Kph pif1 helicase and uses thereof

Abstract

This disclosure pertains to the field of gene sequencing technology and discloses the KphPif1 helicase and its applications. This disclosure relates to a modified helicase, a polynucleotide encoding the helicase, a recombinant expression vector, and a construct capable of expressing the modified helicase and used to construct recombinant cells expressing the helicase. The modified helicase disclosed herein allows target polynucleotides to pass through nanopores in a controllable and stepwise manner, stabilizing polynucleotide movement and preventing detachment from the polynucleotide. Furthermore, it features a wide current range, high decoding accuracy, fast perforation speed, and high sequencing efficiency.

Description

Technical Field

[0001] This disclosure relates to KphPif1 helicase and its applications, and belongs to the field of gene sequencing technology. Background Technology

[0002] Nanopore sequencing technology refers to gene sequencing technology that uses single nucleic acid molecules as measurement units and utilizes nanopores to perform real-time continuous reading of their sequence information. When a single molecule passes through the neck of a nanopore, it blocks a constant ion current (electrical impedance effect), resulting in a decrease in current. The magnitude and duration of this decrease are closely related to the particle size, charge state, and length of the single molecule passing through the nanopore. Therefore, the picoampere-level current change caused by an electric field driving a single molecule through a nanopore can be used to characterize the information of a single molecule. Based on the principle of electrical signal detection, when a nucleic acid chain passes through a nanopore, different bases block the pore channel, generating different pore-blocking current signals. By analyzing the signal characteristics through algorithms, nucleic acid sequence information can be obtained. Currently, bionanopore analysis technology has gradually developed into an important single-molecule analysis method. Its advantages, such as high analytical sensitivity (ultra-high time resolution ~10 μs and current resolution <0.1 pA), speed, low cost, and the absence of molecular modification labels (such as fluorescent labels, radioactive labels) or surface immobilization, make it widely applicable in many fields such as biology and chemistry.

[0003] However, one of the challenges facing this technology is that the polynucleotide perforation speed is too fast, exceeding the current detection limits of electronics, resulting in a current level for a single nucleotide that is too short to be distinguished, leading to low accuracy. This is especially true when dealing with long nucleotide sequences, such as 500 or more nucleotides, where helicases may detach from the nucleic acid sequence and pass through the nanopore uncontrollably and rapidly. Therefore, reducing the nucleic acid perforation speed and preventing polynucleotide detachment are crucial for improving nanopore sequencing accuracy. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] To address the problem of excessively fast polynucleotide perforation speed in existing technologies, a novel modified helicase is provided.

[0006] Solution for solving the problem

[0007] To address the aforementioned technical issues, this disclosure provides a DNA-dependent ATPase (KphE1) helicase mutant derived from Klebsiella pneumoniae phage E1, which overcomes the problem of excessively rapid migration of polynucleotides through nanopores in existing technologies and effectively controls the perforation movement of polynucleotides during chain sequencing.

[0008] This disclosure provides a helicase mutant, wherein, compared to a helicase containing the sequence shown in SEQ ID NO:19, the helicase mutant introduces at least one new amino acid residue or linker molecule into the 1B and 2B domains of the helicase, respectively, such that the 1B and 2B domains are linked.

[0009] The 1B structural domain contains the sequence shown in SEQ ID NO:2; the 2B structural domain contains the sequence shown in SEQ ID NO:3;

[0010] Optionally, cysteine ​​is introduced at one or more of positions 95, 96, 360, 363, 367, 377, 381 and 384 of the sequence shown in SEQ ID NO:19;

[0011] Preferably, two or more introduced cysteine ​​residues are interconnected.

[0012] In some embodiments, the helicase mutant further contains a mutation at at least one of positions 116, 138, and 313 corresponding to the sequence shown in SEQ ID NO:19, and the mutated amino acid is not cysteine.

[0013] In some embodiments, the helicase mutant is selected from any one of the following groups (I)-(V):

[0014] (I) Compared with the sequence shown in SEQ ID NO:19, the helicase mutant has a mutated amino acid at at least one of the following positions corresponding to the sequence shown in SEQ ID NO:19:

[0015] D96C, A363C, C116A, C138A, C313A;

[0016] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:19;

[0017] (III) A mutant encoded by a polynucleotide that can hybridize with the polynucleotide shown in (a) or (b):

[0018] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I);

[0019] (b) A full-length complementary polynucleotide to (a);

[0020] (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses helicase activity;

[0021] (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0022] In some optional embodiments, the helicase mutant, compared to the sequence shown in SEQ ID NO:19, further conforms to SEQ ID NO:19. The sequence shown in NO:19 contains a mutation at at least one of the following positions: positions 1, 42, 56, 57, 85, 100, 116, 152, 158, 160, 196, 212, 214, 223, 241, 353, 357, 361, 364, 389, 418, 201, 203, 196, 218, 179, 204, 381, 103, 104, 94, 362, 361, 364, 158, 57, 212, 223, 353, 249, 193, 357, 350, 214, 187, 171, 203, 218, 226, 263, 326, 191, 187, 202, 230, 128, 136, 146, 168, and 176.

[0023] Preferably, the helicase mutant corresponds to the sequence shown in SEQ ID NO:19 and has a mutated amino acid at at least one of the following positions:

[0024] T42Y, A160F, C116L, D364I, F100W, F357A, K196L, K223E, K361I, K361S, N353E, S57K, T212K, V152H, V152L, V158F, V418R, V418W, V56K, S85K, T214A, T241D, D361A, D364Q, K389M, the first M mutates into two consecutive Gs.

[0025] In some embodiments, the helicase mutant corresponds to the sequence shown in SEQ ID NO:19 and has the following (m1) to (m2) characteristics. 41 Any mutation shown in any of the following:

[0026] (m1) D96C, A363C, C116A, C138A, C313A; (m2) D96C, A363C, C116A, C138A, C313A, the first M mutates into two consecutive Gs; (m3) D96C, A363C, C116A, C138A, C313A, T42Y; (m4) D96C, A363C, C116A, C138A, C313A, V56K; (m5) D96C, A363C, C116 A, C138A, C313A, S57K; (m6) D96C, A363C, C116A, C138A, C313A, S85K; (m7) D96C, A363C, C116A, C138A, C313 A, F100W; (m8) D96C, A363C, C116A, C138A, C313A, C116L; (m9) D96C, A363C, C116A, C138A, C313A, V152H; (m 10 )D96C, A363C, C116A, C138A, C313A, V152L; (m 11 )D96C, A363C, C116A, C138A, C313A, V158F; (m 12 )D96C, A363C, C116A, C138A, C313A, A160F; (m 13 )D96C, A363C, C116A, C138A, C313A, K196L; (m 14 )D96C, A363C, C116A, C138A, C313A, T212K; (m 15 )D96C, A363C, C116A, C138A, C313A, T214A; (m 16 )D96C, A363C, C116A, C138A, C313A, K223E; (m 17 )D96C, A363C, C116A, C138A, C313A, T241D; (m 18 )D96C, A363C, C116A, C138A, C313A, N353E; (m 19 )D96C, A363C, C116A, C138A, C313A, F357A; (m 20 )D96C, A363C, C116A, C138A, C313A, K361I; (m 21 )D96C, A363C, C116A, C138A, C313A, K361S; (m 22)D96C、A363C、C116A、C138A、C313A、D361A;(m 23 )D96C、A363C、C116A、C138A、C313A、D364I;(m 24 )D96C、A363C、C116A、C138A、C313A、D364Q;(m 25 )D96C、A363C、C116A、C138A、C313A、K389M;(m 26 )D96C、A363C、C116A、C138A、C313A、V418R;(m 27 )D96C、A363C、C116A、C138A、C313A、V418W;(m 28 )D96C、A363C、C116A、C138A、C313A、V158F、T42Y;(m 29 )D96C、A363C、C116A、C138A、C313A、V158F、F357A;(m 30 )D96C、A363C、C116A、C138A、C313A、V158F、V418R;(m 31 )D96C、A363C、C116A、C138A、C313A、A160F、K196L、K389M;(m 32 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、A160F;(m 33 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、N353E;(m 34 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、K361I;(m 35 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、K389M;(m 36 )D96C、A363C、C116A、C138A、C313A、N353E、A160F、K196L;(m 37 )D96C、A363C、C116A、C138A、C313A、N353E、K196L;(m 38 )D96C、A363C、C116A、C138A、C313A、N353E、A160F、K361I;(m 39)D96C, A363C, C116A, C138A, C313A, N353E, K361I, K389M; (m 40 )D96C, A363C, C116A, C138A, C313A, N353E, K389M; (m 41 )D96C, A363C, C116A, C138A, C313A, F357A, D364I, K361I.

[0027] This disclosure provides any one of the following (i) to (iii):

[0028] (i) A construct comprising at least one helicase mutant as described above; optionally, the construct further comprises a polynucleotide-binding moiety;

[0029] (ii) encoding a helicase mutant as described above or a polynucleotide of (i);

[0030] (iii) Expressing the helicase mutant as described above or a recombinant expression vector containing (ii).

