A method for detecting protein frameshift mutations in two modes

Abstract

The application provides a protein frame-shift mutation dual-mode detection method, comprising guiding migration of an analyte to be analyzed in a biological nanopore by using an electric field force, and identifying and distinguishing different kinds of analytes by using an electric current signal mode caused by electroosmotic flow and / or electrophoresis effect. The detection method can identify different H1.4 histone frame-shift mutation bodies with higher precision.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for detecting protein frameshift mutations in a dual-modal manner. Background Technology

[0002] HIST1H1E syndrome, also known as Rahman syndrome, is a rare genetic disorder [1-4]. The clinical manifestations of HIST1H1E syndrome are diverse. Patients typically exhibit some typical facial features, such as a high forehead, wide-set eyes, a flat nasal bridge, and a small mouth; facial development may be significantly abnormal. Most patients have mild to severe intellectual disability, which affects their daily life and learning abilities. Developmental delay is also a common symptom; patients usually exhibit delays in intellectual and motor development, and may experience learning difficulties, speech delays, and motor coordination problems. Many patients are short in stature, remaining shorter than their peers even in late adolescence. Other signs may include loose skin, long and thin fingers and toes, and some patients may also exhibit obesity or muscle weakness. Psychologically and behaviorally, some patients may also exhibit hyperactivity or mood swings.

[0003] Research on HIST1H1E syndrome is still in its early stages, especially regarding how histone H1.4 mutations lead to specific clinical symptoms, which scientists are still exploring in depth. This disease is caused by heterozygous mutations in the HIST1H1E gene, which encodes histone H1.4, a key protein that maintains chromatin structure and stability and regulates gene expression [5]. Mutations in the HIST1H1E gene can lead to structural abnormalities in histone H1.4, which affects the structure and function of chromatin and thus the regulation of gene expression [6-7].

[0004] To date, 21 frameshift mutations have been reported in children with HIST1H1E syndrome, all located within the 99-bp region of the H1E CTD domain. Among them, 20 mutations produce new C-terminal truncated mutant proteins containing the same 38 amino acids. In the only case of central sleep apnea, a new 25 amino acid sequence was formed [4]. Compared with wild-type protein, the net positive charge of the C-terminal amino acid sequence of the frameshift mutant H1E is greatly reduced. Whether the newly generated 38 amino acid sequence (hereinafter referred to as 38AA) can affect the function of H1 compressed chromatin or lead to gain-of-function (GOF) is an important scientific question.

[0005] Because the frameshift products share the same C-terminus, these mutant proteins have very similar sequences. Further research into the mechanisms of this disease is essential, and a highly efficient detection method is urgently needed to distinguish different H1.4 frameshift mutants within the same system.

[0006] Currently, there are various methods for protein detection. One of the most common is Edman degradation, a classic chemical method used to progressively cleave amino acids from the N-terminus of proteins or peptides and determine their sequence. Edman degradation offers advantages such as high precision and reliability, making it suitable for analyzing the amino acid sequences of short peptides or small molecules. It also does not rely on known databases and can be used for determining completely unknown sequences. However, its limitations include a limited sequencing length, typically only allowing the determination of sequences within 40-60 amino acids, and a complex and time-consuming process. Furthermore, Edman degradation struggles to identify post-translational modifications in proteins (such as phosphorylation and methylation), limiting its application in modern proteomics.

[0007] Mass spectrometry (MS / MS) is a crucial technique in modern proteomics, particularly tandem mass spectrometry. It determines the amino acid sequence by analyzing the mass of peptides after digestion. MS / MS offers high sensitivity and high throughput, allowing simultaneous analysis of multiple protein fragments in a mixture, making it ideal for complex samples. It can also identify post-translational modifications, which is significant for studying protein function and biological activity. However, MS / MS typically relies on known sequence databases; identification can be challenging if the target protein is not listed. Furthermore, it usually only yields fragmented peptide sequences, making it difficult to directly assemble complete protein sequences. The high cost of MS / MS instruments, maintenance expenses, and the need for specialized personnel limit its widespread adoption.

[0008] De Novo sequencing is also a mass spectrometry-based sequencing method, but unlike conventional mass spectrometry, it does not rely on a database and can be used to infer the sequence of completely unknown proteins. It infers the amino acid sequence from peptide fragment spectra generated by mass spectrometry. De Novo sequencing is suitable for novel proteins not found in databases and can identify post-translational modifications to infer protein function. However, De Novo sequencing algorithms are complex, computationally demanding, and data processing is time-consuming. Sequence reconstruction is also challenging when encountering proteins with repetitive sequences or structural modifications.

[0009] Amino acid analysis is a method for analyzing the amino acid composition of proteins after hydrolysis. This method is suitable for simple compositional analysis, helping to understand the overall characteristics of proteins and detecting the amino acid composition of different proteins in a mixture. The advantages of amino acid analysis are its relatively low cost and simple operation. However, amino acid analysis cannot obtain the specific amino acid sequence; it is only suitable for analyzing amino acid composition and is not suitable for proteins with complex structures or abundant post-translational modifications. Furthermore, this method is destructive to the sample and cannot obtain the original sequence information of the protein.

[0010] In summary, it is easy to see that current methods for detecting histone H1.4 mutations, such as the Edman degradation method and mass spectrometry-based protein sequencing, have shortcomings such as low sequencing throughput, high cost, and limited accuracy. Summary of the Invention

[0011] To address this issue, this invention provides an analyte detection method based on biological nanopores, which solves the problems of low sequencing throughput, high cost, and limited accuracy in existing technologies.

[0012] In one aspect, the present invention provides a method for detecting protein frameshift mutations in a dual-modal manner, comprising the following steps: 1) applying voltages in different directions to both sides of a bio-nanopore chip comprising a single nanoporous protein including a membrane and an intercalation membrane, thereby generating migration of the analyte within the bio-nanopore based on electroosmotic and electrophoretic effects; 2) measuring the ionic current generated by the analyte during migration within the bio-nanopore to provide a current signal pattern; 3) identifying and distinguishing different types of analytes by the current signal pattern induced by the electroosmotic and electrophoretic effects.

