Modification method of double-gated nanopore protein, double-gated nanopore protein for sequencing and application of double-gated nanopore protein

Abstract

The invention relates to a modification method of double-gated nanopore protein, the double-gated nanopore protein for sequencing and application of the double-gated nanopore protein, and belongs to the technical field of biology. The modification method of the double-gated nanopore protein comprises the steps that an x1L2x3L4x5 amino acid sequence and an x6L7x8L9x10 amino acid sequence are inserted into two adjacent beta folded sheets of a beta barrel of a double-gated nanopore respectively, each of the x1L2x3L4x5 amino acid sequence and the x6L7x8L9x10 amino acid sequence comprises 2-20 amino acids, and the amino acids comprise at least two hydrophobic amino acids; and at least two amino acids in the induction region of the double-gated nanopore are substituted by polar amino acids. The double-gated nanopore protein for sequencing obtained by modification can be integrated on a chip on a large scale to form stable and uniform pore channels, and has efficient sequencing performance.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a modification method of a dual-gated nanopore protein, a dual-gated nanopore protein for sequencing, and applications thereof. Background Art

[0002] Single-molecule gene detection based on nanopores is considered a disruptive technology in the field of gene detection. The principle of nanopore technology is similar to the transport of nucleic acids across nuclear pore complexes. It reflects the changes in ion blockage current caused by the molecular sequence passing through the pore. It does not require modification of DNA molecules and can directly detect all information features on the sequence. At present, this technology has been commercially applied in gene assembly and epigenetics, and is also being explored in the detection of peptides, sugar chains, proteins and other fields. It has a wide range of clear applications.

[0003] Although biological nanopore sequencing technology has been commercialized, it still faces many challenges in clinical applications. The biggest challenge is the accuracy of sequencing. Existing nanopores still cannot solve the problem of sequencing more than 5 repeated nucleotides. Double-gated nanopores can theoretically reach 12 bases. However, the hydrophobic area of ​​this type of nanopore interacting with the membrane is 2.3nm, which is much lower than the 3.5nm thickness of the polymer membrane. This makes it difficult for this type of nanopore to adapt to polymer membranes with a thickness greater than 4nm, limiting the large-scale on-chip integration of this type of nanopore, and requires an on-chip integrated solution. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the embodiments of the present application includes providing a modification method of a dual-gated nanopore protein and a dual-gated nanopore protein for sequencing and its application, so as to alleviate the problem that the current dual-gated nanopore is difficult to adapt to a polymer membrane with a thickness greater than 4nm, which limits the large-scale on-chip integration of such nanopores.

[0005] In a first aspect, the present invention provides a method for modifying a dual-gated nanopore protein, comprising: inserting an amino acid sequence x1L2x3L4x5 and an amino acid sequence x6L7x8L9x into two adjacent β-pleated sheets of the β barrel of the dual-gated nanopore, respectively. 10 Amino acid sequence, wherein the amino acid sequence x1L2x3L4x5 and x6L7x8L9x 10 The amino acid sequences all include 2-20 amino acids, including at least two hydrophobic amino acids; and at least two amino acids in the sensing region of the dual-gated nanopore are replaced by polar amino acids.

[0006] The present invention inserts 2-20 amino acid sequences x1L2x3L4x5 and x6L7x8L9x respectively, including at least two hydrophobic amino acids, into two adjacent β-pleated sheets of the β barrel of the dual-gated nanopore. 10Amino acid sequence, and at least two amino acids in the sensing region of the dual-gated nanopore are replaced by polar amino acids. The modified dual-gated nanopore protein for sequencing can be integrated on a large scale on a chip to form a stable and uniform pore with efficient sequencing performance.

[0007] In some embodiments of the present application, the amino acids of L2 and L4 in the x1L2x3L4x5 amino acid sequence include any one of A, L, V, I, M, F, Y, and W.

[0008] In the x1L2x3L4x5 amino acid sequence used in the present application, L2 and L4 are respectively selected from any one of A, L, V, I, M, F, Y, and W hydrophobic amino acids, so that the x1L2x3L4x5 amino acid sequence includes at least two hydrophobic amino acids.

[0009] In some embodiments of the present application, x6L7x8L9x 10 The amino acids of L7 and L9 in the amino acid sequence include any one of A, L, V, I, M, F, Y, and W.

[0010] This application uses x6L7x8L9x 10 In the amino acid sequence, L7 and L9 are selected from any one of A, L, V, I, M, F, Y, and W, respectively, so that x6L7x8L9x 10 The amino acid sequence includes at least two hydrophobic amino acids.

[0011] In some embodiments of the present application, in the x1L2x3L4x5 amino acid sequence, x1 includes 0-3 amino acids, x3 includes 0-5 amino acids, and x5 includes 0-3 amino acids.

[0012] In some embodiments of the present application, the amino acid sequence of x1L2x3L4x5 is: LILL, LLVL, FIFL, FYIL, TLILL, YLLVL, TTLILL, TYLLVL, TTVLILL, TYVLLVL, VTTVLILL or LTYVLLVL.

[0013] In some embodiments of the present application, x6L7x8L9x 10 In the amino acid sequence, x6 includes 0-3 amino acids, x8 includes 0-5 amino acids, and x 10 Includes 0-5 amino acids.

[0014] In some embodiments of the present application, x6L7x8L9x 10The amino acid sequence is: LILL, LLVL, FIFL, FYIL, TLILL, YLLVL, TTLILL, TYLLVL, TTVLILL, TYVLLVL, VTTVLILL or LTYVLLVL.

[0015] In some embodiments of the present application, the sensing region of the dual-gated nanopore includes 3-20 amino acids substituted by 3-20 amino acids including at least two polar amino acids.

[0016] In some embodiments of the present application, the substituted amino acid sequence is: SSS, NNN, NTS, LGDNNN, GSANNN, ATNQSTAVPQSA, LGDNNNNPN, AGSSNSNPN, LGDNNNNPF, ASSSNSNPN, NNQNPFQ, GSTAGSSNSNPN, GSTLGD NNNNPNQN, LGDNNNNSNQN, GSTNNQNPFQT, GSTASSSNSNPNQT, GSTLGDNNNNPFQN, GSTLGDNNNNGNQN, GSTLGDNNNNSNQN, TGATASSSNTNPAQT, TGATASSSNTNGAQT or TGATNQSTAVPQSA.