[0031] This disclosure provides a recombinant host cell, wherein the recombinant host cell comprises a helicase mutant as described above, a polynucleotide as described above, or a recombinant expression vector as described above.

[0032] This disclosure provides a cell culture comprising the recombinant cells as described above.

[0033] This disclosure provides a method for controlling the movement of polynucleotides, comprising contacting the polynucleotides with a helicase mutant as described above or a construct as described above.

[0034] This disclosure provides a method for characterizing a target polynucleotide, comprising:

[0035] (A) Contacting the target polynucleotide with a transmembrane pore and a helicase mutant or construct as described above, such that the helicase controls the movement of the target polynucleotide through the pore; and

[0036] (B) As the polynucleotide moves relative to the pore, one or more measurements are acquired, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide;

[0037] Optionally, the method further comprises the step of applying a potential difference across a pore in contact with the helicase or construct and the target polynucleotide;

[0038] Optionally, the pores are nanopores;

[0039] Preferably, the nanopore includes a biological nanopore, a solid nanopore, or a hybrid of a biological nanopore and a solid nanopore;

[0040] Optionally, the target polynucleotide is single-stranded, double-stranded, or at least partially double-stranded;

[0041] Optionally, the one or more features are selected from at least one of the following: source, length, identity, sequence, secondary structure, or whether the target polynucleotide is modified.

[0042] This disclosure provides a product for characterizing a target polynucleotide, wherein the product comprises a helicase mutant as described above, a construct as described above, a polynucleotide as described above, a recombinant expression vector as described above, a recombinant host cell as described above, or a cell culture as described above, and a well;

[0043] Optionally, the product is selected from reagent kits, devices, or sensors.

[0044] This disclosure provides the use of the helicase mutants, constructs, polynucleotides, recombinant expression vectors, recombinant host cells, cell cultures, or products described above in characterizing target polynucleotides or controlling the movement of target polynucleotides through pores.

[0045] The effects of the invention

[0046] The modified KphE1 helicase disclosed herein can control the movement of polynucleotides through biological nanopores, particularly under the influence of an electric field. The helicase enables the target polynucleotide to move through the nanopore in a controlled and progressive manner.

[0047] The modified KphE1 helicase mutants disclosed herein exhibit enhanced ability to control the translocation of polynucleotides through nanopores. These mutants typically have one or more modifications in the 1B or 2B domains. Therefore, the modified KphE1 helicases disclosed herein, with at least one cysteine ​​or non-natural amino acid insertion, still retain their ability to control polynucleotide movement.

[0048] This disclosure also provides a modified KphE1 helicase in which the 1B and 2B domains are covalently linked by a linker molecule, which improves the stability of the KphE1 helicase bound to polynucleotides. In particular, when the polynucleotide chain length increases, the helicase can still stably control the movement of polynucleotides without detaching from them. Furthermore, it has a wide current range, high decoding accuracy, improved speed distribution consistency, fast pore speed, and improved sequencing efficiency. Attached Figure Description

[0049] Figure 1 This is an SDS-PAGE gel electrophoresis image of the purified KphE1 helicase.

[0050] Figure 2 This is a schematic diagram of fluorescence analysis for detecting helicase activity.

[0051] Figure 3 The 4-10% SDS-PAGE gel electrophoresis image of KphE1-D96C / A363C / C116A / C138A / C313A (SEQ ID NO:1 with the mutation D96C / A363C / C116A / C138A / C313A) is shown.

[0052] Figure 4 The DNA construct X shown is shown.

[0053] Figure 5 An example current trajectory is shown as the KphE1 helicase controls the translocation of the intact DNA construct X through a nanopore, with the X-axis representing time (s) and the Y-axis representing current (nA).

[0054] Figure 6 Showing Figure 6 The image shows an enlarged view of the region where the KphE1 helicase controls the movement of the DNA construct through the X-pore. The X-axis represents time (s) and the Y-axis represents current (nA).

[0055] Figure 7 The DNA construct Y is shown.

[0056] Figure 8A and Figure 8D The perforation rate of the KphE1 helicase mutant was shown.

[0057] Figure 8B and Figure 8E The current noise of the KphE1 helicase mutant is shown.

[0058] Figure 8C and Figure 8F The current range of the KphE1 helicase mutant is shown. Detailed Implementation

[0059] Various exemplary embodiments, features, and aspects of this disclosure will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0060] Furthermore, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the main points of this disclosure.

[0061] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this disclosure should be understood to include systematic errors that are unavoidable in industrial production.

[0062] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0063] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0064] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0065] In this specification, the terms "peptide" and "protein" are used interchangeably herein and refer to an amino acid polymer of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds). The polymer may be linear, branched, or cyclic, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, ubiquitination, glycosylation, amidation of C-terminal amino acids, or any other manipulation, such as conjugation with labeled components).

[0066] In this specification, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids and their abbreviations and English abbreviations in this disclosure are as follows: histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine ​​(Cys, C); lysine (Lys, K); proline (Pro, P).

[0067] In this specification, the term "helicase" refers to a class of enzymes that facilitate the unwinding of the DNA double helix, found in prokaryotes and eukaryotes. These enzymes move directionally along the nucleic acid strand, primarily using single-stranded nucleic acid as their orbital path, utilizing ATP hydrolysis for energy to break hydrogen bonds in the DNA and open the double helix structure, thereby obtaining single-stranded DNA. In some exemplary embodiments of this disclosure, a DNA-dependent ATPase (KphE1) helicase derived from Klebsiella phage E1 contains the amino acid sequence shown in Genbank:QEM42218.1, as shown in SEQ ID NO:19:

[0068] MITFDMLNKGQKKAFDYIISRIKAGKGNHITLNGPAGTGKTTMTKFIVDYLISQGVSGVVLAAPTHAAKKVLSKLSGIEARTIHSLLKINPTTYEDSVTFEQKGDVDVSE LRVVVCDEASMYDRKLFQILMATIPRYCLVIAIGDKAQIRPVEPGSTVPALSPFFSHKDFDQLELDEVMRSNAPIIKVATDIRNGKWIYDHQRDGHGVHGFTSTTALKDFM MKYFEIVKDPEDMFENKMFAFTNKSVDKLNSIIRRRILETEDAFITGEVIVMQEPLIKELEFEGKRFNDLKFNNGQYVRIVSAEYASSFVSCKGVSGEYMLRHWRLTVET YDKDEDYHLETINVISDEQEQNKFQFFLAKAADTYRNWQKGGKAPWKSFWAAKRMFHKVKPLPASTFHKAQGLSVDTSFLYTPCIHVADAELAQQLLYVGTTRGRHDVYYV

[0069] In this specification, the term "fragment" refers to a polypeptide or a catalytic or carbohydrate-binding module that has one or more (e.g., several) amino acids deleted from the amino and / or carboxyl ends of a mature polypeptide or domain. In the technical solutions of this disclosure, the fragment has helicase activity.

[0070] In this specification, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms.

[0071] In this specification, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. Exemplarily, a "mutant" in this disclosure is a polypeptide having enhanced helicase activity.

[0072] In this specification, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In this disclosure, the term "mutation" refers to an alteration of a nucleotide sequence or amino acid sequence. In one specific embodiment, the term "mutation" refers to "substitution."

[0073] In this disclosure, "mutation" can also include the addition, deletion, or substitution of amino acids at one or more positions corresponding to the sequence shown in SEQ ID NO:19 that do not affect the helicase activity. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, does not substantially alter its biological activity; for example, appropriately replacing, adding, or deleting certain amino acids results in sequences that do not affect their activity.

[0074] As used herein, the terms “corresponding” and “corresponding” have the meanings commonly understood by those skilled in the art. Specifically, “corresponding” and “corresponding” refer to the positions in one sequence that correspond to a specified position in another sequence after homology or sequence identity alignment.

[0075] In some embodiments, the term "mutation" in this disclosure may be selected from "conservative mutation." In this disclosure, the term "conservative mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conserved substitution.

[0076] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0077] In this specification, "conservative substitution" generally refers to the exchange of one amino acid at one or more sites in a protein. This substitution can be conserved. Examples of substitutions considered conserved include, for instance, the substitution of Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include: Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. In addition, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin.

[0078] In this specification, the terms "sequence identity" or "percentage of identity" in comparisons of two nucleic acids or peptides refer to the percentage of identical sequences or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed.

[0079] The methods disclosed herein for determining “sequence identity” or “percentage of identity” include, but are not limited to: Computational Molecular Biology, ed. Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, ed. Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, ed. Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, ed. Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0080] As used in this disclosure, the term "very stringent conditions" refers to, for probes at least 100 nucleotides in length, following a standard DNA blotting procedure, pre-hybridizing and hybridizing at 42°C in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0081] In this specification, the term "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides can be in the form of individual fragments or as a component of a larger nucleotide sequence structure, derived from a nucleotide sequence isolated at least once in number or concentration, and capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, T, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0082] In this disclosure, the term "codon optimization" refers to the configuration of a nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0083] In this specification, the term "isolated" means a substance in a form or environment not naturally occurring. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance, including but not limited to any enzyme, mutant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more naturally occurring components associated with it; (3) any substance artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant generation in a host cell; multiple copies of the gene encoding the substance; and the use of a promoter stronger than that naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples. For example, host cells may be genetically modified to express the polypeptides disclosed herein. Fermentation broth from host cells will contain isolated polypeptides. "Recombinant polynucleotide" is a type of "polynucleotide".

