measuring the current flowing through the hole when the polynucleotide moves relative to the hole. Suitable conditions for measuring the ion flow through the transmembrane protein pore are known in the art and disclosed in the examples. The method is usually performed by applying a voltage to the membrane and the hole. The voltage used is usually from +5V to -5V, for example from +4V to -4V, from +3V to -3V or from +2V to -2V. The voltage used is usually from -600mV to +600V or -400mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300nV and +400mV. The voltage used is more preferably in the range of 100mV to 240mV and most preferably in the range of 120mV to 220mV. By using an increased applied potential, the recognition of different nucleotides by the pore can be increased.

[0215] The method is generally carried out in the presence of any charge carrier, such as a metal salt such as an alkali metal salt, a halide salt such as a chloride salt, such as an alkali metal chloride salt. The charge carrier can include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride. In the exemplary device described above, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide are generally used. A mixture of KCl, NaCl and potassium ferrocyanide and potassium ferricyanide is preferred. The charge carrier can be asymmetric on the membrane. For example, the type and / or concentration of the charge carrier can be different on each side of the membrane.

[0216] The concentration of the salt may be saturating. The concentration of the salt may be 3 M or less, and is typically 0.1 to 2.5 M, 0.3 to 1.9 M, 0.5 to 1.8 M, 0.7 to 1.7 M, 0.9 to 1.6 M or 1 M to 1.4 M. The concentration of the salt is preferably 150 mM to 1 M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow the presence of the nucleotide to be identified in the context of normal current fluctuations to be indicated by current.

[0217] The method is typically performed in the presence of a buffer. In the exemplary apparatus described above, the buffer is present in an aqueous solution in the chamber. Any buffer may be used in the methods of the present invention. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES or Tris-HCl buffer. The method is typically performed at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0218] The method can be carried out at a temperature of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C or 20°C to 60°C. The method is typically carried out at room temperature. The method is optionally carried out at a temperature that supports enzyme function, such as about 37°C.

[0219] In one embodiment, the method includes:

[0220] (a) contacting the target analyte, preferably a target polynucleotide, with a pore and a helicase of an embodiment of the present application or a construct of an embodiment of the present application, such that the target analyte, preferably a target polynucleotide, moves through the pore and the helicase or construct controls the movement of the target analyte, preferably a target polynucleotide, through the pore; and

[0221] (b) acquiring a current across the pore as the target analyte, preferably polynucleotide, moves relative to the pore, wherein the current represents one or more characteristics of the target analyte, preferably polynucleotide, and thereby characterizes the target analyte, preferably polynucleotide.

[0222] The method can be implemented using any apparatus suitable for studying membrane / pore systems in which pores are embedded in the membrane. The method can be implemented using an apparatus suitable for sensing pores. For example, the apparatus comprises a chamber containing an aqueous solution and a barrier dividing the chamber into two parts. The barrier typically has a gap in which a membrane containing the pores is formed. Alternatively, the barrier forms a membrane in which the pores are present. The method can be implemented using the apparatus described in International Application No. PCT / GB08 / 000562 (WO 2008 / 102120).

[0223] The method can include measuring the current through the hole as the target analyte, preferably a polynucleotide, moves relative to the hole. Therefore, the device can also include a circuit capable of applying an electric potential across the membrane and the hole and measuring the current signal. The method can be implemented using a patch clamp or a voltage clamp. The method preferably includes using a voltage clamp. The target polynucleotide can be contacted with the helicase or construct and the hole in any order. Preferably, when the target analyte, preferably the target polynucleotide, contacts the helicase or construct and the hole, the target analyte, preferably the target polynucleotide, first forms a complex with the helicase or construct. When a potential is applied across the hole, the target analyte, preferably the target polynucleotide / helicase or construct complex then forms a complex with the hole and controls the target analyte, preferably the polynucleotide, to move through the hole.

[0224] sensor

[0225] The present application embodiment also provides the sensor and method thereof that are formed for characterizing the preferred target polynucleotide of target analyte.Described method is included in the helicase of hole and embodiment, preferably Dda helicase, more preferably forms complex between the construct of T4-Dda helicase, enzyme or the present application embodiment.Described helicase can be the helicase arbitrarily described for the construct of the present application embodiment, comprises the helicase of the present application embodiment and the helicase that is not modified according to the present application embodiment.Can use the Dda helicase of the present application embodiment discussed for the group of the present application embodiment and method above-mentioned embodiment of the present application of any number and combination.