[0013] In one embodiment of the present invention, a positive voltage is applied to obtain a current signal pattern under the electroosmotic effect; preferably, the positive voltage range is 40mV-140mV.

[0014] In one embodiment of the present invention, a negative voltage is applied to obtain the current signal pattern under the electrophoretic effect; preferably, the negative voltage range is -140mV to 40mV.

[0015] In one embodiment of the present invention, the parameters obtained from the current signal pattern analysis include Amp.SD and Interevent intervals(t on ), Δexclude(ΔI / I0*100%) and Dwell time.

[0016] In one embodiment of the present invention, analytes are identified and distinguished by analyzing the current signal patterns induced by electroosmotic flow and electrophoresis effects; preferably, at least one parameter of the current signal pattern induced by electroosmotic flow and at least one parameter of the current signal pattern induced by electrophoresis effects are analyzed; more preferably, two parameters are analyzed: the fluctuation range of the blocking current under the electrophoresis effect (Amp.SD) and the dwell time of the blocking current under the electroosmotic flow effect (Dwell time).

[0017] In one embodiment of the present invention, the nanopore is an MspA homolog or a mutant thereof, preferably an M2 MspA nanopore.

[0018] In one embodiment of the present invention, the analyte is a protein and one or more mutants thereof, wherein at least one mutant is a frameshift mutant.

[0019] In one embodiment of the present invention, the analyte includes linker histone H1.4 and its mutants. In another embodiment, the linker histone H1.4 mutant includes sequences having at least 50%, 60%, 70%, 80%, or 90% identity with linker histone H1.4. In yet another embodiment, the linker histone H1.4 mutant includes a C-terminal truncated mutant protein of linker histone H1.4 and a fusion protein of linker histone H1.4.

[0020] In one embodiment of the present invention, the amino acid sequence of the linker histone H1.4 (H1.4 WT) is shown in SEQ ID NO: 1.

[0021] MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKA ASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTPKKAKKPAAAAGAKKAKSPKKAKAAKPKKAPKSPAKAKAVKPKAAKPKTAKPKAAAKPKKAAAKKK(SEQ ID NO:1)

[0022] In one embodiment of the present invention, the linker histone H1.4 mutant includes the sequence shown in SEQ ID NO: 2-21.

[0023] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTPKKKAKKKPKAAAAGAKKAKSPKKAKAPKSPAKAKAVKPKAAKPKTAKPKAAKKPKAAAKKKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 2,H1.4+38)

[0024] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 3, H1.4 157)

[0025] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTPKKKAKKKPKAAAAGAKISEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 4, H1.4 169)

[0026] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKGRRGQGQEASRSGEEAQEGDGGGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKKGAQEPSEGQSS(SEQ ID NO:5)

[0027] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPASRSGEEAQEGDGGGHPQEREGEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKKGAQEPSEGQSS(SEQ ID NO:6)

[0028] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAIEAQEGDGGGHPQEREGEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:7)

[0029] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKEAQEGDGGGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:8)

[0030] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPQEGDGGGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:9)

[0031] MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKEGDGGGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:10)

[0032] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKAKGGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:11)

[0033] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKAKGVGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:12)

[0034] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGGHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:13)

[0035] MSETAPAAPAAPAAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAGHPQEERQEDPCEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:14)

[0036] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAAHPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:15)

[0037] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAADPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:16)

[0038] MSETAPAAAPAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKLSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO:17)

[0039] MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPQEERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 18)

[0040] MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 19)

[0041] MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKERQEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 20)

[0042] MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAIEDPKEGEEAGCSCWSQKSEKPEKGESSQAKKGAQEPSEGQSS(SEQ ID NO: 21)

[0043] In one embodiment of the present invention, the detection method comprises the following steps:

[0044] (1) preparing the analyte into a test sample;

[0045] (2) Add the test sample to the Cis end of the bio-nanopore test system;

[0046] (3) Apply an electric field to cause the analyte to migrate under the action of electroosmosis or electrophoresis, and capture the current signal generated in the nanopore;

[0047] (4) Analyze the current signal generated during the migration process to identify the characteristics of the analyte;

[0048] (5) Compare the analysis results to achieve the detection and identification of at least four different analytes.

[0049] In one embodiment of the present invention, step (1) includes dissolving the analyte in a test buffer and mixing it thoroughly. In a specific embodiment of the present invention, the test buffer includes a neutral or weakly alkaline test buffer; the pH of the test buffer is not higher than 8.0.

[0050] In one embodiment of the present invention, the bio-nanopore testing system in step (2) includes a bio-nanopore chip and a test buffer.

[0051] In one embodiment of the present invention, the test buffer comprises a neutral or weakly alkaline test buffer; the pH of the test buffer is not higher than 8.0. Specifically, the test buffer comprises 200mM KCl, 150mM potassium ferricyanide, 100mM potassium ferrocyanide, and 25mM PBS, with a pH of 8.0.

[0052] In one embodiment of the present invention, the concentration of the test sample in the bio-nanopore testing system is 10-3000 nM. For example, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 2000 nM, and 3000 nM.

[0053] In one embodiment of the present invention, step (3) includes applying a positive voltage to the Cis terminal to cause the analyte to migrate under the action of electroosmosis and capture the current signal generated by it in the nanopore; and applying a negative voltage to the Cis terminal to cause the analyte to migrate under the action of electrophoresis and capture the current signal generated by it in the nanopore.

[0054] In one embodiment of the present invention, the migration behavior of the analyte is analyzed and the relevant current signal is captured based on the interaction between the size of the analyte and the size of the nanopore confinement region, or the interaction between the size of the analyte and the magnitude of the electric field.

[0055] In one specific embodiment of the present invention, when the size of the analyte is larger than the size of the confinement region of the bio-nanopore or the electric field strength is limited, the analyte is first captured by the vestibular region of the bio-nanopore, and then leaves the vestibular region of the bio-nanopore under Brownian diffusion and generates a current signal of a specific mode; when the size of the analyte added to the Cis end of the bio-nanopore is appropriate and the applied electric field strength reaches a certain level, the analyte will enter the Trans end of the bio-nanopore through the confinement region of the bio-nanopore under the action of the electric field and generate a current signal of a specific mode.