[0084] In this specification, the term "recombinant polynucleotide" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can be used for transformation into a suitable host cell. A host cell containing the recombinant polynucleotide is referred to as a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0085] In this specification, the term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0086] In this specification, the term "construction" refers to a DNA construct, which is an artificially constructed nucleic acid fragment produced by inserting one or more target DNA fragments into a vector backbone, and which can also carry one or more target DNA fragments into target tissues or target cells.

[0087] In this specification, the term "expression vector" refers to a DNA sequence operatively linked to a suitable control sequence to express a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired exogenous polypeptide. Recombinant expression vectors may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into a protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. For example, the expression vectors include, but are not limited to, pET series, Duet series, pGEX series, pHY300, pHY300PLK or pQlink series vectors that can be replicated and expressed in prokaryotic cells.

[0088] In this specification, the term "host cell" refers to any cell type that is easily transformed, transfected, or transduced using a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector containing the present disclosure. The term "recombinant host cell" encompasses a host cell that differs from the parent cell after the introduction of a polynucleotide encoding a mutant polypeptide or a recombinant expression vector; the recombinant host cell is specifically achieved through transformation. The host cell of this disclosure can be a prokaryotic or eukaryotic cell, as long as it is a cell capable of introducing the polynucleotide encoding a helicase-encoding polypeptide or recombinant polypeptide of this disclosure.

[0089] In this specification, the term "cell culture" refers to a combination of cells and cell culture medium, wherein the cells are cultured in a cell culture medium outside the organism.

[0090] In this specification, the terms "transformation," "transfection," and "transduction" have the meanings commonly understood by those skilled in the art, referring to the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0091] The host cell culture disclosed herein can be performed according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0092] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0093] The technical solution of this disclosure is described in detail below:

[0094] helicase mutant

[0095] This disclosure uses a DNA-dependent ATPase (KphE1) helicase derived from Klebsiella pneumoniae phage E1. This enzyme has good helical activity, but it is prone to detachment in the spacer region, making it difficult to effectively control the movement of polynucleotide pores during strand sequencing and failing to detect pore signals.

[0096] Therefore, the inventors predicted the structure of KphE1 helicase using alphafold2 and found that KphE1 helicase contains four domains: 1A, 2A, 1B, and 2B. Domains 1A and 2A are mainly involved in ATP binding and hydrolysis; domain 1B is a large β-hairpin structure; domain 2B adopts an SH-3-like folding pattern, with its orbital-like β-hairpin structure close to domain 1B. Therefore, this disclosure focuses on domains 1B and 2B when reducing the opening size through modification and linkage, tending to use covalent linkage to reduce or close the opening.

[0097] There are two ways to achieve covalent linkage. One is through the natural amino acids themselves, by replacing or inserting new amino acids, such as cysteine ​​or non-natural amino acids. The other is through linker molecules, which tend to link cysteine ​​residues. Linker molecules include BMOE, Bis(PEG)2, and Bis(PEG)3.

[0098] To address the problem of excessively fast polynucleotide perforation and to improve the stability of the KphE1 helicase binding to polynucleotides and reduce its ability to detach from polynucleotides, this disclosure replaces / inserts new amino acids, such as cysteine ​​and non-natural amino acids, into the 1B and 2B domains of the KphE1 helicase, or achieves the linkage between cysteine ​​residues through linker molecules.

[0099] In some optional embodiments, the introduced cysteine ​​is interconnected with each other, with non-natural amino acids, with non-natural amino acids, with non-natural amino acids, with natural amino acids on the helicase, or with natural amino acids on the helicase.

[0100] Two or more introduced cysteines and / or non-natural amino acids of any number and combination can be linked together. For example, 2, 3, 4, 5, 6, 7, 8 or more cysteines and / or non-natural amino acids can be linked together. One or more cysteines can be linked to one or more cysteines. One or more cysteines can be linked to one or more non-natural amino acids such as Faz. One or more non-natural amino acids such as Faz can be linked to one or more non-natural amino acids such as Faz. One or more cysteines can be linked to one or more native amino acids on a helicase. One or more non-natural amino acids such as Faz can be linked to one or more native amino acids on a helicase.

[0101] The connection can be any type, including direct or indirect connection. Preferably, the connection can be a temporary contact or a permanent connection. More preferably, the connection can be a non-covalent connection or a covalent connection.

[0102] In one specific embodiment of this disclosure, the covalent linkage can be achieved using chemical crosslinking agents, linear molecules, or catalysts. The chemical crosslinking agents include, but are not limited to, maleimide (BMOE), active esters, succinimide, azides, alkynes (such as dibenzocyclooctylenol (DIBO or DBCO), difluorocycloalkynes, and linear alkynes), Bis crosslinking agents (Bis(PEG)2, Bis(PEG)3), etc.; the length of the chemical crosslinking agent can vary from one carbon (carbonyl chloride type connector) to multiple angstroms. The linear molecules include, but are not limited to, polyethylene glycol (PEGs), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threonucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, and polyamides. The catalysts include, but are not limited to, any catalyst such as TMAD that can form covalent bonds between cysteine ​​residues, between non-natural amino acids, between cysteine ​​residues and non-natural amino acids, between non-natural amino acids and natural amino acids, or between cysteine ​​residues and natural amino acids.

[0103] In some embodiments, this disclosure introduces cysteine ​​residues into the 1B and 2B domains, respectively. The aforementioned linker molecule typically contains two functional ends that are covalently linked to the cysteine ​​residues to achieve the linking of the 1B and 2B parts, so that the bound polynucleotide will not dissociate from the helicase.

[0104] In some exemplary embodiments, the 1B domain comprises the following amino acids:

[0105] KINPTTYEDSVTFEQKGD(SEQ ID NO:2);

[0106] The amino acids contained in the 2B domain are:

[0107] SDEQEQNKFQFFLAKAADTYRNWQKGGKAPWKSFWAAKRMF (SEQ ID NO: 3).

[0108] Another aspect of this disclosure provides a modified KphE1 helicase that introduces new cysteine ​​residues for achieving the ligation, with the introduction sites including E95, D96, A360, A363, R367, W377, W381, and K384.

[0109] For example, cysteine ​​residues are introduced at sites such as D96 and A363 on the 1B and 2B domains to achieve intramolecular cross-linking of the protein, and further modifications are made to amino acid residues that hinder intramolecular cross-linking, thereby obtaining helicase mutants with enhanced DNA binding ability and effective control over DNA pores. In some embodiments, the amino acid residues that hinder intramolecular cross-linking may be present in and / or between the 1B and 2B domains; the modifications include substituting native amino acid residues with alanine (A) or methionine (M).

[0110] In some exemplary embodiments, this disclosure modifies the three natural cysteines C116, C138 and C313 in the amino acid sequence of KphE1. Preferably, one or more of the natural cysteines are replaced with alanine (A) or methionine (M).

[0111] Building upon this foundation, the remaining amino acid residues are modified to further enhance helicase performance, improve sequencing speed, and increase sequencing accuracy. For example, modifications can be made to protein activity to increase sequencing speed (T42, C116, M1, S57, etc.), reduce protein surface charge to improve protein synergy (A160, D364, F357, K196, K223, K361, N353, T212, V158, etc.), enhance interaction with ssDNA, and improve the resolution between signals of different base combinations (F100, V152, V158, V418, etc.).

[0112] More specifically, in addition to the introduction of cysteine ​​residues, the helicase mutant is further selected from any one of the following groups (I)-(V):

[0113] (I) The helicase mutant contains a mutation at at least one of the positions 96, 363, 116, 138, and 313 corresponding to the sequence shown in SEQ ID NO:19, compared to the sequence shown in SEQ ID NO:19;

[0114] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:19;

[0115] (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions:

[0116] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I);

[0117] (b)(a) full-length complementary polynucleotides;

[0118] (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses helicase activity;

[0119] (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0120] In some embodiments, the helicase mutant corresponds to the sequence shown in SEQ ID NO:19 and has a mutated amino acid at at least one of the following positions:

[0121] D96C, A363C, C116A, C138A, C313A.

[0122] Furthermore, compared to the sequence shown in SEQ ID NO:19, the helicase mutant has the following characteristics in the sequence corresponding to SEQ ID NO:19. The sequence shown in IDNO:19 contains a mutation at at least one of the following positions: positions 1, 42, 56, 57, 100, 116, 152, 158, 196, 212, 223, 260, 364, 353, 357, 361, 418, 201, 203, 196, 218, 179, 204, 381, 103, 104, 94, 362, 361, 364, 158, 57, 212, 223, 353, 249, 193, 357, 350, 214, 187, 171, 203, 218, 226, 263, 326, 191, 187, 202, 230, 128, 136, 146, 168, and 176.