[0226] The complex can be formed by contacting a hole and a helicase or a construct in the presence of a target analyte, preferably a target polynucleotide, and then applying an electric potential to the hole. The applied electric potential can be a chemical potential or a voltage potential as described above. Alternatively, the complex can be formed by being covalently linked to the helicase or the construct through a hole. The method of covalent linkage is known in the art and, for example, disclosed in International Application No. PCT / GB09 / 001679 (publication number WO 2010 / 004265) and PCT / GB10 / 000133 (publication number WO 2010 / 086603). The complex is a sensor for characterizing a target analyte, preferably a target polynucleotide. The method preferably includes forming a complex between a hole derived from Msp and a helicase of the present application embodiment or a construct of the present application embodiment. Any embodiment of the present application embodiment method discussed above is equally applicable to this method. The present application embodiment also provides a sensor prepared using the method of the present application embodiment.

[0227] Reagent test kit

[0228] The present application embodiment also provides the test kit for characterizing the preferred target polynucleotide of target analyte.Described test kit comprises the helicase of (a) hole and (b) embodiment, preferably Dda helicase, more preferably T4-Dda helicase, or the construct of the present application embodiment.Any embodiment discussed above for the embodiment of the present application method is equally applicable to this test kit.Described helicase can be any helicase described for the construct of the embodiment of the present application above, comprises the helicase of the embodiment of the present application and the helicase not modified according to the embodiment of the present application.Described test kit can comprise the helicase of the embodiment of the present application discussed for the group and method of the embodiment of the present application of using any number and combination, preferably Dda helicase, more preferably T4-Dda helicase.

[0229] The test kit may further include components of the membrane, for example, phospholipids required to form an amphiphilic layer such as a lipid bilayer. The test kit of the present application's embodiments may additionally include one or more other reagents or instruments that enable any of the embodiments described above to be implemented. The reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), an instrument for obtaining a sample from a receptor (such as a catheter or an instrument containing a needle), an instrument for amplifying and / or expressing polynucleotides, a membrane as defined above or a voltage clamp or patch clamp device. The reagents present in the test kit can be dry so that the liquid sample resuspends the reagents. The test kit may also optionally include an instrument enabling the test kit to be used in the present application's embodiment methods or instructions for which patients the method may be used for. The test kit optionally includes nucleotides.

[0230] Device

[0231] The present application embodiment also provides a device for characterizing target analyte, preferably target polynucleotide. The device includes multiple holes and multiple helicases, preferably Dda helicases, more preferably T4-Dda helicases, multiple helicases of the present application embodiment or the construct of multiple embodiments of the present application. The device preferably further includes a specification sheet for implementing the method of the present application embodiment. The device can be any conventional device for target analyte, preferably polynucleotide analysis, such as an array or a chip. Any embodiment of the method of the present application embodiment discussed above can be equally applicable to the device of the present application embodiment. The helicase can be any helicase described above for the construct of the present application embodiment, including the helicase of the present application embodiment and the helicase not modified according to the present application embodiment. The device can include the helicase of the present application embodiment discussed above for the group and method of the present application embodiment using any number and combination, preferably Dda helicase, more preferably T4-Dda helicase. The device is preferably assembled into a method for implementing the present application embodiment.

[0232] In one embodiment, the device preferably comprises:

[0233] A sensor device capable of supporting a plurality of pores and operable to characterize a target analyte, preferably a polynucleotide, using the pores and the helicase or construct; and

[0234] At least one port for delivering a material to be characterized.

[0235] Alternatively, the device preferably comprises:

[0236] A sensor device capable of supporting a plurality of pores and operable to characterize a target analyte, preferably a polynucleotide, using the pores and the helicase or construct; and

[0237] At least one memory stores material for characterization.

[0238] The device more preferably comprises:

[0239] a sensor device capable of supporting the membrane and the plurality of pores and operable to characterize a target analyte, preferably a polynucleotide, using the pores and the helicase or construct;

[0240] at least one memory for storing material for characterization;

[0241] A fluidics system configured to controllably supply material from at least one reservoir to the sensor device; and one or more containers for receiving samples, the fluidics system being configured to selectively supply samples from the one or more containers to the sensor device. The device may be any of those described in International Application No. PCT / GB08 / 004127 (published as WO 2009 / 077734), PCT / GB10 / 000789 (published as WO 2010 / 122293), International Application No. PCT / GB10 / 002206 (published as WO 2011 / 067559), or International Application No. PCT / US99 / 25679 (published as WO 00 / 28312).

[0242] Method for preparing helicase

[0243] The present invention also provides a method for preparing the modified helicase of the present invention. The method includes providing a helicase, preferably a Dda helicase, more preferably a T4-Dda helicase to form the modified helicase of the present invention.

[0244] The method preferably further comprises determining whether the helicase can control the movement of the target analyte, preferably a polynucleotide. The analysis performed on it is described above. If the movement of the target analyte, preferably a polynucleotide, can be controlled, then the helicase is correctly modified and the helicase of the present application embodiment is produced. If the movement of the target analyte, preferably a polynucleotide, cannot be controlled, then the helicase of the present application embodiment is not produced.