[0017] The present application replaces 3-20 amino acids in the sensing region of the dual-gated nanopore with 3-20 amino acids including at least two S, T, N, and Q polar amino acids, so that the modified dual-gated nanopore protein for sequencing has efficient sequencing performance.

[0018] In some embodiments of the present application, the dual-gated nanopore includes P45779, A0A553L8Q2, F2GBL1, A0A4R6PPQ3, Q7MPZ6, Q8EKC9, ETEC E3PJ86, A0A369V U29, D4Z3A5, A0A1J1LIE5, A0A1C0VUS7, A0A0F5YGH9, A0A2T1B XK7, A0A1E5QK12, A0A252ECY0, A0A139X416, A0A3S1AFR1, A5G Any one of PC5, E0TBQ0, B1WXU4, B5IQM2, Q8YY21, Q7V469, K9Z6X7, Q115D6, B4D845, D8G3K2, A0A2V3VE55, and W7R0U8, or a dual-gated nanopore having an amino acid sequence homology greater than or equal to 97% with any of the foregoing dual-gated nanopores.

[0019] By adopting any of the above-mentioned dual-gated nanopores, the present application can obtain a modified dual-gated nanopore protein for sequencing through the above-mentioned modification method. The dual-gated nanopore protein for sequencing can be integrated on a large scale on a membrane to form a stable and uniform pore; at the same time, it has efficient sequencing performance.

[0020] In some embodiments of the present application, the dual-gated nanopore P45779 is a dual-gated nanopore having an amino acid sequence as shown in SEQ ID NO.3.

[0021] In a second aspect, the present application provides a dual-gated nanopore protein for sequencing, comprising:

[0022] (a) a dual-gated nanopore protein for sequencing the amino acid sequence as shown in SEQ ID NO.1; or

[0023] (b) a dual-gated nanopore protein for sequencing the amino acid sequence as shown in SEQ ID NO.2; or

[0024] (c) a dual-gated nanopore protein for sequencing, wherein 7 to 10 amino acids are inserted between any two amino acids of G334 to G342 of the amino acid sequence shown in SEQ ID NO. 3, 7 to 10 amino acids are inserted between any two amino acids of T420 to S427, and 5 to 20 amino acids of T460 to V475 are replaced by 5 to 20 amino acids including two polar amino acids; or

[0025] (d) a dual-gated nanopore protein for sequencing, wherein 7-10 amino acids are inserted between any two amino acids of G97-G105 of the amino acid sequence shown in SEQ ID NO.4, 7-10 amino acids are inserted between any two amino acids of T183-S190, and 5-20 amino acids of T223-V238 are replaced by 5-20 amino acids including two polar amino acids; or

[0026] (e) In the A0A553L8Q2, F2GBL1, A0A4R6PPQ3, Q7MPZ6, Q8E KC9, ETEC E3PJ86, A0A369VU29, D4Z3A5, A0A1J1LIE5, A0A1C0V The amino acid sequence of any one of the dual-gated nanopores selected from US7, A0A0F5YGH9, A0A2T1BXK7, A0A1E5QK12, A0A252ECY0, A0A139X416, A0A3S1AFR1, A5GPC5, E0TBQ0, B1WXU4, B5IQM2, Q8YY21, Q7V469, K9Z6X7, Q115D6, B4D845, D8G3K2, A0A2V3VE55, and W7R0U8 is modified by the modification method of the dual-gated nanopore protein provided in the first aspect to obtain a dual-gated nanopore protein for sequencing.

[0027] The dual-gated nanopore protein for sequencing provided by the present application, such as the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, can be integrated on a large scale on a membrane to form a stable and uniform pore; and at the same time, it has efficient sequencing performance. Or the dual-gated nanopore protein for sequencing obtained by modifying the dual-gated nanopore shown in the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4 as described above can be integrated on a large scale on a membrane to form a stable and uniform pore; and at the same time, it has efficient sequencing performance.

[0028] In some embodiments of the present application, an x1L2x3L4x5 amino acid sequence is inserted between any two amino acids G334-G342 of the amino acid sequence shown in SEQ ID NO.3 or between any two amino acids G97-G105 of the amino acid sequence shown in SEQ ID NO.4, wherein the sequence includes three L amino acids, and the amino acids L2 and L4 include any one of L, V, I, F, and Y.

[0029] In some embodiments of the present application, x6L7x8L9x is inserted between any two amino acids of T420-S427 of the amino acid sequence shown in SEQ ID NO.3 or between any two amino acids of T183-S190 of the amino acid sequence shown in SEQ ID NO.4. 10 Amino acid sequence, x6 includes 3 amino acids, one of which is F or Y; x8 includes 2 amino acids, one of which is V or I, and the other is I or L; the amino acids of L7 and L9 include any one of L, V, I, F, and Y.

[0030] In some embodiments of the present application, 10-15 amino acids from T460 to V475 of the amino acid sequence shown in SEQ ID NO.3 are replaced by 10-15 amino acids, or 10-15 amino acids from T223 to V238 of the amino acid sequence shown in SEQ ID NO.4 are replaced by 10-15 amino acids, and the substituted amino acid sequence includes 3 polar amino acids and one negatively charged amino acid D or E.

[0031] In some embodiments of the present application, the substituted amino acid sequence is: LGDNNN, GSANNN, LGDNNNNPN, AGSSNSNPN, LGDNNNNPF, ASSSNSNPN, NNQN PFQ, GSTAGSSNSNPN, GSTLGDNNNNPNQN, GSTASSSNSNPNQT, G STLGDNNNNPFQN, TGATASSSNTNPAQT, TGATASSSNTNGAQT or TGATNQSTAVPQSA.

[0032] In a third aspect, an embodiment of the present application provides a use of the dual-gated nanopore protein for sequencing as provided in the second aspect in nucleic acid sequencing.

[0033] The dual-gated nanopore protein for sequencing provided in the second aspect of the present application has efficient sequencing performance and can therefore be effectively applied to nucleic acid sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is a diagram of the size analysis of the protein pore in Example 1 of the present application; wherein, (a) dual-gated nanopore Vc-WT; (b) dual-gated nanopore protein Vc-E8 for sequencing.