[0123] In some preferred embodiments, the helicase mutant corresponds to the sequence shown in SEQ ID NO:19 having a mutated amino acid at at least one of the following positions:

[0124] M1GG (i.e., the methionine M at position 1 is mutated to two consecutive glycine Gs), T42Y, A160F, C116L, D364I, F100W, F357A, K196L, K223E, K361I, K361S, M1G, N353E, S57K, T212K, V152H, V152L, V158F, V418R, V418W, V56K, S85K, T214A, T241D, D361A, D364Q, K389M.

[0125] By introducing mutations at the aforementioned sites, the interaction between the helicase mutant and single-stranded DNA can be enhanced, while the interaction with porins can be weakened, resulting in improved velocity distribution uniformity and increased pore-passing speed.

[0126] In some preferred embodiments, the helicase mutant corresponds to the sequence shown in SEQ ID NO:19 and has the following (m1) to (m2) sequences. 41 Any mutation shown in any of the following:

[0127] (m1)D96C, A363C, C116A, C138A, C313A; (m2) D96C, A363C, C116A, C138A, C313A, M1GG; (m3) D96C, A363C , C116A, C138A, C313A, T42Y; (m4) D96C, A363C, C116A, C138A, C313A, V56K; (m5) D96C, A363C, C116A, C13 8A, C313A, S57K; (m6) D96C, A363C, C116A, C138A, C313A, S85K; (m7) D96C, A363C, C116A, C138A, C313A, F100W; (m8) D96C, A363C, C116A, C138A, C313A, C116L; (m9) D96C, A363C, C116A, C138A, C313A, V152H; (m 10 )D96C, A363C, C116A, C138A, C313A, V152L; (m 11 )D96C, A363C, C116A, C138A, C313A, V158F; (m 12 )D96C, A363C, C116A, C138A, C313A, A160F; (m 13 )D96C, A363C, C116A, C138A, C313A, K196L; (m 14 )D96C, A363C, C116A, C138A, C313A, T212K; (m 15 )D96C, A363C, C116A, C138A, C313A, T214A; (m 16 )D96C, A363C, C116A, C138A, C313A, K223E; (m 17 )D96C, A363C, C116A, C138A, C313A, T241D; (m 18 )D96C, A363C, C116A, C138A, C313A, N353E; (m19 )D96C、A363C、C116A、C138A、C313A、F357A;(m 20 )D96C、A363C、C116A、C138A、C313A、K361I;(m 21 )D96C、A363C、C116A、C138A、C313A、K361S;(m 22 )D96C、A363C、C116A、C138A、C313A、D361A;(m 23 )D96C、A363C、C116A、C138A、C313A、D364I;(m 24 )D96C、A363C、C116A、C138A、C313A、D364Q;(m 25 )D96C、A363C、C116A、C138A、C313A、K389M;(m 26 )D96C、A363C、C116A、C138A、C313A、V418R;(m 27 )D96C、A363C、C116A、C138A、C313A、V418W;(m 28 )D96C、A363C、C116A、C138A、C313A、V158F、T42Y;(m 29 )D96C、A363C、C116A、C138A、C313A、V158F、F357A;(m 30 )D96C、A363C、C116A、C138A、C313A、V158F、V418R;(m 31 )D96C、A363C、C116A、C138A、C313A、A160F、K196L、K389M;(m 32 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、A160F;(m 33 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、N353E;(m 34 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、K361I;(m 35 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、K389M;(m 36 )D96C、A363C、C116A、C138A、C313A、N353E、A160F、K196L;(m37 )D96C, A363C, C116A, C138A, C313A, N353E, K196L; (m 38 )D96C, A363C, C116A, C138A, C313A, N353E, A160F, K361I; (m 39 )D96C, A363C, C116A, C138A, C313A, N353E, K361I, K389M; (m 40 )D96C, A363C, C116A, C138A, C313A, N353E, K389M; (m 41 )D96C, A363C, C116A, C138A, C313A, F357A, D364I, K361I.

[0128] In other specific embodiments, the mutant described above (m1) comprises the sequence shown in SEQ ID NO:1, further wherein the helicase mutant, compared with the sequence shown in SEQ ID NO:1, has the following characteristics corresponding to SEQ ID NO:1. The sequence shown in NO:1 contains a mutation at at least one of the following positions: 418, 201, 203, 196, 218, 179, 204, 381, 103, 104, 94, 362, 361, 364, 158, 57, 212, 223, 353, 249, 193, 357, 350, 214, 187, 171, 203, 218, 226, 263, 326, 191, 187, 202, 230, 128, 136, 146, 168, and 176. The mutated amino acid residues include any that can improve helicase sequencing performance, such as increasing sequencing speed, improving speed distribution consistency, reducing step noise, improving the resolution between signals of different base combinations, improving the synergy between proteins, and increasing the range of current signals.

[0129] Construct

[0130] This disclosure provides constructs comprising at least one helicase mutant as described herein.

[0131] In some implementations, the construct also includes a polynucleotide binding moiety.

[0132] In some preferred embodiments, the construct has the ability to control polynucleotide movement.

[0133] In some preferred embodiments, the polynucleotide-binding moiety is a portion that can bind to the bases of the polynucleotide, the sugar ring of the polynucleotide, and / or the phosphate group of the polynucleotide. In some optional embodiments, the helicase mutant constituting the construct and the polynucleotide-binding moiety can be prepared separately and then directly ligated.

[0134] Furthermore, the polynucleotide binding portion is a polypeptide capable of binding to polynucleotides, including but not limited to one or more of eukaryotic single-strand binding proteins, bacterial single-strand binding proteins, archaea single-strand binding proteins, viral single-strand binding proteins, or double-strand binding proteins.

[0135] Isolated polynucleotides

[0136] According to some embodiments of this disclosure, this disclosure provides an isolated polynucleotide encoding a helicase mutant or a construct as described above.

[0137] The polynucleotides disclosed herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0138] The polynucleotides encoding the mutants disclosed herein include: a coding sequence that encodes only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and a non-coding sequence.

[0139] Recombinant expression vector

[0140] According to some embodiments of this disclosure, this disclosure provides a recombinant expression vector that expresses the helicase mutant as described above or contains the polynucleotide as described above.

[0141] Recombinant host cells, cell cultures

[0142] According to some embodiments of this disclosure, this disclosure provides a recombinant host cell comprising the helicase mutant, the polynucleotide, or the recombinant expression vector as described above.

[0143] In some alternative embodiments, the recombinant host cell is derived from microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, *Bacillus*, or *Corynebacterium*. In some preferred embodiments, the host cell is derived from *Escherichia*, more preferably *Escherichia coli*, including *Escherichia coli* DH5α, *Escherichia coli* Top10, *Escherichia coli* Trans T1, *Escherichia coli* BL21(DE3), etc.

[0144] Furthermore, this disclosure provides cell cultures comprising the recombinant host cells described above.

[0145] Methods for controlling polynucleotide migration

[0146] According to some embodiments of this disclosure, this disclosure provides a method for controlling the movement of polynucleotides, which includes contacting the polynucleotides with a helicase mutant as described above or a construct as described above.

[0147] In some preferred embodiments, the control of polynucleotide movement refers to the control of the movement of polynucleotides through a pore. The pore is a nanopore, specifically a transmembrane pore.

[0148] More preferably, the nanopore comprises a biological nanopore, a solid nanopore, or a hybrid of a biological nanopore and a solid nanopore.

[0149] In some exemplary embodiments, the nanopores are CsgG nanopores.

[0150] In some optional embodiments, the method is used to control the movement of polynucleotides through a pore.

[0151] In some alternative embodiments, the polynucleotide may also include any number of spacer regions, such as one, two, three, four, five, six, or more spacer regions. Preferably, the spacer regions are selected from iSpC3 spacer regions and iSpC18 spacer regions.

[0152] Methods for characterizing target polynucleotides

[0153] According to some disclosed embodiments, this disclosure provides a method for characterizing polynucleotides, the method comprising:

[0154] (A) Contacting the target polynucleotide with a transmembrane pore and a helicase mutant or construct as described above, such that the helicase controls the movement of the target polynucleotide through the pore; and

[0155] (B) As the polynucleotide moves relative to the pore, one or more measurements are acquired, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide.

[0156] In some preferred embodiments, any number of helicase mutants described herein may be used in the method, preferably at least one, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. Two or more helicase mutants described herein may be identical or different. Furthermore, two or more helicase mutants may be linked together or arranged by binding to polynucleotides to exert their function of controlling polynucleotide movement.

[0157] In some preferred embodiments, when a force (e.g., voltage) is applied to the pore, the rate at which the target polynucleotide passes through the pore is controlled by a helicase mutant or construct, thereby obtaining a recognizable and stable current level for characterizing the target polynucleotide.

[0158] In some optional implementations, steps (A) and (B) are repeated once or more.

[0159] In some alternative embodiments, the method further includes the step of applying a potential difference across the pores in contact with the helicase or construct and the target polynucleotide.

[0160] In some exemplary embodiments, the pore is a nanopore, specifically a transmembrane pore. The transmembrane pore provides a channel for the movement of the target polynucleotide. Preferably, the nanopore comprises a biological nanopore, a solid-state nanopore, or a hybrid of a biological nanopore and a solid-state nanopore.