[0056] In one embodiment of the present invention, the magnitude of the positive voltage is 40mV-140mV, preferably 90mV. In another embodiment of the present invention, the magnitude of the negative voltage is -140mV-40mV, preferably -90mV.

[0057] In one embodiment of the present invention, the parameters analyzed in step (4) include the fluctuation range of the blocking current, Amp.SD, and the event intervals (t). on The blocking amplitude Δexclude(ΔI / I0*100%) and the dwell time of the blocking current are also considered.

[0058] In one embodiment of the present invention, step (5) includes comparing two parameters: the fluctuation range of the blocking current under the electrophoretic effect and the residence time of the blocking current under the electroosmotic effect. Different H1.4 histone mutants are identified by setting different thresholds for Amp.SD and Dwell time.

[0059] In one embodiment of the present invention, under the electrophoretic effect at a voltage of -90mV, the difference in the fluctuation range of the blocking current between samples is 0.2-4.0pA, preferably 0.3-3.3pA; under the electroosmotic effect at a voltage of 90mV, the difference in the residence time of the blocking current between samples is 20-300ms, preferably 29.4-245.3ms.

[0060] In a second aspect, the present invention provides a bio-nanopore analysis device for implementing the above-described detection method, the device comprising:

[0061] (a) A bio-nanopore chip, comprising a membrane and individual nanopore proteins inserted into the membrane;

[0062] (b) An electric field application device for applying a specific electric field to a nanopore, the electric field application device having a voltage switching element, thereby applying voltages in different directions to the nanopore to achieve dual-mode detection of the electroosmotic and electrophoretic effects of the analyte;

[0063] (c) A current signal detection device for detecting the current signal generated when an analyte passes through a biological nanopore;

[0064] (d) Signal analysis system, used to perform pattern recognition on the detected current signal to identify the types and characteristics of different analytes.

[0065] In one embodiment of the present invention, the membrane separates a first conductive liquid medium from a second conductive liquid medium. The membrane may be an organic membrane (e.g., a lipid bilayer) or a synthetic membrane (e.g., a membrane formed from a polymer material).

[0066] In one embodiment of the present invention, the nanoporous protein is an MspA homolog or a mutant thereof, preferably an M2 MspA nanopore.

[0067] In one embodiment of the present invention, the current signal is the signal generated by the ions of the nanoporous protein when the protein to be tested is captured by the nanoporous protein and then escapes.

[0068] In one embodiment of the present invention, the parameters of the signal include the fluctuation range of the blocking current, Amp.SD, and the event intervals (t). on The parameters are: the blockage amplitude Δexclude(ΔI / I0*100%) and the dwell time of the blockage current; preferably, the parameters are the fluctuation range of the blockage current under the electrophoretic effect and the dwell time of the blockage current under the electroosmotic effect.

[0069] In one embodiment of the present invention, the signal analysis system analyzes at least one parameter of the current signal pattern induced by electroosmotic flow and at least one parameter of the current signal pattern induced by electrophoresis; more preferably, it analyzes two parameters: the fluctuation range Amp.SD of the blocking current under the electrophoresis effect and the dwell time Dwell time of the blocking current under the electroosmotic flow effect.

[0070] In a third aspect, the present invention provides the application of the above-described detection method or analytical apparatus in the detection and identification of different proteins and their mutants.

[0071] Preferably, the detection method or analytical device can distinguish between a protein and one or more of its mutants, wherein at least one mutant is a frameshift mutant; more preferably, the analyte is H1.4 protein and its frameshift mutant.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] This invention utilizes the electrophoretic effect and electroosmotic effect of MspA for testing. By comprehensively utilizing the two parameters of the fluctuation range of the blocking current under the electrophoretic effect and the residence time of the blocking current under the electroosmotic effect, different H1.4 histone frameshift mutants can be identified with higher accuracy by setting different thresholds for Amp.SD and Dwell time. Attached Figure Description

[0074] Figure 1 The histone amino acid sequences are linked at H1.4 WT, H1.4+38, H1.4 157, and H1.4 169.

[0075] Figure 2 SDS-PAGE results for histones H1.4, H1.4+38, H1.4 157, and H1.4 169.

[0076] Figure 3 This is a schematic diagram illustrating the principle of using bio-nanopores to detect and identify four linker histones H1.4 WT, H1.4+38, H1.4 157, and H1.4169.

[0077] Figure 4 The signal is H1.4 WT detected at -90mV.

[0078] Figure 5 The signal is H1.4 WT detected at 90mV.

[0079] Figure 6 The H1.4+38 detection signal is at -90mV.

[0080] Figure 7 The H1.4+38 detection signal is at 90mV.

[0081] Figure 8 The signal of the H1.4 157 via at -90mV.

[0082] Figure 9 The signal of the H1.4 157 via at 90mV.

[0083] Figure 10 The H1.4 169 detection signal is at -90mV.

[0084] Figure 11 The H1.4 169 detection signal is at 90mV.

[0085] Figure 12 This is a schematic diagram showing the detection signal parameters of different histone H1.4 and its frameshift mutants under different negative voltages.

[0086] Figure 13This is a schematic diagram showing the detection signal parameters of different histone H1.4 and its frameshift mutants under different forward voltages. Detailed Implementation

[0087] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0088] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0089] The terms "MspA," "MspA porin," and "MspA nanopore" are used interchangeably to refer to Mycobacterium smegmatis porin A (MspA). As known to those skilled in the art, MspA porin may comprise two or more MspA monomers (e.g., eight monomers) that bind together to form a channel, wherein each monomer may be the same or different. MspA may be an octamer MspA. The MspA porin used in this invention should be capable of forming nanopores. Any MspA monomer forming the MspA porin may be selected from the group consisting of wild-type MspA monomers, mutant MspA monomers, wild-type MspA paralogs or homologs, or mutant MspA paralogs or homologs. In some embodiments, all monomers in the MspA porin are identical, such as the same mutant MspA monomer.