[0036] Figure 2 Schematic diagram of the structure of the dual-gated nanopore in Example 1 of the present application.

[0037] Figure 3 This is a diagram of the electrophoresis results of the four proteins purified in Example 3 of the present application; wherein, M-Marker; 1-dual-gated nanopore Vc-WT; 2-dual-gated nanopore protein Vc-E8 for sequencing; 3-dual-gated nanopore S1; 4-dual-gated nanopore protein S1-E8 for sequencing.

[0038] Figure 4 This is a diagram of the detection electrical signal results of the on-chip integration of the protein in Experimental Example 1 of the present application; wherein, (a) dual-gated nanopore Vc-WT; (b) dual-gated nanopore protein Vc-E8 for sequencing.

[0039] Figure 5 This is a graph showing the number of channels integrated on the chip for the four proteins in Experimental Example 1 of this application.

[0040] Figure 6 This is a conductivity result diagram of dual-gated nanopore protein sequencing in Experimental Example 2 of this application.

[0041] Figure 7 This is a graph showing the current noise results of dual-gated nanopore protein sequencing in Experimental Example 2 of this application.

[0042] Figure 8 This is a current signal diagram of dual-gated nanopore protein sequencing in Experimental Example 2 of this application.

[0043] Fig. 9 This is a current signal diagram of dual-gated nanopore protein sequencing in Experimental Example 2 of this application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0045] The present invention provides a method for modifying a dual-gated nanopore protein, comprising: inserting an amino acid sequence x1L2x3L4x5 and an amino acid sequence x6L7x8L9x into two adjacent β-pleated sheets of the β barrel of the dual-gated nanopore, respectively. 10 Amino acid sequence, wherein the amino acid sequence x1L2x3L4x5 and x6L7x8L9x 10 The amino acid sequences all include 2-20 amino acids, including at least two hydrophobic amino acids; and at least two amino acids in the sensing region of the dual-gated nanopore are replaced by polar amino acids.

[0046] Insert 2-20 amino acid sequences x1L2x3L4x5 and x6L7x8L9x respectively, including at least two hydrophobic amino acids, into two adjacent β-pleated sheets of the β barrel of the dual-gated nanopore. 10Amino acid sequence, and at least two amino acids in the sensing region of the dual-gated nanopore are replaced by polar amino acids. The modified dual-gated nanopore protein for sequencing can be integrated on a large scale on a chip to form a stable and uniform pore with efficient sequencing performance.

[0047] As an example, the amino acid sequence x1L2x3L4x5 and x6L7x8L9x 10 The length of the amino acid sequence can be the same or different, x1L2x3L4x5 amino acid sequence and x6L7x8L9x 10 The length of the amino acid sequences did not differ by more than 3 amino acids.

[0048] In some embodiments of the present application, the amino acids L2 and L4 in the x1L2x3L4x5 amino acid sequence include any one of A, L, V, I, M, F, Y, and W. In the adopted x1L2x3L4x5 amino acid sequence, L2 and L4 are selected as any one of A, L, V, I, M, F, Y, and W hydrophobic amino acids, respectively, so that the x1L2x3L4x5 amino acid sequence includes at least two hydrophobic amino acids.

[0049] In some embodiments of the present application, x6L7x8L9x 10 The amino acids of L7 and L9 in the amino acid sequence include any one of A, L, V, I, M, F, Y, and W. 10 In the amino acid sequence, L7 and L9 are selected from any one of A, L, V, I, M, F, Y, and W, respectively, so that x6L7x8L9x 10 The amino acid sequence includes at least two hydrophobic amino acids.

[0050] In some embodiments of the present application, in the x1L2x3L4x5 amino acid sequence, x1 includes 0-3 amino acids, x3 includes 0-5 amino acids, and x5 includes 0-3 amino acids.

[0051] Further, in the amino acid sequence of x1L2x3L4x5, x1 is an uncharged amino acid. As an example, the amino acid of x1 in the amino acid sequence of x1L2x3L4x5 may include, but is not limited to, V, I, T, L, S, A, F or Y.

[0052] Further, in the amino acid sequence of x1L2x3L4x5, x3 includes 2-3 amino acids. As an example, in the amino acid sequence of x1L2x3L4x5, the amino acid of x3 may include, but is not limited to, L or I.

[0053] Furthermore, in the x1L2x3L4x5 amino acid sequence, x5 includes 0 amino acids.

[0054] In some embodiments of the present application, the amino acid sequence of x1L2x3L4x5 is: LILL, LLVL, FIFL, FYIL, TLILL, YLLVL, TTLILL, TYLLVL, TTVLILL, TYVLLVL, VTTVLILL or LTYVLLVL.

[0055] In some embodiments of the present application, x6L7x8L9x 10 In the amino acid sequence, x6 includes 0-3 amino acids, x8 includes 0-5 amino acids, and x 10 Includes 0-5 amino acids.

[0056] Furthermore, x6L7x8L9x 10 In the amino acid sequence, x6 is an uncharged amino acid. 10 The amino acid of x6 in the amino acid sequence may include, but is not limited to, V, I, T, L, S, A, F or Y.

[0057] Furthermore, x6L7x8L9x 10 In the amino acid sequence, x8 includes 2-3 amino acids. For example, x6L7x8L9x 10 The amino acid of x8 in the amino acid sequence may include, but is not limited to, L or I.

[0058] Furthermore, x6L7x8L9x 10 The amino acid sequence x 10 Includes 0-3 amino acids. As an example, x6L7x8L9x 10 The amino acid sequence x 10 The amino acids may include, but are not limited to, S or A.

[0059] In some embodiments of the present application, x6L7x8L9x 10 The amino acid sequence is: LILL, LLVL, FIFL, FYIL, TLILL, YLLVL, TTLILL, TYLLVL, TTVLILL, TYVLLVL, VTTVLILL or LTYVLLVL.

[0060] In some embodiments of the present application, the sensing region of the dual-gated nanopore includes 3-20 amino acids substituted by 3-20 amino acids including at least two polar amino acids.