[0161] In some embodiments, the membrane can be any membrane existing in the prior art, preferably an amphoteric molecular layer, that is, a layer formed by amphoteric molecules such as phospholipids having at least one hydrophilic portion and at least one lipophilic or hydrophobic portion, the amphoteric molecules being either naturally occurring or artificially synthesized; preferably, the membrane is a lipid bilayer membrane.

[0162] In some exemplary embodiments, the nanopores are CsgG nanopores.

[0163] In some embodiments, the target polynucleotide is single-stranded, double-stranded, or at least partially double-stranded. Optionally, the target polynucleotide may be modified by tagging, spacers, methylation, oxidation, or damage.

[0164] In some alternative embodiments, the target polynucleotide may also include any number of spacer regions, such as one, two, three, four, five, six, or more spacer regions. Preferably, the spacer regions are selected from iSpC3 spacer regions and iSpC18 spacer regions.

[0165] In some specific implementations, the helicase mutant binds to the internal nucleotides of a single-stranded polynucleotide.

[0166] In some specific implementations, the one or more features are selected from at least one of the following: the source, length, identity sequence, secondary structure, or whether the target polynucleotide is modified.

[0167] In some alternative implementations, one or more features are measured by electrical and / or optical measurements. Preferably, electrical and / or optical signals are generated by electrical and / or optical measurements, with each nucleotide corresponding to a signal level, and the electrical and / or optical signals are then converted into features of the nucleotide.

[0168] In some specific implementations, the electrical measurements include, but are not limited to, current measurements, impedance measurements, tunnel measurements, wind tunnel measurements, or field-effect transistor measurements; the electrical signals are selected from measured values ​​of current, voltage, resistance, tunneling, potential, conductivity, or lateral electrical measurements.

[0169] In some specific implementations, the electrical signal is a current passing through the hole.

[0170] Products characterizing target polynucleotides

[0171] According to some embodiments of this disclosure, this disclosure provides a product for characterizing a target polynucleotide, the product comprising a helicase mutant as described above, a construct as described above, a polynucleotide as described above, a recombinant expression vector as described above, a recombinant host cell as described above, or a cell culture as described above, and a well.

[0172] In some alternative implementations, the product comprises one or more helicase mutants and / or constructs.

[0173] In some alternative embodiments, the product comprises one or more pores, exemplarily nanopores, which are transmembrane pores. The transmembrane pores provide a channel for the movement of the target polynucleotide. Preferably, the nanopores include biological nanopores, solid nanopores, or a hybrid of biological and solid nanopores.

[0174] In some exemplary embodiments, the nanopores are CsgG nanopores.

[0175] In some preferred embodiments, the product comprises a reagent kit, device, or sensor.

[0176] Example

[0177] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0178] Example 1: Preparation of KphE1 helicase protein

[0179] Based on the amino acid sequence of the wild-type KphE1 protein (SEQ ID NO:24), its nucleic acid sequence was obtained through in vitro gene synthesis. Then, using *E. coli* as the host, the relevant codons were optimized by replacing them with commonly used *E. coli* codons, resulting in the optimized KphE1 protein nucleic acid sequence. This sequence was then ligated into the pET28 expression vector via two restriction enzyme sites, NdeⅠ and XhoⅠ. After sequence verification, a recombinant expression plasmid containing the KphE1 helicase was finally obtained.

[0180] The recombinant plasmid was transformed into the BL21(DE3) *E. coli* expression host via heat shock. During the induction of expression, the host bacteria containing the plasmid were first cultured overnight at 37°C in LB medium supplemented with kanamycin. Then, the culture was scaled up at 37°C at a 1:100 ratio until the OD(600) value reached 0.4-0.6. The culture was then stopped and the bacteria were cooled to 4°C for 1 hour. Subsequently, 0.5 mM isopropyl β-D-thiogalactoside (IPTG) was added, and expression was induced at 16°C for 12-16 hours. The bacteria were then collected by centrifugation at 15,000 rpm at 4°C. The cells were then autoclaved at 4°C, and the supernatant was collected by centrifugation at 4°C. The target protein was then purified step-by-step using nickel columns, heparin columns, Q columns, and molecular sieves to obtain a large quantity of high-purity wild-type helicase protein.

[0181] Example 2: Fluorescence assay to analyze the helical activity of KphE1 helicase

[0182] like Figure 2 As shown in (1), the fluorescent substrate chain (final concentration 100 nM, d, SEQ ID NO:8) has a 20-base single-stranded DNA portion at the 5' end and an 18-base hybridized double-stranded DNA portion, and its 3' end has a fluorescent group (Cy3, f). The complementary short chain above it (c, SEQ ID NO:9) has a fluorescence quenching group (BHQ-1, e) at the 5' end. When the two chains are complementary, the fluorescence of Cy3(f) is quenched by BHQ-1(e), and the substrate is essentially non-fluorescent.

[0183] SEQ ID NO:8: 5'-GAACAACAACAACAACCATGGAAAATCAAAACTAAAAC-3'

[0184] SEQ ID NO:9:5'-GTTTTAGTTTTGATTTTC-3'

[0185] During the experiment, such as Figure 2 As shown in (2), KphE1 helicase (a) binds to the 5' end single-stranded DNA portion of the fluorescent substrate. After the addition of ATP (2mM) and MgCl2 (2mM), it shifts along the 5'-3' direction and unwinds the double-stranded portion.

[0186] Subsequently, as Figure 2 As shown in (3), the excess capture chain (b, SEQ ID NO:10) preferentially pairs with the short chain (c) to prevent re-annealing between the initial substrates, and the released substrate backbone (d) emits fluorescence.

[0187] SEQ ID NO:10:5'-GAAAATCAAAACTAAAAC-3'

[0188] The results showed that the substrate changed from essentially non-fluorescent to fluorescent.

[0189] Example 3: Preparation of KphE1-D96C / A363C / C116A / C138A / C313A

[0190] KphE1-D96C / A363C / C116A / C138A / C313A is an intramolecular linker of SEQ ID NO:1 with the mutant combination D96C / A363C / C116A / C138A / C313A, formed via a disulfide bond catalyzed by N,N,N',N'-tetramethylazodicarbonamide (TMAD). Specifically, a covalent link is formed between cysteine ​​residues at positions 96 and 363 in the KphE1-D96C / A363C / C116A / C138A / C313A sequence via TMAD catalysis.

[0191] SEQ ID NO:1(KphE1-D96C / A363C / C116A / C138A / C313A)

[0192] MITFDMLNKGQKKAFDYIISRIKAGKGNHITLNGPAGTGKTTMTKFIVDYLISQGVSGVVLAAPTHAAKKVLSKLSGIEARTIHSLLKINPTTYE

[0193] CSVTFEQKGDVDVSELRVVVADEASMYDRKLFQILMATIPRYALVIAIGDKAQIRPVEPGSTVPALSPFFSHKDFDQLELDEVMRSNAPIIKVATD

[0194] IRNGKWIYDHQRDGHGVHGFTSTTALKDFMMKYFEIVKDPEDMFENKMFAFTNKSVDKLNSIIRRRILETEDAFITGEVIVMQEPLIKELEFEG

[0195] KRFNDLKFNNGQYVRIVSAEYASSFVSAKGVSGEYMLRHWRLTVETYDKDEDYHLETINVISDEQEQNKFQFFLAKACDTYRNWQKGGKAP

[0196] WKSFWAAKRMFHKVKPLPASTFHKAQGLSVDTSFLYTPCIHVADAELAQQLLYVGTTRGRHDVYYV

[0197] Method for constructing the combined mutant KphE1-D96C / A363C / C116A / C138A / C313A:

[0198] Based on the amino acid sequence of the wild-type KphE1 protein (SEQ ID NO:24), its nucleic acid sequence was obtained through in vitro gene synthesis. Then, using *E. coli* as the host, the relevant codons were optimized by replacing them with commonly used *E. coli* codons, resulting in the optimized KphE1 protein nucleic acid sequence. This sequence was then ligated into the pET28 expression vector via two restriction enzyme sites, NdeⅠ and XhoⅠ. After sequence verification, the recombinant expression plasmid of the KphE1 helicase was finally obtained. Subsequently, site-directed mutagenesis was performed using overlap PCR to obtain the nucleic acid sequence encoding the mutant combination D96C / A363C / C116A / C138A / C313A.

[0199] The mutated recombinant plasmid was transformed into the BL21(DE3) *E. coli* expression host via heat shock. During the induction of expression, the host bacteria containing the expression plasmid were first cultured overnight at 37°C in LB medium supplemented with kanamycin. Then, the culture was scaled up at 37°C at a 1:100 ratio until the OD(600) value reached 0.4-0.6. The culture was then stopped and the bacteria were cooled to 4°C for 1 hour. Subsequently, isopropyl β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM was added, and expression was induced at 16°C for 12-16 hours. The bacteria were then collected by centrifugation at 15,000 rpm at 4°C. The cells were then autoclaved at 4°C, and the supernatant was collected by centrifugation at 4°C. The target protein was then purified step-by-step using nickel columns, heparin columns, Q columns, and molecular sieves to obtain a large quantity of high-purity helicase mutant protein, the sequence of which is shown in SEQ ID NO:1.