[0245] Methods of making constructs

[0246] The present invention also provides a method for preparing the construct. The method comprises linking, preferably covalently linking, a helicase of the present invention, preferably a Dda helicase, more preferably a T4-Dda helicase to an adapter. Any helicase and adapter as described above can be used in this method.

[0247] Said method preferably further comprises determining whether said construct can control the movement of target analyte, preferably polynucleotide.The analysis carried out thereof is described above.If the movement of target analyte, preferably polynucleotide can be controlled, said helicase and adapter are correctly connected, and the construct of the present application embodiment is obtained.If the movement of target analyte, preferably polynucleotide cannot be controlled, then the construct of the present application embodiment is not obtained.

[0248] The above-mentioned prior art is incorporated herein by reference in its entirety.

[0249] The following examples are used to illustrate the present invention but are not intended to limit the present invention.

[0250] Example 1.1: Enzyme cross-linking mediated by different catalytic systems

[0251] Main strand S1: 5'-33333333333333333333333333333-GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT (SEQ ID NO: 1)-8888- T(SEQ ID NO:2)-3'

[0252] Antisense strand S2: 5'-CGACTTCTACCGTTTGACTCCGC (SEQ ID NO: 3)-3'

[0253] Connecting chain YB: (SEQ ID NO: 4) / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeC / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeC / / i2OMeC / / i2OMeG / / i2OMeA / / i2OMeC / / i2OMeC / -3': The main strand S1, antisense strand S2, and linker strand YB were synthesized separately and mixed at a ratio of 1:1.1:1.1 and annealed to form an adapter. The annealing treatment was performed by slowly cooling the temperature from 95°C to 25°C with a cooling rate of no more than 0.1°C / s.

[0254] Four reaction systems were prepared, each containing 1 μM adapter and 7.5-fold the amount of T53 (mutant T4 DDA-E94Y / N365Y, whose wild-type T4 DDA sequence is shown in SEQ ID NO: 5). Alternatively, the reaction mixtures were added without catalyst, with 100 μM H₂O₂, 50 μM Hemin / 100 μM H₂O₂, and 0.1 U HRP / 100 μM H₂O₂, respectively. The mixtures were incubated at 30°C for 18 hours. Cross-linking was then analyzed using a TBE PAGE gel run at 160 V for 40 minutes. The results are shown in Figure 1. As shown in Figure 1, the nucleic acid-protein complex is a complex of adapter and helicase. Effective cross-linking products were formed after the addition of H₂O₂, and the HRP enzyme catalysis was significantly superior to that of other catalysts.

[0255] Two reaction systems were prepared, each with 1 μM adapter and 5-fold the amount of Q1 (mutant T4DDA-E93Q) protein added. Alternatively, 0.1 U of microbial transglutaminase was added as a catalyst, and the mixture was incubated at 37°C for 36 hours. The enzyme cross-linking was then detected using a TBE PAGE gel run at 160 V for 40 minutes. The results, shown in Figure 2, indicate that the nucleic acid-protein complex is a complex of the adapter and the helicase. The addition of microbial transglutaminase enabled the formation of effective cross-linked products.

[0256] Two reaction systems were prepared, each with 1 μM adapter and a 5-fold increase in the amount of Q11 (mutant T4DDA-E94G) protein. Alternatively, the reaction mixtures were incubated at 30°C for 4 hours with 5 mM EDC as a catalyst, followed by TBE PAGE run at 160 V for 40 minutes to assess enzymatic cross-linking. As shown in Figure 3, the nucleic acid-protein complex is a complex of adapter and helicase. Effective cross-linking products were formed after the addition of EDC.

[0257] Example 1.2: Ru-catalyzed Tyr-amino lock enzyme reaction

[0258] Main chain S1: 5'-333333333333333333333333333333-GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT(SEQ ID NO:1)-8888-ACTGCTCATTCGGTCCTGCTGAC T(SEQ ID NO:2)-3'

[0259] Antisense strand S2: 5'-Ru-CGACTTCTACCGTTTGACTCCGC (SEQ ID NO: 3)-3'

[0260] Linking chain YB: 5'P-GTCAGCAGGACCGAATGA (SEQ ID NO:4) / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeC / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeC / / i2OMeC / / i2OMeG / / i2OMeA / / i2OMeC / / i2OMeC / -3'

[0261] The main strand S1, antisense strand S2, and linker strand YB were synthesized separately, mixed at a ratio of 1:1.1:1.1, and annealed to form the adapter. The annealing step was to slowly cool the reaction mixture from 95°C to 25°C at a rate of no more than 0.1°C / s.