[0061] Further, the sensing region of the dual-gated nanopore comprises 5-20 amino acids substituted by 5-20 amino acids including at least two polar amino acids. As an example, at least 2 amino acids in the sensing region of the dual-gated nanopore are substituted by polar side chains of uncharged amino acids S, T, N or Q. As an example, among the substituted amino acids in the sensing region of the dual-gated nanopore, F amino acid is substituted by A, S, N or T.

[0062] In some embodiments of the present application, the substituted amino acid sequence is: SSS, NNN, NTS, LGDNNN, GSANNN, ATNQSTAVPQSA, LGDNNNNPN, AGSSNSNPN, LGDNNNNPF, ASSSNSNPN, NNQNPFQ, GSTAGSSNSNPN, GSTLGD NNNNPNQN, LGDNNNNSNQN, GSTNNQNPFQT, GSTASSSNSNPNQT, GSTLGDNNNNPFQN, GSTLGDNNNNGNQN, GSTLGDNNNNSNQN, TGATASSSNTNPAQT, TGATASSSNTNGAQT or TGATNQSTAVPQSA.

[0063] In the sensing region of the dual-gated nanopore, 3-20 amino acids are replaced by 3-20 amino acids including at least two S, T, N, Q polar amino acids, so that the modified dual-gated nanopore protein for sequencing has efficient sequencing performance.

[0064] In some embodiments of the present application, the dual-gated nanopore includes P45779, A0A553L8Q2, F2GBL1, A0A4R6PPQ3, Q7MPZ6, Q8EKC9, ETEC E3PJ86, A0A369V U29, D4Z3A5, A0A1J1LIE5, A0A1C0VUS7, A0A0F5YGH9, A0A2T1B XK7, A0A1E5QK12, A0A252ECY0, A0A139X416, A0A3S1AFR1, A5G PC5, E0TBQ0, B1WXU4, B5IQM2, Q8YY21, Q7V469, K9Z6X7, Q115D6, B4D845, D8G3K2, A0A2V3VE55, W7R0U8, or a dual-gated nanopore having an amino acid sequence homology of 97% or more to any of the above dual-gated nanopores. Using any of the above dual-gated nanopores, the modified dual-gated nanopore protein for sequencing can be obtained through the above modification, and the dual-gated nanopore protein for sequencing can be integrated on a large scale on the membrane to form a stable and uniform pore; at the same time, it has efficient sequencing performance. The above dual-gated nanopores are specifically shown in Table 1.

[0065] Table 1 Dual-gated nanopores

[0066] Serial No.S Protein Accessions Species origin 1 P45779 Vibrio cholerae 2 A0A553L8Q2 Aliiglaciecola sp.M165 3 F2GBL1 Alteromonas mediterranea 4 A0A4R6PPQ3 Idiomarina aquatica 5 Q7MPZ6 Vibrio vulnificus 6 Q8EKC9 Shewanella oneidensis 7 ETEC E3PJ86 Escherichia coli O78:H11 8 A0A369VU29 Sphingomonas aracearum 9 D4Z3A5 Sphingobium japonicum 10 A0A1J1LIE5 Planktothrix tepida 11 A0A1C0VUS7 Oscillatoriales cyanobacterium 12 A0A0F5YGH9 Limnoraphis robusta 13 A0A2T1BXK7 Merismopedia glauca 14 A0A1E5QK12 Desertifilum sp. 15 A0A252ECY0 Nostoc sp.T09 16 A0A139X416 Scytonema hofmannii 17 A0A3S1AFR1 Trichormus variabilis 18 A5GPC5 Synechococcus sp. 19 E0TBQ Parvularcula bermudensis 20 B1WU Chondrocystis sp. 21 B5IQM2 Cyanobium sp. 22 Q8YY21 Nostoc sp. 23 Q7V469 Prochlorococcus marinus 24 K9Z6X7 Cyanobacterium aponinum 25 Q115D6 Trichodesmium erythraeum 26 B4D845 Chthoniobacter flavus 27 D8G3K2 Kamptonema sp. 28 A0A2V3VE55 Blastomonas natatoria 29 W7R0U8 Catenovulum agarivorans

[0067] In some embodiments of the present application, the dual-gated nanopore P45779 is a dual-gated nanopore having an amino acid sequence as shown in SEQ ID NO.3.

[0068] The present application embodiment provides a dual-gated nanopore protein for sequencing, comprising:

[0069] (a) a dual-gated nanopore protein for sequencing the amino acid sequence as shown in SEQ ID NO.1; or

[0070] (b) a dual-gated nanopore protein for sequencing the amino acid sequence as shown in SEQ ID NO.2; or

[0071] (c) a dual-gated nanopore protein for sequencing, wherein 7 to 10 amino acids are inserted between any two adjacent amino acids of G334 to G342 of the amino acid sequence shown in SEQ ID NO. 3, 7 to 10 amino acids are inserted between any two adjacent amino acids of T420 to S427, and 5 to 20 amino acids of T460 to V475 are replaced by 5 to 20 amino acids including two polar amino acids; or

[0072] (d) a dual-gated nanopore protein for sequencing, wherein 7-10 amino acids are inserted between any two amino acids of G97-G105 of the amino acid sequence shown in SEQ ID NO.4, 7-10 amino acids are inserted between any two amino acids of T183-S190, and 5-20 amino acids of T223-V238 are replaced by 5-20 amino acids including two polar amino acids; or

[0073] (e) In the A0A553L8Q2, F2GBL1, A0A4R6PPQ3, Q7MPZ6, Q8E KC9, ETEC E3PJ86, A0A369VU29, D4Z3A5, A0A1J1LIE5, A0A1C0V The amino acid sequence of any one of the dual-gated nanopores selected from US7, A0A0F5YGH9, A0A2T1BXK7, A0A1E5QK12, A0A252ECY0, A0A139X416, A0A3S1AFR1, A5GPC5, E0TBQ0, B1WXU4, B5IQM2, Q8YY21, Q7V469, K9Z6X7, Q115D6, B4D845, D8G3K2, A0A2V3VE55, and W7R0U8 is modified by the modification method of the dual-gated nanopore protein provided in the first aspect to obtain a dual-gated nanopore protein for sequencing.