[0090] In this invention, the term "mutant MspA" refers to a mutant of wild-type MspA. Wild-type MspA consists of wild-type MspA monomers. Mutant MspA may include at least one mutant MspA monomer, and the remaining monomers in the mutant MspA may be selected from the group consisting of wild-type MspA monomers, mutant MspA monomers, wild-type MspA paralogs or homologs, or mutant MspA paralogs or homologs. Mutant MspA may include two or more MspA monomers (e.g., eight monomers), and mutant MspA may be an octamer MspA.

[0091] “M2 MspA” is derived from Mycobacterium smegmatis porin A (MspA). MspA is a natural nanoporin with a unique octamer structure, which can form stable nanopore channels. M2MspA was obtained by genetically engineering MspA to mutate (D90N / D91N / D93N / D118R / D134R / E139K) to optimize its pore characteristics, making it more suitable for single-molecule detection and sequencing applications. This mutant was obtained by recombinant expression using the method disclosed in Single-molecule DNA detection with an engineered MspA protein nanopore December 30, 2008 105(52)20647-20652https: / / doi.org / 10.1073 / pnas.0807514106.

[0092] The "nanoporous chip" comprises a lipid bilayer membrane or a polymer membrane and individual nanoporous proteins intercalated within the membrane. The nanoporous proteins are divided into cis-terminals (Cis, corresponding to the conductive dielectric chamber being electrically grounded) and trans-terminals (Trans). When a protein to be tested is placed at the cis-terminal of the nanoporous protein, and a voltage (positive voltage) with the same charge as the protein is applied to the trans-terminal of the nanoporous protein, the protein migrates primarily within the nanoporous protein under the influence of electroosmosis. Conversely, when a voltage (negative voltage) with the opposite charge is applied to the trans-terminal of the nanoporous protein, the protein migrates primarily within the nanoporous protein under the influence of electrophoresis. The membrane separates a first conductive liquid medium from a second conductive liquid medium; in specific embodiments of the invention, the first and second conductive liquid media may be the same or different.

[0093] The "vestibular region" refers to the conical portion within the Msp porin, whose diameter typically decreases from one end to the other along the central axis. The narrowest part of the vestibule connects to the restrictive region. For an example of the vestibule of the MspA porin, see [link to relevant documentation]. Figure 3 The vestibular and restrictive regions together define the channels, or MspA nanopores, of the MspA pore protein.

[0094] It should be understood that because the vestibule is conical in shape, its diameter varies along the path of the central axis, with the diameter being larger at one end than at the other. The diameter can range from about 2 nm to about 6 nm. Optionally, the diameter is at least about or at most about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or can come from any range therebetween. The length of the central axis can range from about 2 nm to about 6 nm. Optionally, the length is at least about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or may come from any range therebetween. When “diameter” is referred to herein, the diameter can be determined by measuring the center-to-center distance or the surface-to-surface distance of an atom.

[0095] The "restricted region" refers to the narrowest portion of the Msp porin channel in terms of diameter, which connects to the vestibule. The restricted region of MspA porin is shown in... Figure 3 The region is a restricted area (labeled "MspA restricted area"). The length of the restricted area can range from about 0.3 nm to about 2 nm. Optionally, the length is at most about or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or 3 nm, or any range thereof. The diameter of the restricted area can range from about 0.3 nm to about 2 nm. Optionally, the diameter is at most about or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or 3 nm, or any range thereof.

[0096] The term "nanopore" refers to a central hollow portion defined by the vestibule and confinement region, through which gases, liquids, ions, or analytes can pass. The term "nanopore" includes biological (e.g., M2 MspA nanopores) or artificial nanopores. Current nanopores can vary in size; for example, they can have diameters between approximately 0.5 nm and 10 nm. For example, diameters can be approximately 0.5 nm, 1 nm, 1.25 nm, 1.5 nm, 1.75 nm, 2 nm, 2.25 nm, 2.5 nm, 2.75 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, or any size in between. Biological nanopores can be created from pore proteins. Artificial nanopores can be generated through microforming or drilling. They can also be generated by etching slightly larger pores (tens of nanometers) in a silicon wafer and then gradually filling them in an ion beam engraving method, resulting in pores of much smaller diameters.

[0097] "Cis end" refers to the side through which the analyte enters the nanopore or moves across the nanopore on its surface, and the corresponding conductive dielectric chamber is electrically grounded.

[0098] "Trans end" refers to the side through which the analyte (or fragment thereof) exits the nanopore or through which the analyte does not move across the nanopore on its surface.

[0099] The term "frameshift mutation" refers to a change in the coding sequence following a normal DNA molecule when a base is deleted or added at a number other than three, resulting in a shift error.

[0100] The term "truncation mutation" refers to a mutation in the DNA sequence bases that causes the stop codon to appear prematurely, resulting in incomplete translation of the protein sequence and loss of protein function.

[0101] Electrophoresis refers to the phenomenon of charged substances migrating along the direction of an electric field under its influence. Specifically, the electrophoretic effect involves charged substances (such as proteins, DNA, and RNA) undergoing directional movement driven by an applied electric field. For proteins, the direction and speed of migration in the electric field are determined by their charge properties (positive or negative). In one embodiment of this invention, the charged substances include histones H1.4WT, H1.4+38, H1.4 157, and H1.4 169, etc., and a neutral or weakly alkaline test buffer is selected. When the pH of the test buffer is not higher than 8.0, the protein to be tested carries a positive charge.

[0102] Electroosmotic flow refers to the phenomenon where solvent molecules in a charged liquid solution flow along the direction of an applied electric field. During electroosmotic flow, the electric field acts on ions in the solution, causing the ion layer near the charged interface (such as the surface of a nanopore) to move. These ions carry water molecules or solvent molecules along with the flow, forming electroosmotic flow. The velocity of electroosmotic flow is related to factors such as the electric field strength, the conductivity of the medium, and the ion concentration of the solution. In nanopore experiments, electroosmotic flow affects molecular migration in the solution through the flow of the liquid under the influence of the electric field, and also affects the current signal passing through the nanopore.