[0200] Specifically, 1 μL of 1M DTT was added to 100 μL of KphE1-D96C / A363C / C116A / C138A / C313A (SEQ ID NO:1 with the mutant D96C / A363C / C116A / C138A / C313A, stored in 25 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% glycerol) and incubated at room temperature for 30 minutes.

[0201] The buffer was replaced with PBS buffer (pH 7.0) using a 0.5 mL Zeba desalting column (7 kWh MWCO) to obtain 100 μL of sample. 1 μL of 10 mM TMAD was added, and the sample was incubated at 20 rpm for 1 hour at room temperature. The crosslinking results were then analyzed using a 4-10% polyacrylamide gel electrophoresis.

[0202] Figure 3The 4-10% SDS-PAGE gel electrophoresis image of KphE1-D96C / A363C / C116A / C138A / C313A (SEQ ID NO:1 with the mutation D96C / A363C / C116A / C138A / C313A) is shown.

[0203] It is clear from the gel that the cross-linking reaction of the KphE1 mutant achieved a yield of almost 100%.

[0204] Example 4: Using a gel migration assay to measure the DNA binding capacity of modified KphE1 helicase.

[0205] The DNA substrates required for gel migration experiments were prepared by annealing (SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 were annealed at a molar ratio of 1:1.1:1.1 to a final concentration of 10 μM). Then, they were incubated with wild-type KphE1 (GenBank: QEM42218.1) and KphE1-D96C / A363C / C116A / C138A / C313A (SEQ ID NO:1) at molar ratios of 1:1, 1:3, and 1:5 in buffer (25 mM Tris-HCl pH 7.0, 100 mM NaCl, 10% glycerol, 1 mM EDTA) at room temperature for half an hour to obtain a final DNA concentration of 500 nM and a total reaction volume of 20 μL.

[0206] TMAD was added to the corresponding helicase mutant sample to a final concentration of 8 μM and incubated at room temperature for 1 hour. Subsequently, the sample was tested with a 4%–10% TBE gel, run at 120V for 1.5 hours, and then DNA bands were observed under UV light by Gel Red staining.

[0207] The results showed the effect of KphE1 helicase on DNA binding ability after modification, that is, the binding ability of modified KphE1 to polynucleotides was significantly enhanced.

[0208] SEQ ID NO:11:

[0209] 5'-GGCGTCTGCTTGGGTGTTTAACCTTTTTTTTTTCCACAACTTCGTTCAGTTACGTATTGCT-3'

[0210] SEQ ID NO:12:5'-GCAATACGTAACTGAACGAAGTTGTGG-3'

[0211] SEQ ID NO:13:5'-GGTTAAACACCCAAGCAGACGCC-3'

[0212] Example 5: KphE1-D96C / A363C / C116A / C138A / C313A has control over the X-ray penetration of the intact DNA construct. The ability of nanopores to move

[0213] Among them, KphE1-D96C / A363C / C116A / C138A / C313A is K1 with the mutation combination D96C / A363C / C116A / C138A / C313A.

[0214] Preparation such as Figure 4 The DNA construct X shown is shown.

[0215] First, primers containing sequences A, C, D, E, and F were designed. Then, a 1000-base sequence (G) on λDNA was amplified using these primers. The obtained PCR product was purified and then annealed and hybridized with sequence H at a molar ratio of 1:1.1 to obtain the final DNA construct X. Here, A represents 50 T (thymine nucleotides), B represents the helicase KphE1-D96C / A363C / C116A / C138A / C313A constructed in Example 3, C represents one iSpC18 spacer region, D represents SEQ ID NO:4, E represents two iSpC18 spacers, F represents SEQ ID NO:5, G represents SEQ ID NO:6, and H represents cholesterol modification at the 3' end of SEQ ID NO:7.

[0216] The prepared DNA construct X (final concentration 0.1 nM) and KphE1-D96C / A363C / C116A / C138A / C313A (final concentration 10 nM) were pre-incubated for 30 minutes in a buffer solution (10 mM Hepes, pH 8.0, 100 mM KCl, 10% glycerol) at room temperature. TMAD was added to the corresponding helicase mutant sample to a final concentration of 8 μM, and the sample was incubated at room temperature for 1 hour to obtain the pre-incubated sample.

[0217] SEQ ID NO:4: 5'-GGCGTCTGCTTGGGTGTTTAACC-3'

[0218] SEQ ID NO:5:5'-TTGACCTTTCTCTCCCATATTGCA-3'

[0219] SEQ ID NO:6

[0220] TTGACCTTTCTCTCCCATATTGCAGTCGCGGCACGATGGAACTAAATTAATAGGCATCACCGAAAATTCAGGATAATGTGCAATAGGAAGAAAATGATCTATATTTTTTGTCTGTCCTATATCACCACCAAAATGGACATTTTTCACCTGATGAAACAAGCATGTCATCGTAATATGTTCTAGCGGGTTTGTTTTTATCTCGGAGATTATTTTCATAAAGCTTTTCTAATTTAACCTTTGTCAGGTTACCAACTACTAAGGTTGTAGGCTCAAGAGGGTGTGTCCTGTCGTAGGTAAATAACTGACCTGTCGAGCTTAATATTCTATATTGTTGTTCTTTCTGCAAAAAAGTGGGGAAGTGAGTAATGAAATTATTTCTAACATTTATCTGCATCATACCTTCCGAGCATTTATTAAGCATTTCGCTATAAGTTCTCGCTGGAAGAGGTAGTTTTTTCATTGTACTTTACCTTCATCTCTGTTCATTATCATCGCTTTTAAAACGGTTCGACCTTCTAATCCTATCTGACCATTATAATTTTTTAGAATGGTTTCATAAGAAAGCTCTGAATCAACGGACTGCGATAATAAGTGGTGGTATC CAGAATTTGTCACTTCAAGTAAAAAACACCTCACGAGTTAAAACACCTAAGTTCTCACCGAATGTCTCAATATCCGGACGGATAATATTTATTGCTTCTCTTGACCGTAGGACTTTCCACATGCAGGATTTTGGAACCTCTTGCAGTACTACTGGGGAATGAGTTGCAATTATTGCTACACCATTGCGTGCATCGAGTAAGTCGCTTAATGTTCGTAAAAAAGCAGAGAGCAAAGGTGGATGCAGATGAACCTCTGGTTCATCGAATAAAACTAATGACTTTTCGCCAACGACATCTACTAATCTTGTGATAGTAAATAAAACAATTGCATGTCCAGAGCTCATTCGAAGCAGATATTTCTGGATATTGTCATAAAACAATTTAGTGAATTTATCATCGTCCCACTTGAATCTGTGGTTCATTACGT

[0221] SEQ ID NO:7:5'-GGTTAAACACCCAAGCAGACGCCTTTGAGGCGAGCGGTCAA-3'

[0222] Electrochemical signals were measured from CsgG nanopores embedded in a diaphytylphosphatidylcholine (DPhPC) phospholipid bilayer in a buffer solution (600 mM KCl, 75 mM K3[Fe(CN)6], 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0). After achieving single-pore insertion into the phospholipid bilayer, the system was purged with 2 ml of buffer solution (600 mM KCl, 75 mM K3[Fe(CN)6], 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0) to remove any remaining excess nanopores. The pre-incubated sample, ATP (final concentration 2 mM), and MgCl2 (final concentration 10 mM) were then fed into a single nanopore experimental system (total volume 100 μL), and the signal was measured for 6 h at a constant voltage of +180 mV (including a potential 2 s -180 mV voltage reversal).

[0223] Figure 5 The mutant K1 was shown to be able to control the movement of the intact DNA construct X through the nanopore. Figure 6 This shows a magnified view of a portion of the region where it controls DNA movement.

[0224] Example 6: Comparative analysis of the electrical activity generated by different KphE1 mutants controlling the target DNA to pass through CsgG nanopores streaming signal

[0225] The helicase mutants tested in this embodiment have at least one amino acid substitution that may interact with nanopores or target single-stranded DNA. By extracting and analyzing different parameters of the target DNA perforation signal controlled by these mutants, it was found that the amino acid substitutions that interact with nanopores or target single-stranded DNA can affect the speed, noise, and accuracy of nucleic acid sequencing. As a result, the inventors also obtained several mutants that can improve the speed and accuracy of nucleic acid sequencing.

[0226] Preparation such as Figure 7 DNA construct Y shown:

[0227] The 5' end of region D is connected to 23 iSpC3 spacer regions (region I), the 3' end of which is connected to the 5' end of 10 T molecules (for binding helicase (B)). The 3' end of the 10 T molecules is connected to one iSpC18 spacer region (region C), which is connected to the 5' end of region J. Regions D and J hybridize with regions H (which contain cholesterol modification at the 3' end of the sequence shown in SEQ ID NO:16) and L (SEQ ID NO:17), respectively.