[0074] The dual-gated nanopore protein for sequencing with an amino acid sequence such as SEQ ID NO.1 or SEQ ID NO.2 can be integrated on a large scale on a membrane to form a stable and uniform pore; and at the same time, it has efficient sequencing performance. Or the dual-gated nanopore protein for sequencing obtained by modifying the dual-gated nanopore with an amino acid sequence such as SEQ ID NO.3 or SEQ ID NO.4 can be integrated on a large scale on a membrane to form a stable and uniform pore; and at the same time, it has efficient sequencing performance.

[0075] In some embodiments of the present application, an x1L2x3L4x5 amino acid sequence is inserted between any two amino acids G334-G342 of the amino acid sequence shown in SEQ ID NO.3 or between any two adjacent amino acids G97-G105 of the amino acid sequence shown in SEQ ID NO.4, wherein the sequence includes three L amino acids, and the amino acids L2 and L4 include any one of L, V, I, F, and Y.

[0076] In some embodiments of the present application, x6L7x8L9x is inserted between any two amino acids of T420-S427 of the amino acid sequence shown in SEQ ID NO.3 or between any two adjacent amino acids of T183-S190 of the amino acid sequence shown in SEQ ID NO.4. 10 Amino acid sequence, x6 includes 3 amino acids, one of which is F or Y; x8 includes 2 amino acids, one of which is V or I, and the other is I or L; the amino acids of L7 and L9 include any one of L, V, I, F, and Y.

[0077] In some embodiments of the present application, 10-15 amino acids of T460-V475 of the amino acid sequence shown in SEQ ID NO.3 are replaced by 10-15 amino acids, or 10-15 amino acids of T223-V238 of the amino acid sequence shown in SEQ ID NO.4 are replaced by 10-15 amino acids, and the replaced amino acid sequence includes 3 polar amino acids and a negatively charged amino acid D or E. This causes the sensing region of the dual-gated nanopore to change from a blunt end to a flat end, promotes the interaction between DNA and the tip sensing region, and enables the modified dual-gated nanopore protein for sequencing to have efficient sequencing performance.

[0078] In some embodiments of the present application, the substituted amino acid sequence is: LGDNNN, GSANNN, LGDNNNNPN, AGSSNSNPN, LGDNNNNPF, ASSSNSNPN, NNQN PFQ, GSTAGSSNSNPN, GSTLGDNNNNPNQN, GSTASSSNSNPNQT, G STLGDNNNNPFQN, TGATASSSNTNPAQT, TGATASSSNTNGAQT or TGATNQSTAVPQSA.

[0079] The present application provides an application of the above-mentioned dual-gated nanopore protein for sequencing in nucleic acid sequencing. The above-mentioned dual-gated nanopore protein for sequencing has efficient sequencing performance, and thus can be effectively applied to nucleic acid sequencing.

[0080] SEQ ID NO.1 sequence is: NEFSASFKGTDIQEFINIVGRNLEKTIIVDP SVRGKVDVRSFDTLNEEQYYSFFLSVLEVYGFAVVEMDNGVLKVIKSKDAKTSAIPVLSGEERANGDEVITQVVAVKNVSVRELSPLLRQLIDNAGAGNVVHYDPANIILITGRAAVVNRLAE IIRRVDQAGDKEIEVVELNNASAAEMVRIVEALNKTTDAQNTPEFLKPKFVADERTNSILISGDPKVRERLKRLIKQLDVEMAAKGNNRVVYLKYAKAEDLVEVLKGVSENLQAEKGTGQPTTS KRNEVMIAAHADTNSLVLTAPQDIMNAMLEVIGQLDIRRAQVLIEALIVEMAEGDGINLGTYVLLVLVQWGSLESGSVIQYGNTGASIGNVMIGLEEAKDTTQTKAVYDTNNNFLRNETTTTK GDYTKLASALSSIQGAAVSIAMGDWTALITTVLILLNAVSNDSSSNILSSPSITVMDNGEASFIVGEEVPVITGSTLGDNNNNPNQTVDRKEVGIKLKVVPQINEGNSVQLNIEQEVSNVLGAN GAVDVRFAKRQLNTSVMVQDGQMLVLGGLIDERALESESKVPLLGDIPLLGQLFRSTSSQVEKKNLMVFIKPTIIRDGVTADGITQRKYNYIRAEQLFRAEKGLRLLDDASVPVLPKFGDDRRHSPEIQAFI

[0081] The sequence of SEQ ID NO.2 is: AKGNNRVVYLKYAKAEDLVEVLKGVSEN LQAEKGTGQPTTSKRNEVMIAAHADTNSLVLTAPQDIMNAMLEVIGQLDIRRAQVLIEALIVEMAEGDGINLGTYVLLVLVQWGSLESGSVIQYGNTGASIGNVMIGLEEAKDTTQTKAVYDTNNNFLRNETTTTKGDYTKLASALSSIQGAAVSIAMGDWTALITTVLILLNAVSNDSSSNILSSPSITVMDNGEASFIVGEEVPVITGSTLGDNNNNPNQTVDRKEVGIKLKVVPQINEGNSVQLNIEQEVSNVLGANGAVDVRFAKRQLNTSVMVQDGQMLVLGGLIDERALESESKVPLLGDIPLLGQLFRSTSSQVEKKNLMVFIKPTIIRDGVTADGITQRKYNYIRAEQLFRAEKGLRLLDDASVPVLPKFGDDRRHSPEIQAFI

[0082] The sequence of SEQ ID NO.3 is: NEFSASFKGTDIQEFINIVGRNLEKTIIVDPSV RGKVDVRSFDTLNEEQYYSFFLSVLEVYGFAVVEMDNGVLKVIKSKDAKTSAIPVLSGEERANGDEVITQVVAVKNVSVRELSPLLRQLIDNAGAGNVVHYDPANIILITGRAAVVNRLAEIIRRVDQAGDKEIEVVELNNASAAEMVRIVEALNKTTDAQNTPEFLKPKFVADERTNSILISGDPKVRERLKRLIKQLDVEMAAKGNNRVVYLKYAKAEDLVEVLKGVSENLQAEKGTGQPTTSKRNEVMIAAHADTNSLVLTAPQDIMNAMLEVIGQLDIRRAQVLIEALIVEMAEGDGINLGVQWGSLESGSVIQYGNTGASIGNVMIGLEEAKDTTQTKAVYDTNNNFLRNETTTTKGDYTKLASALSSIQGAAVSIAMGDWTALINAVSNDSSSNILSSPSITVMDNGEASFIVGEEVPVITGSTAGSNNDNPFQTVDRKEVGIKLKVVPQINEGNSVQLNIEQEVSNVLGANGAVDVRFAKRQLNTSVMVQDGQMLVLGGLIDERALESESKVPLLGDIPLLGQLFRSTSSQVEKKNLMVFIKPTIIRDGVTADGITQRKYNYIRAEQLFRAEKGLRLLDDASVPVLPKFGDDRRHSPEIQAFIEQMEAKQ