[0103] Example 1: Recombinant Expression and Purification of Histone H1.4WT and its Mutants H1.4+38, H1.4157, and H1.4169

[0104] This study purified wild-type H1.4 and three mutant proteins in vitro to further elucidate their pathological functions. H1.4 157fs* is the C-terminus-closest mutant to produce 38AA, representing a significant candidate for studying the acquired function of 38AA. To investigate whether 38AA possesses a specific function, a fusion protein of H1.4 + 38AA was designed. Meanwhile, H1.4 169fs*, as the only mutant without 38AA, is crucial for disease research as its function and protein properties are analyzed. This embodiment utilizes bio-nanopore detection technology to detect and identify four different types of linker histone H1.4. The four linker histones are wild-type linker histone H1.4 WT, wild-type linker histone mutant H1.4+38, wild-type linker histone mutant H1.4 157, and wild-type linker histone mutant H1.4 169. The amino acid sequences of the wild-type and different mutant forms of these four linker histones H1.4 are as follows: Figure 1 As shown, H1.4 WT, H1.4+38, H1.4 157, and H1.4 169 linker histones were all obtained through recombinant expression.

[0105] 1. Construction of histone H1.4 and its mutant vector

[0106] Wild-type H1.4 and H1.4+38aa genes were synthesized by the company. After synthesis, the genes were constructed into the pET-28a vector by restriction enzyme digestion and ligation. A 6×his protein was added to the N terminus as an affinity purification tag. The 157fs* and 169fs* mutants were obtained by site-directed mutagenesis on the wild-type plasmid.

[0107] 1) The construction steps of wild-type H1.4 (SEQ ID NO:1) and H1.4+38aa (SEQ ID NO:2) vectors are as follows:

[0108] Using the synthesized gene as a template, the target gene fragment was amplified by PCR using forward and reverse primers (primer H1.4-F (SEQ ID NO:22): CGCGGCAGCCATATGATGTCCGAGACTGCGCCTG / primer H1.4-R (SEQ ID NO:23): GGAGCTCGAATTCCTACTTTTTCTTGGCTGCCGCC; primer H1.4-F (SEQ ID NO:22): CGCGGCAGCCATATGATGTCCGAGACTGCGCCTG / primer 38aa-R (SEQ ID NO:24): GGAGCTCGAATTCCTAACTGCTTTGGCCTTCGCTGG). After recovery, the fragments were double-digested with RcoR1 and Nde1, digested at 37℃ for 1 h, and the products were recovered and ligated into a similarly double-digested linear vector. The ligation was then performed into DH5α competent cells for screening positive clones. Two clones from each vector were selected for sequencing. After successful sequencing, the constructed plasmids were stored at -20℃ for later use.

[0109] The PCR system is as follows (50 μL):

[0110]

[0111] The PCR procedure is as follows:

[0112]

[0113] The enzyme digestion system (20 μL) is as follows:

[0114]

[0115] The product was recovered after reacting at 37°C for 1 hour.

[0116] The vector was also digested using the same method.

[0117] The connection system (10 μL) is as follows:

[0118]

[0119] After reacting overnight at 16°C, the product was recovered, transformed into DH5α cells, and sequenced.

[0120] 2) The construction steps of the 157fs*(SEQ ID NO:3) and 169fs*(SEQ ID NO:4) mutant vectors are as follows:

[0121] Using pET-28a-H1.4 as a template, two pairs of forward and reverse primers were used respectively:

[0122] primer 157-F (SEQ ID NO:25): AAGACCCCcAAAGAAGGCGAAGAAG / primer 157-R (SEQ ID NO:26): TTCTTTgGGGGTCTTCTTGGCGCTC;

[0123] Primer 169-F (SEQ ID NO:27): CCAAAAtAAGCGAAAAGCCCGAAAAAGGCG / Primer 169-R (SEQ ID NO:28): TCGCTTaTTTTGGCTCCAGCAGCTGC. The target gene fragment was amplified by PCR. After cleaning and recovery of the product, it was digested with Dpn1 enzyme at 37℃ for 1 hour, and then transformed into DH5α competent cells for screening of positive clones. Two clones from each cell were selected for sequencing. After successful sequencing, the constructed plasmid was stored at -20℃ for later use.

[0124] The PCR system is as follows (20 μL):

[0125]

[0126] The PCR procedure is as follows:

[0127]

[0128] The template digestion system (10 μL) is as follows:

[0129] 10× enzyme digestion buffer 1 μL

[0130] Dpn1(NEB#R0176L) 1μL

[0131] 8 μL of recovered product (20 ng / μL)

[0132] After reacting at 37℃ for 1 hour, the product was recovered, transformed into DH5α cells, and sequenced.

[0133] 2. Purification of histone H1.4 and its mutant proteins

[0134] Histone H1.4 and mutant proteins H1.4+38, H1.4157, and H1.4169, obtained using recombinant expression methods, showed high protein purity after His-beads enrichment, Hi-Trap Heparin HP column purification, and hydroxyapatite column purification. Figure 2 ).

[0135] The expression and purification steps are as follows:

[0136] 1) pET-28a-H1.4 and the mutant recombinant vector were introduced into Escherichia coli BL21(DE3) to obtain strains containing the recombinant vector.

[0137] 2) Pick a single clone and inoculate it into 100 mL of LB liquid medium. Incubate at 37°C and 220 rpm for 12 hours.

[0138] 3) Inoculate the bacterial culture at a ratio of 1:50 into six 750mL LB liquid medium for expansion culture (37℃, 220rpm). When the OD=0.6, add IPTG (final concentration 0.5mM) for induction for 4 hours.

[0139] 4) Centrifuge the bacterial culture, discard the supernatant, resuspend the bacterial culture in 100 mL PBS and centrifuge (4℃, 4000 rpm, 30 min), discard the supernatant.