[0228] D region: 5'-GGCGTCTGCTTGGGTGTTTAACC-3' (SEQ ID NO:14)

[0229] J region: 5'-AATGTACTTCGTTCAGTTACGTATTGCT-3'(SEQ ID NO:15)

[0230] H region: 5'-GGTTAAACACCCAAGCAGACGCCTTTGAGGCGAGCGGTCAA-3' (SEQ ID NO: 16)

[0231] L region: 5'-GCAATACGTAACTGAACGAAGTACATT-3' (SEQ ID NO: 17)

[0232] Fragments consisting of 100 μM I, D, C, J, and 10 T segments were synthesized and linked together in the order shown in the figure. These fragments, along with H and L segments, were added to annealing buffer (20 mM HEPES pH 7.5, 100 mM KCl) at a ratio of 1:1.1:1.1. Annealing was performed at 95 °C for 5 min, -2 °C / min, for 36 cycles.

[0233] The prepared DNA construct Y and different KphE1 mutants were incubated at 25°C for 30 minutes in a buffer solution (25 mM Tris, pH 7.0, 100 mM NaCl, 10% glycerol, 1 mM EDTA) at a ratio of 1:5. Then, 10 μM TMAD was added, and the reaction was catalyzed at room temperature for 1 hour. After Q-column purification, the purified DNA construct / enzyme complex and a 1 kb end-repaired de Bleuin sequence (K region, SEQ ID NO: 21) were added to the rapid ligation reaction system of T4 DNA ligase (Yisheng Biotechnology) at a molar ratio of 4:1. Rapid ligation was performed at 20°C for 30 minutes. The ligation product was purified by magnetic beads and then used in subsequent sequencing.

[0234] SEQ ID NO:18 (DEBRAIN sequence):

[0235] AAAAGGGGGTGGTGTGGGTTGTGTTGGTTTGTTTTTGGGGCTGGGCGTGGCTTGGCGGTGCTGTGCGTTGCTTTGCGGCGCTGCGCGGGTCTGGTCGTGTCTTGTCGCGTCGGTTCTGTTCGTTTCTTTTCGCTTCGGCTCTGCTCGTCTCTTCTCGGGCCTGGCCGTGCCTTGCCGCGCCTCGCCGGTCCTGTCCGTTCCTTTCCGCTCCTCTCCGGCCCTGCCCGTCCCTTCCCGCCCCTCCCCCGGGGATGGGACGGGAGTGGATTGGACTGGAGCGGATCGGACCGGAGGTGATGTGACGTGAGTTGATTTGACTTGAGCTGATCTGACCTGAGGCGATGCGACGCGAGTCGATTCGACTCGAGCCGATCCGACCCGAGGAGATGAGACGAGAGGGTATGGTACGGTAGTGTATTGTACTGTAGCGTATCGTACCGTAGAGTAGGTTATGTTACGTTAGTTTATTTTACTTTAGCTTATCTTACCTTAGATTAGGCTATGCTACGCTAGTCTATTCTACTCTAGCCTATCCTACCCTAGACTAGGATATGATACGATAGTATATTATACTATAGGGCATGGCACGGCAGTGCATTGCACTGCAGCGCATCGCACCGCAGAGCATAGCAGGTCATGTCACGTCAGTTTCATTTCACTTCAGCTCATCTCACCTCAGATCATATCAGGCCATGCCACGCCAGTCCATTCCACTCCAGCCATCCCACCCCAGACCATACCAGGACATGACACGACAGTACATTACACTACAGCACATCACACCACAGGGAATGGAACGGAAGTGAATTGAACTGAAGCGAATCGAACCGAAG AGAATAGAACAGAAGGTAATGTAACGTAAGTTAATTTAACTTAAGCTAATCTAACCTAAGATAATATAACATAAGGCAATGCAACGCAAGTCAATTCAACTCAAGCCAATCCAACCCAAGACAATACAACACAAGGAAATGAAACGAAAGTAAATTAAACTAAAGCAAATCAAACCAAAGAAAATAAAACAAAAA

[0236] Electrochemical measurements were obtained from CsgG nanopores embedded in the DPhPC phospholipid bilayer in a buffer solution (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0). After achieving single-pore insertion into the phospholipid bilayer, the system was circulated through 2 ml of buffer solution (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0) to remove any remaining excess nanopores. The purified sample, ATP (final concentration 30 mM), and MgCl2 (final concentration 10 mM) were then fed into a single nanopore experimental system (total volume 20 μL). The signal was measured at a constant voltage of +180 mV for 6 h (including a potential 2 s -180 mV voltage reversal) to monitor and record the current signal controlling the helicase to pass the target DNA through the nanopore.

[0237] By analyzing the current characteristics of different mutants controlling the passage of a 1kb de Bryin sequence (K region) through nanopores, including median velocity, step noise, pore velocity distribution, and current signal range, the inventors theoretically believe that mutants with larger median pore velocity and smaller current signal range, and smaller step noise and pore velocity distribution, generate better quality current signals controlling the movement of target nucleic acids. Based on this information, the inventors evaluated the impact of different mutants on the pore signal, screened mutants that could efficiently and stably control the passage of target nucleic acids through nanopores, and then trained base recognition algorithms on these mutants to verify the impact of the differences in the above parameters on the final sequencing accuracy.

[0238] Example 7: Construction and performance testing of KphE1 helicase mutants K2-K41

[0239] The construction method follows that in Example 3. The nucleotide sequence encoding KphE1-D96C / A363C / C116A / C138A / C313A was ligated into the pET28 expression vector using two restriction enzyme sites, NdeⅠ and XhoⅠ. After sequence verification, the recombinant expression plasmid of KphE1 helicase was obtained. Then, site-directed mutagenesis was performed by PCR to obtain recombinant plasmids with nucleotide sequences of mutants K2–K27 (specific mutation sites are shown in Table 1). The helicase mutants were then expressed according to the protein expression method described in Example 3.

[0240] Table 1

[0241] K2 K1-HMGG (M at position 1 mutates to GG) K15 K1-T214A K3 K1-T42Y K16 K1-K223E K4 K1-V56K K17 K1-T241D K5 K1-S57K K18 K1-N353E K6 K1-S85K K19 K1-F357A K7 K1-F100W K20 K1-K361I K8 K1-C116L K21 K1-K361S K9 K1-V152L K22 K1-K361A K10 K1-V152H K23 K1-D364I K11 K1-V158F K24 K1-D364Q K12 K1-A160F K25 K1-K389M K13 K1-K196L K26 K1-V418W K14 K1-T212K K27 K1-V418R

[0242] The obtained mutants were analyzed for via speed, current noise, and current range.

[0243] The results are as follows Figures 8A to 8C As shown.

[0244] Theoretically, the larger the current range (i.e., the difference between the maximum and minimum current) caused by nucleic acid perforation, the better. A larger current range means greater differences between different current values, which translates to higher current resolution caused by different bases, and thus higher decoding accuracy. In the KphPif1 mutant, relative to K1, from... Figure 8C The results show that mutations such as N353E, K361I, K389M, and A160F significantly improved the current range. Furthermore, in terms of perforation speed, faster perforation speed means higher sequencing throughput can be achieved in the same amount of time. Figure 8AAs can be seen, the via speeds of V56K, S57K, S85K, V158F, K196L, K361S, and V418R, among others, have all been significantly improved. Regarding current noise, lower current noise results in a higher signal-to-noise ratio, meaning higher decoding accuracy. Figure 8B As can be seen, the current noise of most mutants did not show significant differences compared to K1; Figure 8C Among them, the current range of sudden changes in HMGG, S57K, S85K, V158F, A160F, K196L, T212K, T214A, T241D, N353E, K361I, K361A, K389M, etc. has also been significantly improved.

[0245] Based on the above results, this disclosure further performed combined mutations. Referring to the construction method of Example 3, a recombinant plasmid containing the nucleotide sequence of mutants K28 to K41 (specific mutation sites are shown in Table 2) was constructed, and the helicase mutant was obtained according to the protein expression method in Example 3.

[0246] Table 2

[0247] K28 K1-V158F / T42Y K35 K1-S57K / S85K / K389M K29 K1-V158F / F357A K36 K1-N353E / A160F / K196L K30 K1-V158F / V418R K37 K1-N353E / K196L K31 K1-A160F / K196L / K389M K38 K1-N353E / A160F / K361I K32 K1-S57K / S85K / A160F K39 K1-N353E / K361I / K389M K33 K1-S57K / S85K / N353E K40 K1-N353E / K389M K34 K1-S57K / S85K / K361I K41 K1-F357A / D364I / K361I

[0248] Following the above method, the perforation speed, noise, and current range of each mutant were measured, and the results are as follows: Figures 8D to 8F As shown:

[0249] from Figure 8D As can be seen, except for K1-V158F / T42Y, K1-S57K / S85K / A160F, and K1-S57K / S85K / N353E, the via speeds of other mutation combinations all showed varying degrees of improvement; however, no significant difference was observed in terms of noise. Figure 8E In terms of current range, K1-N353E / K361I / K389M, K1-N353E / K196L, K1-N353E / K389M, K1-S57K-S85K-K389M, and K1-N353E / A160F / K361I all show significant improvements. Figure 8F ).