[0083] SEQ ID NO.4 sequence: AKGNNRVVYLKYAKAEDLVEVLKGVSENL QAEKGTGQPTTSKRNEVMIAAHADTNSLVLTAPQDIMNAMLEVIGQLDI RRAQVLIEALIVEMAEGDGINLGVQWGSLESGSVIQYGNTGASIGNVMIGLEEAKDTTQTKAVYDTNNNFLRNETTTTKGDYTKLASALSSIQGAAVSIAMGDWTALINAVSNDSSSNILSSPSITVMDNGEASFIVGEEVPVITGSTAGSNNDNPFQTVDRKEVGI KLKVVPQINEGNSVQLNIEQEVSNVLGANGAVDVRFAKRQLNTSVMVQDGQMLVLGGLIDERALESESKVPLLGDIPLLGQLFRSTSSQVEKKNLMVFIKPTIIRDGVTADGITQRKYNYIRAEQLFRAEKGLRLLDDASVPVLPKFGDDRRHSPEIQAFIEQMEAKQ

[0084] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0085] Example 1

[0086] Design and structural analysis of S1 protein:

[0087] The structure of S1 dual-gated nanopore P45779 (PDB 5WQ8) was obtained from the PDB database, and visualized with the help of ChimeraX software. Its hydrophobic region was displayed by the Mlp command. Its amino acid sequence is shown in SEQ ID NO.3. The TYLLVL sequence was inserted above the alpha helix of the hydrophobic region, that is, between G338 and V339; the TTLILL sequence was inserted between I423 and N424; the sequence GSTAGSNNDNPFQT between T460 and V475 was replaced with GSTLGDNNNNPN QN, and the amino acid sequence of the modified dual-gated nanopore protein for sequencing was obtained as shown in SEQ ID NO.1. The structure of the modified dual-gated nanopore protein for sequencing was predicted by AlphaFold and the size of its pore was analyzed by Hole 2.0, as shown in the following. Figure 1 The results shown show that after modification, the length of the hydrophobic region of the dual-gated nanopore increased to 35 angstroms.

[0088] The amino acid sequence of S1 dual-gated nanopore P45779 (Vc-WT) shown in SEQ ID NO.3 is truncated with N1-N2 (such as Figure 2 The amino acid sequence of the peptide is shown in SEQ ID NO.4 (S1).

[0089] The amino acid sequence is shown in SEQ ID NO.1. The double-gated nanopore protein (Vc-E8) is used for sequencing by cutting off N1-N2 (such as Figure 2 The amino acid sequence of the peptide is shown in SEQ ID NO.2 (S1-E8).

[0090] Or the TYLLVL sequence is inserted between G103 and V104 of the truncated S1 dual-gated nanopore as shown in the amino acid sequence of SEQ ID NO.4; the TTLILL sequence is inserted between I188 and N189; the sequence GSTAGSNNDNPFQT between T223 and V238 is replaced with GSTLGD NNNNPNQN, and the amino acid sequence of the modified dual-gated nanopore protein for sequencing is obtained as shown in SE Q ID NO.2.

[0091] Example 2

[0092] Design and structural analysis of protein seq2-29:

[0093] The sequences of the dual-gated nanopore proteins for dual-gated nanopore sequencing of S2-29 were downloaded from the Uniprot database, and the monomer structure of the sequence of S2-29 was predicted by AlphaFold 2. The obtained monomer structure was compared with the structure of the S1 dual-gated nanopore P45779 by TM-Align to obtain a sequence with the same structure; the obtained sequence was predicted to have a 15-mer structure by AlphaFold 3, and then the structure was compared. According to the method of Example 1, 1-10 amino acid sequences were respectively substituted on the two adjacent β-folded sheets inserted into the β barrel of each dual-gated nanopore, and at least two amino acids in the sensing region of each dual-gated nanopore were substituted with polar amino acids. The same method as in Example 1 was used to predict the structure of each modified dual-gated nanopore protein for sequencing using AlphaFold and the size of the pore of each modified dual-gated nanopore protein for sequencing was analyzed by Hole 2.0, and the results shown in Table 2 were obtained.

[0094] Table 2 Modified results of each double-gated nanopore No. S2-29

[0095]

[0096]

[0097] Example 3

[0098] Expression and purification of dual-gated nanopore proteins for sequencing:

[0099] Protein expression: Synthesize the gene sequence of the dual-gated nanopore protein shown in SEQ ID NO.1 for coding sequencing, and transform it into the E. coli C43 expression strain with the strep II-tag sequence (sequence: WSHPQFEK) at the N-terminus of the gene sequence. Select a single colony on an LB medium agar plate (Shanghai Biotechnology) containing 100 μg / mL antibiotics; pick a single colony and culture it at 200 rpm at 37°C until the OD 600 The culture was expanded at 1:200 (seed solution / LB liquid medium (Shanghai Biotech)). 600 After the pH value reached 0.6, IPTG was added and the temperature was lowered to 20°C and culture was continued for 14 hours. The bacteria were collected by centrifugation at 4000 g and washed once with phosphate buffer at pH 7.4.