[0262] Prepare two reaction systems, each with 2 μM adapter, add 4 times the amount of T49 (mutant T4 DDA-E94Y) or 4 times the amount of T53 protein / 1 mM 1,4-diamine, mix and incubate at 30°C for 30 min, then add 1.25 mM sodium persulfate and place under LED 450 nm light for crosslinking (light power 50 mw / cm 2 The sample was then run on a TBE PAGE gel at 160V for 40 minutes to examine enzyme cross-linking, as shown in Figure 4. The nucleic acid-protein complex consists of an adaptor and helicase complex. Under Ru photocatalysis, enzyme cross-linking was clearly observed (Y94-K364 in T49; Y94 and N365Y in T53 were cross-linked with 1,4-butanediamine).

[0263] Example 1.3: HRP cross-linking reaction of different Tyr-Tyr mutants

[0264] Three reaction systems were prepared, each with 1 uM adapter, 7.5 times the amount of T52 (mutant T4DDA-E94Y / G357Y) / T53 / T61 (mutant T4DDA-A372Y) proteins, and 0.1 U HRP / 100 uM H2O2 added, mixed, and incubated at 30°C for 18 hours. Afterwards, TBE PAGE gel was run at 160 V for 40 minutes and the enzyme cross-linking was detected. The results are shown in FIG5 . For the three selected Tyr-Tyr mutants, good enzyme cross-linking effects were observed under the catalysis of HRP / H2O2.

[0265] Example 1.4: Preparation of nucleic acid-protein complexes based on Tyr-Tyr crosslinking

[0266] The enzyme cross-linking product catalyzed by HRP / H2O2 in Example 1.1 was added to a DNAPac PA200 column and purified with an elution buffer to remove impurities such as the enzyme that was not bound to the adapter and the incompletely reacted EDC from the column.

[0267] The nucleic acid-protein complex was then eluted with a mixture of 10 column volumes of buffer A and buffer B. The main elution peaks were then pooled, their concentrations measured, and run on a TBE PAGE gel at 160V for 40 minutes. Buffer A: 20mM Na-CHES, 250mM NaCl, 4% (W / V) glycerol, pH 8.6; buffer B: 20mM Na-CHES, 1M NaCl, 4% (W / V) glycerol, pH 8.6. The elution process is shown in Figure 6. The individual peaks during the elution process were collected and detected on a TBE PAGE gel run at 160V for 40 minutes. The results are shown in Figure 7, indicating that E3 is the desired nucleic acid-protein complex.

[0268] Example 1.5: On-machine testing of nucleic acid-protein complexes based on Tyr-Tyr crosslinking

[0269] A 10 kb library was prepared by end repair, and the nucleic acid protein complex in Example 1.4 was connected to the library to build a library. Finally, sequencing was performed on the QNome-3841 of Qi Carbon Technology Co., Ltd., and its actual sequencing signal is shown in Figure 8. The nucleic acid protein complex can normally generate sequencing signals, and the signal steps are clear for subsequent base inference. The complete library pass-through signal can be seen. This shows that the helicase that forms the Tyr-Tyr ectopic bond between the 1A domain and the 2A domain can control the movement of the target polynucleotide and can be used for nanopore sequencing of polynucleotides. We subsequently analyzed its sequencing signal and found that it is superior to the traditional disulfide bond cross-linking (referring to the nucleic acid protein complex formed by T4Dda-E94C / A360C) in terms of noise and signal-to-noise ratio. The analysis results are shown in Table 3.

[0270] Example 1.6: Analysis of sequencing accuracy based on Tyr-Tyr crosslinking

[0271] A randomly fragmented human genomic library was prepared using end-repair and ligated with the nucleic acid-protein complex described in Example 1.4 or the disulfide-crosslinked SAC-M1 (nucleic acid-protein complex formed by T4Dda-E94C / A360C). Sequencing was performed on a QNome-3841 sequencing system from Qitan Technology Co., Ltd. Using a similarly small training dataset, the accuracy achieved using Tyr-Tyr crosslinking was higher than that achieved using traditional disulfide crosslinking. The analysis results are shown in Table 4.

[0272] Table 3 - Sequencing signals of nucleic acid-protein complexes based on Tyr-Tyr crosslinking

[0273] Table 4 - Analysis of sequencing accuracy based on Tyr-Tyr cross-linking

[0274] The meanings of the parameters in Table 3 and Table 4 are as follows:

[0275] Normalized noise: The signal value of each read is normalized by the Median Absolute Deviation (MAD). The signal and noise are separated by median filtering. The root mean square (RMS) value of the noise is calculated as an indicator of normalized noise. The smaller the normalized noise value, the less noise interference the signal level is affected by, that is, the clearer the signal is.