[0140] 5) Resuspend the obtained bacterial pellet in 80 mL of Lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA) in a beaker, add lysozyme (final concentration 0.2 mg / mL), incubate at 4 °C for 10 min, then sonicate in an ice-water bath for 3 s, pause for 5 s, power 200 W, and then centrifuge at high speed (4 °C, 18,000 rpm, centrifuge for 30 min) and collect the supernatant.

[0141] 6) Incubate the supernatant with 2 mL His-beads at 4 °C for 3 hours. After incubation, transfer the supernatant to an empty Bio-rad column and collect the flow-through.

[0142] 7) Wash the beads with 100 mL of wash buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0).

[0143] 8) Elute the protein with different concentrations of imidazole in elution buffer (20mM Tris, 300mM NaCl, 100mM / 300mM / 500mM / 1M imidazole, pH 8.0) and collect the eluent.

[0144] 9) SDS-PAGE gel electrophoresis was used to detect the purity and concentration of proteins in the eluent.

[0145] 10) H1.4 and mutants were purified using a Hi-Trap Heparin HP column. Low-salt Buffer A (50mM Tris, pH 7.5, 500mM NaCl) was used to attach the column. Low-salt Buffer A and high-salt Buffer B (50mM Tris, 2M NaCl, pH 8.0) were mixed to form different salt concentrations for elution of the protein. The eluent was collected and the purification results were detected by SDS-PAGE gel electrophoresis.

[0146] 11) Use a hydroxyapatite column to remove nucleic acids from the protein. Use low-salt buffer A (200mM K2HPO4, KH2PO4, pH 6.8) to attach the protein to the column. Mix low-salt buffer A and high-salt buffer B (1M K2HPO4, KH2PO4, pH 6.8) to form different salt concentrations for elution of the protein. Collect the eluent and detect the purification results by SDS-PAGE gel electrophoresis.

[0147] 12) Collect the target protein and dialyze it into BC 100 Buffer (100mM NaCl, 20mM Tris, pH 8.0, 20% Glycerol).

[0148] 13) The concentrations of the target proteins were determined using the dithiothreonine blue (BCA) method, with the four proteins ranging from 0.1 to 1 mg / ml. To obtain higher concentrations, the target protein samples were concentrated using a concentration tube method, yielding four linker histones at concentrations of 0.5 to 2.5 mg / ml. The purity of the four linker histones was determined using SDS-PAGE, and the results are as follows. Figure 2 As shown. The above four types of linker histones need to be refrigerated or aliquoted and stored at -80°C after expression, purification, and concentration.

[0149] Example 2: Measurement of H1.4WT and its mutants H1.4+38, H1.4157, and H1.4169 using bio-nanopores.

[0150] The detection and identification of four linker histones H1.4 WT, H1.4+38, H1.4 157, and H1.4 169 using bio-nanopores is based on the following principle: Figure 3As shown. One, two, three, or all of the four different linker proteins H1.4 WT, H1.4+38, H1.4 157, and H1.4 169 were diluted with the corresponding folds using a bio-nanopore assay buffer (containing 200 mM KCl, 150 mM potassium ferricyanide, 100 mM potassium ferrocyanide, and 25 mM PBS, pH 8.0, with the test proteins being positively charged) and thoroughly mixed to obtain the test sample. This test sample was then added to the Cis end of the bio-nanopore assay system, while the same assay buffer was added to the trans end. A specific electric field was applied to the bio-nanopore assay system. Under the influence of this electric field, the analytes added to the Cis end migrated from the Cis end to the trans end of the bio-nanopore. When the size of the analyte was much larger than the confinement region of the bio-nanopore or the electric field was limited, the analyte was first captured by the vestibular region of the bio-nanopore, and then migrated through Brownian diffusion. The analyte exits the vestibular region of the bio-nanopore and generates a specific pattern of current signal. By comparing the patterns or statistical characteristics of the detection signals corresponding to different analytes, the detection and identification of four different linker histones can be achieved. When the analyte added to the Cis terminal of the bio-nanopore is of appropriate size and the applied electric field strength reaches a certain level, the analyte will pass through the confinement region of the bio-nanopore and enter the Trans terminal of the bio-nanopore under the action of the electric field force, thereby generating a specific pattern of detection signal. Similarly, by comparing the patterns or statistical characteristics of the detection signals corresponding to different analytes through the pore, the detection and identification of four different linker histones can be achieved. The specific steps include the following:

[0151] 1. Preparation of M2 MspA nanopores

[0152] The concentration of the M2 mutant biomolecular nanopore MspA (hereinafter referred to as M2 MspA) pore protein monomer obtained by recombinant expression was 0.1-1 mg / ml. It was diluted 10 μL with conductive buffer for biomolecular nanopore detection (containing 200 mM KCl, 150 mM potassium ferricyanide, 100 mM potassium ferrocyanide, 25 mM PBS, pH = 8.0). 7 -10 8 Add 100 μL of the above-mentioned embedding protein solution to the Cis terminal of M2 MspA, apply a voltage of 180-320 mV, and switch the voltage to zero when a sudden change in ion current is observed. If the ion current is observed to be around 200 pA after applying a voltage of 180 mV, it proves that the embedding of a single M2 MspA is successful. Then, use conductive buffer to replace the embedding protein solution at the Cis terminal of the bio-nanopore to avoid embedding multiple M2 MspA.

[0153] 2. Use M2 MspA to perform signal detection on H1.4 WT.

[0154] The applied voltage was switched to zero, and 0.1-10 μl of H1.4 WT sample was added to the Cis terminal of the M2 MspA and mixed well. At this point, the final concentration of H1.4 WT sample at the Cis terminal of the M2 MspA nanopore was 10-3000 nM. Subsequently, different positive voltages (40 mV-140 mV) were applied at room temperature to obtain the detection signal of H1.4 WT using MspA under electroosmotic flow. In addition, different negative voltages (-140 mV-40 mV) were applied at room temperature to obtain the detection signal of H1.4 WT using MspA under electrophoretic effect.