[0250] Example 8: Construction and performance testing of KphE1 helicase mutants

[0251] Referring to the construction method in Example 7, mutations were performed on H201, R203, K196, K218, M179, D204, W381, K103, G104, Y94, A362, K361, D364, V158, S57, T212, K223, N353, K249, R193, F357, Q350, T214, K187, D171, R203, K218, E226, D263, R326, D191, K187, D202, D230, Q128, R136, K146, K168, and D176 based on the K1 mutant to obtain the corresponding helicase mutants.

[0252] The obtained mutants were analyzed for perforation speed, current noise, and current range. The results showed that the obtained helicase mutants exhibited improved uniformity in speed distribution and increased perforation speed.

Claims

1. Helicase mutants, among which, Compared to a helicase containing the sequence shown in SEQ ID NO:19, the helicase mutant introduces at least one new amino acid residue or linker molecule into the 1B and 2B domains of the helicase, respectively, so that the 1B and 2B domains are linked. The 1B structural domain contains the sequence shown in SEQ ID NO:2; The 2B domain contains the sequence shown in SEQ ID NO:3; Optionally, cysteine ​​is introduced at one or more of positions 95, 96, 360, 363, 367, 377, 381 and 384 of the sequence shown in SEQ ID NO:19; Preferably, two or more introduced cysteine ​​residues are interconnected.

2. The helicase mutant according to claim 1, wherein, The helicase mutant further contains a mutation at at least one of the positions 116, 138, and 313 corresponding to the sequence shown in SEQ ID NO:19, and the mutated amino acid is not cysteine.

3. The helicase mutant according to claim 1 or 2, wherein, The helicase mutant is selected from any one of the following groups (I)-(V): (I) The amino acid sequence corresponding to SEQ ID NO:19 has a mutation at at least one of the following positions: D96C, A363C, C116A, C138A, C313A; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:19; (III) A mutant encoded by a polynucleotide that can hybridize with the polynucleotide shown in (a) or (b): (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I); (b) A full-length complementary polynucleotide to (a); (IV) A fragment of a mutant as shown in any one of (I), (II) or (III), and the fragment still possesses helicase activity; (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

4. The helicase mutant according to any one of claims 1 to 3, wherein, Compared to the sequence shown in SEQ ID NO:19, the helicase mutant further modifies the sequence corresponding to SEQ ID NO:

19. The sequence shown in NO:19 contains a mutation at at least one of the following positions: positions 1, 42, 56, 57, 85, 100, 116, 152, 158, 160, 196, 212, 214, 223, 241, 353, 357, 361, 364, 389, 418, 201, 203, 196, 218, 179, 204, 381, 103, 104, 94, 362, 361, 364, 158, 57, 212, 223, 353, 249, 193, 357, 350, 214, 187, 171, 203, 218, 226, 263, 326, 191, 187, 202, 230, 128, 136, 146, 168, and 176. Optionally, the helicase mutant corresponds to the sequence shown in SEQ ID NO:19 and has a mutated amino acid at at least one of the following positions: T42Y, A160F, D364I, F100W, F357A, K196L, K223E, K361I, K361S, N353E, S57K, T212K, V152H, V152L, V158F, V418R, V418W, V56K, S85K, T214A, T241D, D361A, D364Q, K389M, the first M mutates into two consecutive Gs.

5. The helicase mutant according to any one of claims 1 to 4, wherein, The helicase mutant corresponds to the sequence shown in SEQ ID NO:19 and has the following (m1)~(m) characteristics. 41 Any mutation shown in any of the following: (m1)D96C, A363C, C116A, C138A, C313A; (m2)D96C, A363C, C116A, C138A, C313A, the first M mutates into two consecutive Gs; (m3)D96C, A363C, C116A, C138A, C313A, T42Y; (m4)D96C, A363C, C116A, C138A, C313A, V56K; (m5)D96C, A363C, C116A, C138A, C313A, S57K; (m6)D96C, A363C, C116A, C138A, C313A, S85K; (m7)D96C, A363C, C116A, C138A, C313A, F100W; (m8)D96C, A363C, C116A, C138A, C313A, C116L; (m9)D96C, A363C, C116A, C138A, C313A, V152H; (m 10 )D96C、A363C、C116A、C138A、C313A、V152L; (m 11 )D96C、A363C、C116A、C138A、C313A、V158F; (m 12 )D96C、A363C、C116A、C138A、C313A、A160F; (m 13 )D96C、A363C、C116A、C138A、C313A、K196L; (m 14 )D96C、A363C、C116A、C138A、C313A、T212K; (m 15 )D96C、A363C、C116A、C138A、C313A、T214A; (m 16 )D96C、A363C、C116A、C138A、C313A、K223E; (m 17 )D96C、A363C、C116A、C138A、C313A、T241D; (m 18 )D96C, A363C, C116A, C138A, C313A, N353E; (m 19 )D96C, A363C, C116A, C138A, C313A, F357A; (m 20 )D96C、A363C、C116A、C138A、C313A、K361I; (m 21 )D96C、A363C、C116A、C138A、C313A、K361S; (m 22 )D96C、A363C、C116A、C138A、C313A、D361A; (m 23 )D96C、A363C、C116A、C138A、C313A、D364I; (m 24 )D96C、A363C、C116A、C138A、C313A、D364Q; (m 25 )D96C、A363C、C116A、C138A、C313A、K389M; (m 26 )D96C、A363C、C116A、C138A、C313A、V418R; (m 27 )D96C、A363C、C116A、C138A、C313A、V418W; (m 28 )D96C、A363C、C116A、C138A、C313A、V158F、T42Y; (m 29 )D96C、A363C、C116A、C138A、C313A、V158F、F357A; (m 30 )D96C、A363C、C116A、C138A、C313A、V158F、V418R; (m 31 )D96C、A363C、C116A、C138A、C313A、A160F、K196L、K389M; (m 32 )D96C、A363C、C116A、C138A、C313A、S57K、S85K、A160F; (m 33 )D96C, A363C, C116A, C138A, C313A, S57K, S85K, N353E; (m 34 )D96C, A363C, C116A, C138A, C313A, S57K, S85K, K361I; (m 35 )D96C, A363C, C116A, C138A, C313A, S57K, S85K, K389M; (m 36 )D96C、A363C、C116A、C138A、C313A、N353E、A160F、K196L; (m 37 )D96C, A363C, C116A, C138A, C313A, N353E, K196L; (m 38 )D96C、A363C、C116A、C138A、C313A、N353E、A160F、K361I; (m 39 )D96C, A363C, C116A, C138A, C313A, N353E, K361I, K389M; (m 40 )D96C, A363C, C116A, C138A, C313A, N353E, K389M; (m 41 )D96C, A363C, C116A, C138A, C313A, F357A, D364I, K361I.

6. Any one of (i) to (iii) below: (i) a construct comprising at least one helicase mutant as described in any one of claims 1 to 5; optionally, the construct further comprises a polynucleotide-binding moiety; (ii) encoding a helicase mutant as described in any one of claims 1 to 5 or a polynucleotide of (i); (iii) Expressing the helicase mutant as described in any one of claims 1 to 5 or a recombinant expression vector containing (ii).

7. A recombinant host cell, wherein, The recombinant host cell comprises the helicase mutant as described in any one of claims 1 to 5, the polynucleotide as described in claim 6, or the recombinant expression vector as described in claim 6.

8. A cell culture comprising the recombinant cells as described in claim 7.

9. A method for controlling the movement of a polynucleotide, comprising contacting the polynucleotide with a helicase mutant as described in any one of claims 1 to 5 or a construct as described in claim 6.

10. A method for characterizing a target polynucleotide, comprising: (A) Contacting the target polynucleotide with a transmembrane pore and a helicase mutant as described in any one of claims 1-5 or a construct as described in claim 6, such that the helicase controls the movement of the target polynucleotide through the pore; and (B) As the polynucleotide moves relative to the pore, one or more measurements are acquired, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide; Optionally, the method further comprises the step of applying a potential difference across a pore in contact with the helicase or construct and the target polynucleotide; Optionally, the pores are nanopores; Preferably, the nanopore includes a biological nanopore, a solid nanopore, or a hybrid of a biological nanopore and a solid nanopore; Optionally, the target polynucleotide is single-stranded, double-stranded, or at least partially double-stranded; Optionally, the one or more features are selected from at least one of the following: source, length, identity, sequence, secondary structure, or whether the target polynucleotide is modified.

11. A product characterizing a target polynucleotide, wherein, The product comprises the helicase mutant as described in any one of claims 1 to 5, the construct as described in claim 6, the polynucleotide as described in claim 6, the recombinant expression vector as described in claim 6, the recombinant host cell as described in claim 7, or the cell culture as described in claim 8, and a well; Optionally, the product is selected from reagent kits, devices, or sensors.

12. The use of the helicase mutant of any one of claims 1 to 5, the construct of claim 6, the polynucleotide of claim 6, the recombinant expression vector of claim 6, the recombinant host cell of claim 7, the cell culture of claim 7, or the product of claim 11 in characterizing the target polynucleotide or controlling the movement of the target polynucleotide through the pore.

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