[0276] Noise: Perform median filtering on the signal value of each read to obtain the filtered noise signal. Calculate the root mean square (RMS) value of the noise signal as a noise indicator, reflecting the relative intensity of the noise. A smaller noise value indicates less noise interference.

[0277] Signal-to-noise ratio: The signal value of each read is normalized by the median absolute deviation (MAD). The signal and noise are separated by median filtering. The signal-to-noise ratio is expressed as the ratio of signal power to noise power. A larger signal-to-noise ratio indicates better signal quality and less noise interference.

[0278] Train loss: It is the error between the predicted results of the neural network model and the actual results during the training of the training data set.

[0279] Val loss: It is the error between the predicted result of the validation dataset through the neural network model and the actual result.

[0280] ACC: It is the accuracy of the comparison between the predicted results of the neural network model and the actual results of the validation dataset.

[0281] Example 2.1: Preparation of nucleic acid-protein complexes based on Tyr-Tyr linkage

[0282] The main strand S1, antisense strand S2 and connecting strand YB were synthesized respectively, and their specific sequences are shown below:

[0283] Main strand S1: 5'-33333333333333333333333333333-GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCTGACT-3'

[0284] Antisense strand S2: 5'-Ru-CGACTTCTACCGTTTGACTCCGC-3'

[0285] Connection chain YB: 5'-GTCAGCAGGACCGAATGA / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeA / / i2OMeG / / i2OMeU / / i2O MeC / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeC / / i2OMeC / / i2OMeG / / i2OMeA / / i2OMeC / / i2OMeC / -3'

[0286] in:

[0287] The main strand S1 sequentially comprises the leader sequence iSpC3 (i.e., a nucleotide lacking a sugar and a base, indicated as 3), which is linked to the 5' end of SEQ ID NO: 1. The 3' end of SEQ ID NO: 1 is sequentially linked to the blocking strand iSpC18 (indicated as 8888) and the 5' end of SEQ ID NO: 2. iSpC3 and iSpC18 were purchased from Integrated DNA Technologies.

[0288] The sequence of the antisense strand S2 is shown in SEQ ID NO:3.

[0289] The connecting chain YB comprises the sequence shown in SEQ ID NO: 4, and its 3' end is connected to multiple methoxy modifications, including i2OMeA, i2OMeC, i2OMeG, and i2OMeU.

[0290] The main strand S1, antisense strand S2, and linker strand YB were mixed and annealed at a ratio of 1:1.1:1.1 to form an adapter. The annealing treatment was performed by slowly cooling the temperature from 95°C to 25°C, with the cooling rate not exceeding 0.1°C / s.

[0291] 2 μM adapter was mixed with 4 times the amount of wild-type T4 Dda protein (SEQ ID NO: 5) and 1 mM 1,3-propanediamine and 1,4-butanediamine, respectively, and incubated at 30°C for 0.5 h. 2.5 mM sodium persulfate was then added and the cells were placed under a blue LED 450 nm light source (distance 15 cm, radiation power 50 mW / cm 2 ) for 1 min, and then run TBE PAGE gel at 160 V for 40 min to detect the enzyme cross-linking. The results are shown in Figure 9. The results show that tyrosine can be oxidized under the catalysis of Ru to form an effective nucleic acid-protein complex with the amino linker.

[0292] Example 2.2: Preparation of Lys-Glu-linked Nucleic Acid-Protein Complex

[0293] The main strand S1, antisense strand S2 and connecting strand YB were synthesized respectively, and their specific sequences are shown below:

[0294] Main strand S1: 5'-33333333333333333333333333333-GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCTGACT-3'

[0295] Antisense strand S2: 5'-CGACTTCTACCGTTTGACTCCGC-3'

[0296] Connection chain YB: 5'-GTCAGCAGGACCGAATGA / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeU / / i2OMeA / / i2OMeG / / i2OMeU / / i2O MeC / / i2OMeC / / i2OMeA / / i2OMeG / / i2OMeC / / i2OMeA / / i2OMeC / / i2OMeC / / i2OMeG / / i2OMeA / / i2OMeC / / i2OMeC / -3'

[0297] in:

[0298] The main strand S1 sequentially comprises the leader sequence iSpC3 (i.e., a nucleotide lacking a sugar and a base, indicated as 3), which is linked to the 5' end of SEQ ID NO: 1. The 3' end of SEQ ID NO: 1 is sequentially linked to the blocking strand iSpC18 (indicated as 8888) and the 5' end of SEQ ID NO: 2. iSpC3 and iSpC18 were purchased from Integrated DNA Technologies.

[0299] The sequence of the antisense strand S2 is shown in SEQ ID NO:3.

[0300] The connecting chain YB comprises the sequence shown in SEQ ID NO: 4, and its 3' end is connected to multiple methoxy modifications, including i2OMeA, i2OMeC, i2OMeG, and i2OMeU.