[0100] Protein purification: The washed cells were added to buffer A, and the mass concentration of the cells in buffer A (containing 300 mM NaCl and 50 mM Tris-HCl, pH 8.0) was 0.1 g / mL; the cells were then lysed by ultrasonic disruption, centrifuged at 4000 g to remove cell debris, and then centrifuged at 200000 g (Beckman, 70Ti rotor) for 1.5 h; buffer B (containing 300 mM NaCl, 50 mM Tris-HCl, 0.001 g / mL GDN (glycoenzyme) and 0.01 g / mL DDM (dodecyl-β-D-maltoside), pH 8.0) was added to the centrifuged system to fully dissolve, mixed at 4°C for 1 h, and filtered with a 0.22 μm filter to obtain the supernatant; the supernatant was then injected into Strep-Tactin XT The resin chromatography column was washed with solution C (containing 300 mM NaCl, 50 mM Tris-HCl and 0.01 g / mL GDN), and an eluent (containing 300 mM NaCl, 50 mM Tris-HCl, 0.01 g / mL GDN and 5 mM desthiobiotin) was added to collect the dual-gated nanopore protein for sequencing. The collected dual-gated nanopore protein for sequencing was further separated into polymers and monomers by gel chromatography molecular sieve to obtain pure dual-gated nanopore protein Vc-E8 for sequencing.

[0101] The dual-gated nanopores Vc-WT, S1 and dual-gated nanopore protein S1-E8 for sequencing were purified in the same manner as above.

[0102] The above-mentioned dual-gated nanopore proteins Vc-E8 and S1-E8 for sequencing were stored in a buffer (containing 300 mM NaCl, 50 mM Tris-HCl and 0.05% detergent) for use. The above-mentioned dual-gated nanopore Vc-WT and S1 proteins were stored in a buffer (containing 50 mM NaCl, 15 mM Tris-HCl and 0.01% GDN) for use.

[0103] The above four proteins were tested by electrophoresis, and the results were as follows Figure 3 shown.

[0104] Test Example 1

[0105] In this test example, the on-chip integration efficiency of the four proteins Vc-E8, Vc-WT, S1, and S1-E8 obtained in Example 3 was tested respectively. The specific method is:

[0106] Vc-WT and Vc-E8 were diluted to the same concentration with a solution containing 350 mM KCl. A layer of phospholipid was coated on a 100 μm biochip (Element, Italy) and a buffer solution with a pH of 8.0 (containing 1 M KCl and 50 mM Tris-HCl) was added. 0.01 μg / mL of the above four proteins were added, and a voltage of 180 mV was applied. The electrical signals were detected by an electrophysiological instrument. The results are shown in Figure 2. Figure 4 shown.

[0107] Depend on Figure 4 The results show that the wild-type dual-gated nanopore is difficult to form a step electrical signal, while the modified dual-gated nanopore protein for sequencing provided in the present application has a step transition, which indicates that the modified protein provided in the present application is more likely to form a channel in phospholipids.

[0108] Furthermore, the above four proteins were arrayed on a 256-channel chip (provided by BGI Life Sciences Institute), and the same concentration of the above four proteins was added to count the number of channels. Then, the number of channels generated per milligram of protein was calculated by concentration conversion. The results are as follows: Figure 5 shown.

[0109] Depend on Figure 5 The results show that the dual-gated nanopore proteins Vc-E8 and S1-E8 for sequencing provided by the present application have significantly increased on-chip integration efficiency, that is, the dual-gated nanopore proteins for sequencing provided by the present application can be integrated on a large scale on a chip.

[0110] Test Example 2

[0111] In this test example, the sequencing obtained in Example 3 was evaluated using the dual-gated nanopore protein Vc-E8. The specific method is:

[0112] The sequencing dual-gated nanopore protein was diluted 1:1000 using a buffer containing 500mM KCl and added to a 256-channel sequencing chip (provided by BGI Life Sciences Institute). The wells were inserted at a gradient voltage of 0-140mV. After the wells were inserted, the excess wells were washed with the above buffer to remove the excess wells. The chip was rinsed twice with a sequencing mixture at pH 8.0 (containing 500mM KCl, 25mM HEPES, 11mM ATP and 10mM MgCl2). The prepared DNA samples with characteristic sequences were sequenced, and 1μg of DNA analyte was mixed and connected with 40nM adapter mixture (provided by BGI Life Sciences Institute). The sequencing samples were obtained by magnetic bead purification, and the purified sequencing samples were added to the sequencing chip and sequenced at a voltage of 180mV for 6 hours. The results are shown in Figure 6-Figure 9 shown.

[0113] Depend on Figure 6 The results show that the conductivity of most of the pores of the dual-gated nanopore proteins for sequencing is about 1 nS, indicating that the pores formed by the dual-gated nanopore proteins for sequencing provided by the present application are uniform. Figure 7 The results show that the opening current of the dual-gated nanopore protein for sequencing is 220pA, and the noise of the dual-gated nanopore protein for sequencing is 1.49pA, indicating that the pore formed by the dual-gated nanopore protein for sequencing provided by the present application is stable. Figure 8 The results show that the blocking current ratio generated by sequencing is 0.75 when the 3.5 kbp sequence is used for library construction test. Fig. 9 As a result, the current change caused by the base is about 60pA. Based on the above results, it is shown that the sequencing performance of the dual-gated nanopore protein for sequencing provided by the present application is consistent with the sequencing performance of MspA and CsgG reported in the prior art. That is, the dual-gated nanopore protein for sequencing provided by the present application can be integrated on a large scale on a chip to form a stable and uniform pore; at the same time, it has efficient sequencing performance.

[0114] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A method for modifying a dual-gated nanopore protein, characterized in that: include: The amino acid sequences x1L2x3L4x5 and x6L7x8L9x are inserted into the two adjacent β-pleated sheets of the β-barrel of the dual-gated nanopore, respectively. 10 Amino acid sequence, wherein the amino acid sequence x1L2x3L4x5 and x6L7x8L9x 10 The amino acid sequences all include 2-20 amino acids, including at least two hydrophobic amino acids; and at least two amino acids in the sensing region of the dual-gated nanopore are replaced by polar amino acids.

2. The method for modifying a dual-gated nanopore protein according to claim 1, characterized in that: The amino acids L2 and L4 in the x1L2x3L4x5 amino acid sequence include any one of A, L, V, I, M, F, Y, and W; and / or, the x6L7x8L9x 10 The amino acids of L7 and L9 in the amino acid sequence include any one of A, L, V, I, M, F, Y, and W.

3. The method for modifying a dual-gated nanopore protein according to claim 2, characterized in that: In the x1L2x3L4x5 amino acid sequence, x1 includes 0-3 amino acids, x3 includes 0-5 amino acids, and x5 includes 0-3 amino acids; Preferably, the x1L2x3L4x5 amino acid sequence is: LILL, LLVL, FIFL, FYIL, TLILL, YLLVL, TTLILL, TYLLVL, TTVLILL, TYVLLVL, VTTVLILL or LTYVLLVL.