[0155] Figure 4 , Figure 5 At voltages of -90mV and 90mV respectively, H1.4WT samples with a Cis end concentration of 1μM in the M2 MspA nanopores were captured by the M2 MspA vestibular region under the effects of electrophoresis and electroosmosis, and escaped the ion current signal generated by the M2 MspA nanopore vestibular region under the effect of Brownian diffusion.

[0156] 3. Use M2 MspA to perform signal detection on H1.4+38.

[0157] The applied voltage was switched to zero, and 0.1-10 μL of H1.4+38 sample was added to the Cis terminal of the M2 MspA and mixed well. At this point, the final concentration of H1.4+38 sample at the Cis terminal of the M2 MspA nanopore was 10-3000 nM. Subsequently, different positive voltages (40 mV-140 mV) were applied at room temperature to obtain the detection signal of H1.4+38 using MspA under electroosmotic flow. In addition, different negative voltages (-140 mV-40 mV) were applied at room temperature to obtain the detection signal of H1.4+38 using MspA under electrophoretic effect.

[0158] Figure 6 , Figure 7 At voltages of -90mV and 90mV respectively, H1.4+38 samples with a Cis end concentration of 1uM in the M2 MspA nanopore were captured by the M2 MspA vestibular region under the effects of electrophoresis and electroosmosis, and escaped the ion current signal generated by the M2 MspA nanopore vestibular region under the action of Brownian diffusion.

[0159] 4. Use M2 MspA to perform signal detection on H1.4 157.

[0160] The applied voltage was switched to zero, and 0.1-10 μL of H1.4 157 sample was added to the Cis terminal of the M2 MspA and mixed well. At this point, the final concentration of H1.4 157 sample at the Cis terminal of the M2 MspA nanopores was 10-3000 nM. Subsequently, different positive voltages (40 mV-140 mV) were applied at room temperature to obtain the detection signal of H1.4 157 using MspA under electroosmotic flow. In addition, different negative voltages (-140 mV-40 mV) were applied at room temperature to obtain the detection signal of H1.4 157 using MspA under electrophoretic effect.

[0161] Figure 9 , Figure 10 The H1.4157 sample with a Cis end concentration of 1 μM in the M2 MspA nanopore was captured by the M2 MspA vestibular region under the effects of electrophoresis and electroosmosis, and escaped the ion current signal generated by the M2 MspA nanopore vestibular region under the action of Brownian diffusion, respectively, at voltages of -90 mV and 90 mV.

[0162] 5. Use M2 MspA to perform signal detection on H1.4 169.

[0163] The applied voltage was switched to zero, and 0.1-10 μL of H1.4 169 sample was added to the Cis terminal of the M2 MspA and mixed well. At this point, the final concentration of H1.4 169 sample at the Cis terminal of the M2 MspA nanopores was 10-3000 nM. Subsequently, different positive voltages (40 mV-140 mV) were applied at room temperature to obtain the detection signal of H1.4 169 using MspA under electroosmotic flow. In addition, different negative voltages (-140 mV-40 mV) were applied at room temperature to obtain the detection signal of H1.4 169 using MspA under electrophoretic effect.

[0164] Figure 10 , Figure 11 The H1.4169 sample with a Cis end concentration of 1 μM in the M2 MspA nanopore was captured by the M2 MspA vestibular region under the effects of electrophoresis and electroosmosis, and escaped the ion current signal generated by the M2 MspA nanopore vestibular region under the action of Brownian diffusion, respectively, at voltages of -90 mV and 90 mV.

[0165] 6. Signal Analysis

[0166] Whether it's the detection signal of histone samples under the electrophoresis effect test mode or the detection signal of histone samples under the electroosmotic effect test mode, the Amp.SD and Interevent intervals (t) are used to measure the detection signal. onThe parameters Δexclude(ΔI / I0*100%) and Dwell time are closely related to the structural characteristics, distribution, and quantity of charged amino acids of the protein molecule to be detected. Therefore, by extracting and statistically analyzing the relevant parameters of the detection signal, the detection and identification of different H1.4 histone frameshift mutants can be effectively realized. Furthermore, by analyzing the above characteristic parameters under dual modalities, the accuracy of identifying different histone samples can be further improved.

[0167] 1) By processing the detection signals of different histone H1.4 and its mutants under different intensities of electrophoresis, a series of statistical parameters characterizing the permeation behavior of specific histone samples can be obtained, such as Amp.SD, Interevent intervals (t). on ), Δexclude(ΔI / I0*100%), and Dwell time, the relevant representations are shown in [link to relevant documentation]. Figure 12 .

[0168] 2) By processing the detection signals of different histone H1.4 and its mutants under different intensities of electroosmotic flow in another mode, a series of statistical parameters characterizing specific histone samples can be obtained, such as Amp.SD, Interevent intervals (ton), Δexclude (ΔI / I0*100%), and Dwell time. See relevant representations. Figure 13 .

[0169] Tables 1 and 2 compare the statistical mean values ​​of Amp.SD and Dwell time parameters corresponding to the dual-modal detection signals obtained from tests at -90mV and 90mV for four different histone H1.4 proteins. By selecting the statistical characteristics of two or more parameters obtained from single or multiple tests at different voltages in two modalities for different histone H1.4 proteins and their mutants, the aim is to accurately identify four different histone H1.4 proteins.

[0170] The results show that when testing different samples at a test voltage of 90mV, the differences in the fluctuation range of the blocking current corresponding to the test data of the four different samples are small, with the largest difference being only 1.42. However, the differences in the residence time of the blocking current corresponding to the test data of the four different samples are large, with the largest difference reaching 245.3. Conversely, when testing the four different test samples at a voltage of -90mV, the differences in the residence time of the blocking current corresponding to the test data of the four different samples are much smaller than 245.3, but the fluctuation range of the blocking current corresponding to the test data of the four different samples is much larger than 1.42.

[0171] Therefore, in actual operation, four different samples were tested using voltages of -90mV and 90mV respectively, that is, the electrophoretic effect and electroosmotic effect of MspA were used for testing respectively. By comprehensively utilizing the two parameters of the fluctuation range of the blocking current under the electrophoretic effect and the residence time of the blocking current under the electroosmotic effect, based on this, different H1.4 histone frameshift mutants can be identified with higher accuracy by setting different thresholds for Amp.SD and Dwell time.