[0301] The main strand S1, antisense strand S2, and linker strand YB were mixed and annealed at a ratio of 1:1.1:1.1 to form an adapter. The annealing treatment was performed by slowly cooling the temperature from 95°C to 25°C, with the cooling rate not exceeding 0.1°C / s.

[0302] 1 μM adapter was mixed with 0, 1, 2, and 5 times the amount of wild-type T4 Dda protein (SEQ ID NO: 5) and 5 mM EDC, and incubated at 30°C for 4 h. The reaction was terminated by adding 20 mM β-ME. The enzyme cross-linking was then detected by TBE PAGE gel run at 160 V for 40 min. The results are shown in Figure 10. The results show that the proportion of nucleic acid-protein complexes gradually increased with increasing protein concentration, and effective nucleic acid-protein complexes can be formed.

[0303] Example 2.3: Purification and quality control of nucleic acid-protein complexes

[0304] The incubation binding product with 5 times the amount of substance added in Example 2.2 was added to a DNAPac PA200 column and purified with buffer A and buffer B to remove impurities such as enzymes that were not bound to the adapter and incompletely reacted EDC from the column.

[0305] The nucleic acid-protein complex was then eluted with a mixture of 10 column volumes of buffer A and buffer B. The main elution peaks were then pooled, their concentrations were measured, and the gel was run on a TBE PAGE gel at 160 V for 40 min.

[0306] The components of buffer A are: 20 mM Na-CHES, 250 mM NaCl, 4% (W / V) glycerol, pH 8.6; the components of buffer B are: 20 mM Na-CHES, 1 M NaCl, 4% (W / V) glycerol, pH 8.6.

[0307] The elution process is shown in FIG11 . The peaks in the elution process were collected and detected by TBE PAGE gel running at 160 V for 40 min. The results are shown in FIG12 . E2 is the desired nucleic acid-protein complex.

[0308] The eluted main peak was quantified using Qubit, and 10 ng was taken for quality control. 1*Seq buffer (10 mM HEPES 7.0, 600 mM KCl, 25 mM ATP, 25 mM MgCl2), NF-H20 (control group, a certain amount of ultrapure water was added to make up the reaction system), or 0.1% SDS was added, treated at 35°C for 30 minutes, and then analyzed on a TBE PAGE gel run at 160V for 40 minutes. The results are shown in Figure 13. The results show that the spacer can successfully block the helicase in the complex, and the amount of nucleic acid released by the quality control enzyme is relatively small. No residual nucleic acid-protein complex was observed in the SDS-treated group, indicating that the constructed lock-enzyme complex did not form a covalent crosslink with the nucleic acid.

[0309] Example 2.4: ATP consumption detection of nucleic acid-protein complexes

[0310] Approximately 1 ng of the purified nucleic acid-protein complex from Example 2.3 was added to 200 μL of 1*Seq Buffer (10 mM HEPES 7.0, 600 mM KCl, 25 mM ATP, 25 mM MgCl2). A control of enzyme-free water without the addition of nucleic acid-protein complex was used. The ADP content was measured by HPLC at 0 h, 4 h, and 8 h following elution on a C18 column based on the peak elution time. The results are shown in Figure 14. The results demonstrate that under this locked enzyme regime, the nucleic acid-protein complex retains ATPase activity and the protein continues to function normally.

[0311] Example 2.5: Sequencing function test of nucleic acid-protein complex

[0312] A 10 kb library was prepared using end-repair and ligated with the nucleic acid-protein complex prepared in Example 2.2 using the QLK-V1.1.1 library construction kit from Qitan Technology. Sequencing was performed on a QNome-3841 sequencer from Qitan Technology. The actual sequencing signal is shown in Figure 15. The results show that the nucleic acid-protein complex can generate sequencing signals normally, with clear signal steps sufficient for subsequent base inference.

[0313] Comparative Example 2.1: Sequencing Function Test of Nucleic Acid-Protein Complex Without Crosslinking

[0314] The method for preparing the nucleic acid-protein complex without forming cross-links is similar to that in Example 2.2, except that no EDC is added during the preparation process, that is, the enzyme does not form cross-links.

[0315] The library was constructed and sequenced using the same method as in Example 2.5, and the results are shown in Table 5. The results showed that, compared with Example 2.5, the enzyme in the complex easily fell off during sequencing when the enzyme was not cross-linked, making it impossible to obtain effective sequencing signals, let alone a complete 10 kb sequencing signal.

[0316] Table 5 Test results of Example 2.5 and Comparative Example 2.1

[0317] Note: A valid signal is a signal that can be subsequently identified by basecalling and converted into a sequence.