4. The method for modifying a dual-gated nanopore protein according to claim 2, characterized in that: x6L7x8L9x 10 In the amino acid sequence, x6 includes 0-3 amino acids, x8 includes 0-5 amino acids, and x 10 Includes 0-5 amino acids; Preferably, the x6L7x8L9x 10 The amino acid sequence is: LILL, LLVL, FIFL, FYIL, TLILL, YLLVL, TTLILL, TYLLVL, TTVLILL, TYVLLVL, VTTVLILL or LTYVLLVL.

5. The method for modifying a dual-gated nanopore protein according to claim 1, characterized in that: The sensing region of the dual-gated nanopore comprises 3-20 amino acids substituted with 3-20 amino acids comprising at least two polar amino acids; Preferably, the substituted amino acid sequence is: SSS, NNN, NTS, LGDNNN, GSANNN, ATNQSTAVPQSA, LGDNNNNPN, AGSSNSNPN, LGDNNNNP F, ASSSNSNPN, NNQNPFQ, GSTAGSSNSNPN, GSTLGDNNNNPNQN, LGDNNNNSNQN, GSTNNQNPFQT, GSTASSSNSNPNQT, GSTLGDNN NNPFQN, GSTLGDNNNNGNQN, GSTLGDNNNNSNQN, TGATASSSNT NPAQT, TGATASSSNTNGAQT or TGATNQSTAVPQSA.

6. The method for modifying a dual-gated nanopore protein according to claim 1, characterized in that: The dual-gated nanopores include P45779, A0A553L8Q2, F2GBL1, A0A4R6PPQ3, Q7MPZ6, Q8EKC9, ETEC E3PJ86, A0A369VU29, D4Z3A5, A0A1J1LI Any one of A0A1E5QK12, A0A252ECY0, A0A139X416, A0A3S1AFR1, A5GPC5, E0TBQ0, B1WXU4, B5IQM2, Q8YY21, Q7V469, K9Z6X7, Q115D6, B4D845, D8G3K2, A0A2V3VE55, and W7R0U8, or a dual-gated nanopore having an amino acid sequence homology of greater than or equal to 97% with any of the above dual-gated nanopores; Preferably, the dual-gated nanopore P45779 is a dual-gated nanopore having an amino acid sequence as shown in SEQ ID NO.

3.

7. A dual-gated nanopore protein for sequencing, characterized in that: include: (a) a dual-gated nanopore protein for sequencing the amino acid sequence as shown in SEQ ID NO.1; or (b) a dual-gated nanopore protein for sequencing the amino acid sequence as shown in SEQ ID NO.2; or (c) a dual-gated nanopore protein for sequencing, wherein 7 to 10 amino acids are inserted between any two amino acids of G334-G342 of the amino acid sequence shown in SEQ ID NO.3, 7 to 10 amino acids are inserted between any two amino acids of T420-S427, and 5 to 20 amino acids of T460-V475 are replaced by 5 to 20 amino acids including two polar amino acids; or (d) a dual-gated nanopore protein for sequencing, wherein 7-10 amino acids are inserted between any two amino acids of G97-G105 of the amino acid sequence shown in SEQ ID NO.4, 7-10 amino acids are inserted between any two amino acids of T183-S190, and 5-20 amino acids of T223-V238 are replaced by 5-20 amino acids including two polar amino acids; or (e) In the A0A553L8Q2, F2GBL1, A0A4R6PPQ3, Q7MPZ6, Q8EKC9, ETEC The amino acid sequence of any one of the dual-gated nanopores E3PJ86, A0A369VU29, D4Z3A5, A0A1J1LIE5, A0A1C0VUS7, A0A0F5YGH9, A0A2T1BXK7, A0A1E5QK12, A0A252ECY0, A0A139X416, A0A3S1AFR1, A5GPC5, E0TBQ0, B1WXU4, B5IQM2, Q8YY21, Q7V469, K9Z6X7, Q115D6, B4D845, D8G3K2, A0A2V3VE55, and W7R0U8 is modified by the modification method of the dual-gated nanopore protein according to any one of claims 1 to 6 to obtain a dual-gated nanopore protein for sequencing.

8. The dual-gated nanopore protein for sequencing according to claim 7, characterized in that: An x1L2x3L4x5 amino acid sequence is inserted between any two amino acids of G334-G342 of the amino acid sequence shown in SEQ ID NO.3 or between any two amino acids of G97-G105 of the amino acid sequence shown in SEQ ID NO.4, wherein the amino acid sequence includes three L amino acids, and the amino acids L2 and L4 both include any one of L, V, I, F, and Y; And / or, x6L7x8L9x is inserted between any two amino acids of T420-S427 of the amino acid sequence shown in SEQ ID NO.3 or between any two amino acids of T183-S190 of the amino acid sequence shown in SEQ ID NO.4 10 Amino acid sequence, x6 includes 3 amino acids, one of which is F or Y; x8 includes 2 amino acids, one of which is V or I, and the other is I or L; the amino acids of L7 and L9 include any one of L, V, I, F, and Y.

9. The dual-gated nanopore protein for sequencing according to claim 7, characterized in that: 10-15 amino acids from T460 to V475 of the amino acid sequence shown in SEQ ID NO.3 are replaced by 10-15 amino acids, or 10-15 amino acids from T223 to V238 of the amino acid sequence shown in SEQ ID NO.4 are replaced by 10-15 amino acids, and the replaced amino acid sequence includes 3 polar amino acids and one negatively charged amino acid D or E; Preferably, the substituted amino acid sequence is: LGDNNN, GSANNN, LGDNNN NPN, AGSSNSNPN, LGDNNNNPF, ASSSNSNPN, NNQNPFQ, GSTAG SSNSNPN, GSTLGDNNNNPNQN, GSTASSSNSNPNQT, GSTLGDNNNN PFQN, TGATASSSNTNPAQT, TGATASSSNTNGAQT or TGATNQSTAVP QSA.

10. Use of the dual-gated nanopore protein for sequencing according to any one of claims 7 to 9 in nucleic acid sequencing.

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