Claims

1. A helicase, wherein (a) comprising a polynucleotide binding domain and two RecA-like domains, wherein two or more amino acid residues on the surface of the helicase surrounding the polynucleotide binding domain are connected by an ectopic bond, so that the polynucleotide bound to the polynucleotide binding domain is surrounded by the structure formed after the connection; or (b) comprising a polynucleotide binding domain and two RecA-like domains, wherein two or more amino acid residues on the surface of the helicase surrounding the polynucleotide binding domain can be connected by an ectopic bond, so that the polynucleotide bound in the polynucleotide binding domain is surrounded by the connected structure, and at least one of the two or more amino acid residues is a mutant amino acid residue; or (c) comprises a polynucleotide binding domain, wherein the polynucleotide binding domain comprises an opening in at least one conformational state through which a polynucleotide can be debound by the helicase, wherein the helicase forms an ectopic bond between two or more amino acids at the opening, and wherein the helicase retains its ability to control the movement of the target polynucleotide; or (d) comprising a polynucleotide binding domain, wherein the polynucleotide binding domain comprises an opening in at least one conformational state, through which a polynucleotide can be debound by the helicase, wherein the helicase is modified so that it can form an ectopic bond between two or more amino acids at the opening, and wherein the helicase retains its ability to control the movement of the target polynucleotide.

2. The helicase according to claim 1, wherein the ectopic bond does not include an ectopic bond formed with the participation of cysteine, and the ectopic bond includes an ectopic bond formed with the participation of any natural amino acid except cysteine, The arbitrary natural amino acid except cysteine ​​is selected from the group consisting of the following amino acids: Ile, Val, Leu, Phe, Met, Ala, Gly, Thr, Ser, Trp, Tyr, Pro, His, Glu, Gln, Asp, Asn, Lys, Arg, Preferably, the ectopic bond comprises Lys-His, Lys-Ser, Lys-Thr, Lys-Tyr, Lys-Lys, Lys-Glu, Lys-Asp, Lys-Gln, Lys-Arg, Arg-Glu, Lys-Met, Arg-Asp, Arg-Arg, Tyr-Tyr, Tyr-Trp, Met-Met ectopic bond or a combination thereof; and / or, the helicase consists of one or more monomers, And / or, the two amino acids forming the isopeptide bond are located in the same domain or in different domains.

3. The helicase according to any one of the preceding claims, wherein the helicase is derived from a member of the natural or modified helicase family of the following group: Dda helicase, Pifl-like helicase, Upfl-like helicase, UvrD / Rep helicase, Ski-like helicase, Rad3 / XPD helicase, NS3 / NPH-II helicase, DEAD helicase, DEAHi RHA helicase, RecG-like helicase, REcQ-like helicase, T1R-like helicase, Swi / Snf-like helicase and Rig-I-like helicase, preferably Dda helicase, more preferably T4-Dda helicase; And / or, the helicase is derived from a natural or modified member of the helicase family of the following group: RecD helicase, Upfl helicase, PcrA helicase, Rep helicase, UvrD helicase, Hel308 helicase, Mtr4 helicase, XPD helicase, NS3 helicase, Mssl 16 helicase, Prp43 helicase, RecG helicase, RecQ helicase, T1R helicase, RapA helicase and Hef helicase.

4. The helicase according to any one of the preceding claims, wherein the helicase is derived from a T4-Dda helicase and comprises: (1) Tyr-Tyr heterotopic bond formed by x1 and x2; (2) Lys-Gln heterotopic bond formed by x3 and x4; (3) Tyr-Lys heterotopic bond formed by x1 and x3; (4) Tyr-Gln heterotopic bond formed by x1 and x4; (5) Tyr-Lys heterotopic bond formed by x2 and x3; (6) Tyr-Gln heterotopic bond formed by x2 and x4; (7) Lys-Glu ectopic bond formed by x3 and x5; or (8)Any combination of the above.

5. A helicase according to any of the preceding claims, wherein the helicase comprises any mutant of the 1A domain and / or any mutant of the 2A domain, as long as the helicase retains its ability to control the movement of polynucleotides.

6. A polypeptide comprising the 1A domain and the 2A domain that form an ectopic bond, and a polynucleotide binding domain from the helicase according to any one of the preceding claims, and excluding other domains of the helicase.

7. A construct comprising a helicase or polypeptide as described in any one of claims 1-6, and a target polynucleotide bound to its polynucleotide binding domain, wherein two or more amino acid residues on the surface of the helicase surrounding the polynucleotide binding domain are connected by an isopeptide bond, and the bound target polynucleotide is surrounded by the structure after the isopeptide bond connection. 8 . The construct according to claim 7 , comprising an adapter, the target polynucleotide being connected to the adapter, and the construct having the ability to control the movement of the target polynucleotide.

9. The construct according to claim 7, wherein the construct comprises two or more helicases or polypeptides according to any one of claims 1-6.

10. The construct according to claim 8, wherein the target polynucleotide in the adapter is linked to a target analyte, and the construct has the ability to control the movement of the target analyte.

11. A polynucleotide comprising a sequence encoding the helicase according to any one of claims 1 to 5, the polypeptide according to claim 6, or the construct according to any one of claims 7 to 10, or consisting of a sequence thereof.

12. A vector comprising the polynucleotide of claim 11 operably linked to a promoter.

13. A host cell comprising the vector according to claim 12.

14. A method for preparing the helicase according to any one of claims 1 to 5, the polypeptide according to claim 6, or the construct according to any one of claims 7 to 10, comprising expressing the polynucleotide according to claim 11, transfecting cells with the vector according to claim 12, or culturing the host cell according to claim 13.

15. A method for controlling the movement of a target analyte, comprising contacting the target analyte with the helicase or polypeptide described in any one of claims 1-6, or the construct described in any one of claims 7-10, and thereby controlling the movement of the target analyte.

16. The method of claim 15, wherein the method is used to control the movement of a target analyte through a pore.

17. A method for characterizing a target analyte, comprising: (a) contacting a target analyte with a pore and a helicase or polypeptide according to any one of claims 1 to 6 or a construct according to any one of claims 7 to 10, such that the helicase controls the movement of the target analyte through the pore; and (b) obtaining one or more measurements as the target analyte moves relative to the pore, wherein the measurements represent one or more characteristics of the target analyte and thereby characterize the target analyte.

18. The construct according to claim 10, or the method according to 15-17, wherein the target analyte is selected from one or more of polynucleotides, polypeptides, polysaccharides and lipids, preferably polynucleotides or polypeptides, polysaccharides and lipids linked to polynucleotides, more preferably single-stranded polynucleotides, double-stranded polynucleotides or partially double-stranded polynucleotides; or The target analyte is a target polynucleotide, and the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide; and (v) whether the target polynucleotide is modified.

19. The method of any one of claims 17-18, wherein one or more characteristics of the target analyte are measured by electrical and / or optical measurements.

20. The method of claim 19, wherein the electrical measurement is a current measurement, an impedance measurement, a tunneling measurement, or a field effect transistor measurement.

21. A method according to any one of claims 17 to 20, wherein the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the helicase or construct.

22. The method according to any one of claims 17 to 21, wherein the pore is a nanopore or a transmembrane pore, or the pore is selected from a biological pore, a solid-state pore or a hybrid biological and solid-state pore.

23. The method of claim 22, wherein the biological pore is derived from hemolysin, leukocidin, CsGG, Mycobacterium smegmatis porin A (MspA), porin B, porin C, porin D, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria autotransporter, and WZA; The solid-state pores are derived from graphene nanopores, MoS2 nanopores, BN nanopores or PA63 nanopores.

24. A method of forming a sensor for characterizing a target analyte, comprising forming a complex between (a) a pore and (b) a helicase or polypeptide as described in any one of claims 1 to 6 or a construct as described in any one of claims 7 to 10 and thereby forming a sensor for characterizing the target analyte.

25. A sensor for characterizing a target analyte, comprising a complex between (a) a pore and (b) a helicase or polypeptide according to any one of claims 1 to 6, or a construct according to any one of claims 7 to 10.

26. Use of a helicase or polypeptide as claimed in any one of claims 1 to 6, or a construct as claimed in any one of claims 7 to 10, for controlling the movement of a target analyte through a pore.

27. A kit for characterizing a target analyte, comprising (a) a pore and (b) a helicase or polypeptide according to any one of claims 1 to 6, or a construct according to any one of claims 7 to 10.

28. A device for characterizing a target analyte, comprising (a) a plurality of pores and (b) a plurality of helicases or polypeptides according to any one of claims 1-6, or constructs according to any one of claims 7-10.

29. A method for preparing the helicase according to any one of claims 1 to 5, comprising: (a) providing a helicase, preferably a Dda helicase, more preferably a T4-Dda helicase; and (b) forming an ectopic bond within the protein of the helicase to prepare the helicase according to any one of claims 1 to 5; Preferably, in step (b), the step of contacting the helicase with the polynucleotide is also included. Preferably, the ectopic bond is formed by catalysis using H2O2, Hemin and H2O2, HRP enzyme and H2O2, transglutaminase, EDC, or Ru. More preferably, HRP enzyme and H2O2 are used. 2、 or EDC.

30. A method for preparing the construct of any one of claims 7-10, comprising connecting the helicase or polypeptide of any one of claims 1-6 to the adaptor and thereby preparing the construct.

31. A group of two or more helicases attached to a target analyte, wherein at least one of the two or more helicases is the helicase described in any one of claims 1-5.

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