[0172] Table 1. Comparative analysis of four different histone H1.4 values ​​at -90mV voltage.

[0173]

[0174] Table 2. Comparative analysis of four different histone H1.4 levels at 90 mV.

[0175]

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting dual-modal protein frameshift mutations, characterized in that: The steps include: 1) Applying voltages in different directions to both sides of a bio-nanopore chip containing a single nanoporous protein, including a membrane and an intercalation membrane, to induce the migration of the analyte within the bio-nanopore based on electroosmotic and electrophoretic effects; 2) Measure the ionic current generated by the analyte during migration in the bio-nanopore to provide current signal patterns; 3) Identify and distinguish different types of analytes by current signal patterns induced by electroosmotic and electrophoretic effects.

2. The method for detecting bimodal protein frameshift mutations as described in claim 1, characterized in that: A positive voltage is applied to obtain the current signal pattern under the electroosmotic effect; preferably, the positive voltage range is 40mV-140mV. Preferably, a negative voltage is applied to obtain the current signal pattern under the electrophoretic effect; more preferably, the negative voltage range is -140mV to 40mV.

3. The method for detecting bimodal protein frameshift mutations as described in claim 1, characterized in that: The parameters obtained from the current signal pattern analysis include Amp.SD and Interevent intervals(t). on ), Δexclude(ΔI / I0*100%) and Dwell time; Preferably, the analytes are identified and distinguished by analyzing the current signal patterns induced by electroosmotic flow and electrophoresis effects; preferably, at least one parameter of the current signal pattern induced by electroosmotic flow and at least one parameter of the current signal pattern induced by electrophoresis effects are analyzed; more preferably, two parameters are analyzed: the fluctuation range of the blocking current under the electrophoresis effect (Amp.SD) and the residence time of the blocking current under the electroosmotic flow effect (Dwell time).

4. The method for detecting bimodal protein frameshift mutations as described in claim 1, characterized in that: The analyte is a protein and one or more mutants thereof, wherein at least one mutant is a frameshift mutant; Preferably, the analyte comprises linker histone H1.4 and its mutants; Preferably, the linker histone H1.4 mutant includes a sequence having at least 50%, 60%, 70%, 80%, or 90% identity with linker histone H1.4; more preferably, the linker histone H1.4 mutant includes a C-terminal truncated mutant protein of linker histone H1.4 and a fusion protein of linker histone H1.

4.

5. The method for detecting bimodal protein frameshift mutations as described in claim 1, characterized in that: The detection method includes the following steps: (1) Prepare test samples from the analytes; (2) Add the test sample to the Cis end of the bio-nanopore test system; (3) Apply an electric field to cause the analyte to migrate under the action of electroosmosis or electrophoresis, and capture the current signal generated in the nanopore; (4) Analyze the current signal generated during the migration process to identify the characteristics of the object to be analyzed; (5) Compare the analysis results to achieve the detection and identification of at least four different analytes; Preferably, step (1) includes dissolving the analyte in a test buffer and mixing it; preferably, the test buffer is a neutral or weakly alkaline test buffer; the pH of the test buffer is not higher than 8.0; Preferably, the bio-nanopore testing system in step (2) includes a bio-nanopore chip and a test buffer. Preferably, the concentration of the test sample in the bio-nanopore testing system is 10-3000 nM; Preferably, step (3) includes applying a positive voltage to the Cis terminal to cause the analyte to migrate under the action of electroosmosis and capture the current signal generated in the nanopore; applying a negative voltage to the Cis terminal to cause the analyte to migrate under the action of electrophoresis and capture the current signal generated in the nanopore. Preferably, the migration behavior is analyzed and relevant current signals are captured based on the interaction between the size of the analyte and the size of the nanopore confinement region, or the interaction between the size of the analyte and the magnitude of the electric field. Preferably, step (5) includes comparing two parameters: the fluctuation range of the blocking current under the electrophoretic effect and the residence time of the blocking current under the electroosmotic effect. Different H1.4 histone mutants can be identified by setting different thresholds for Amp.SD and Dwell time.

6. The method for detecting bimodal protein frameshift mutations as described in claim 3, characterized in that: At a voltage of -90mV, the difference in the fluctuation range of the blocking current between samples under the electrophoretic effect is 0.2-4.0pA, preferably 0.3-3.3pA; at a voltage of 90mV, the difference in the residence time of the blocking current between samples under the electroosmotic effect is 20-300ms, preferably 29.4-245.3ms.

7. A bio-nanopore analysis device for implementing the detection method according to any one of claims 1-6, the device comprising: (a) A bio-nanopore chip, comprising a membrane and individual nanopore proteins inserted into the membrane; (b) An electric field application device for applying a specific electric field to a nanopore, the electric field application device having a voltage switching element, thereby applying voltages in different directions to the nanopore to achieve dual-mode detection of the electroosmotic and electrophoretic effects of the analyte; (c) A current signal detection device for detecting the current signal generated when an analyte passes through a biological nanopore; (d) Signal analysis system, used to perform pattern recognition on the detected current signal to identify the types and characteristics of different analytes.

8. The bio-nanopore analysis device as described in claim 7, characterized in that: The membrane separates the first conductive liquid medium from the second conductive liquid medium. The membrane may be an organic membrane (e.g., a lipid bilayer) or a synthetic membrane (e.g., a membrane formed from a polymer material). Preferably, the current signal is the signal generated by the ions of the nanoporous protein when the protein to be tested is captured by the nanoporous protein and then escapes; Preferably, the parameters of the signal include the fluctuation range of the blocking current (Amp.SD) and the event interval (Interevent intervals). on The parameters are: the blockage amplitude Δexclude(ΔI / I0*100%) and the dwell time of the blockage current; more preferably, the parameters are the fluctuation range of the blockage current under the electrophoretic effect and the dwell time of the blockage current under the electroosmotic effect. Preferably, the signal analysis system analyzes at least one parameter of the current signal pattern induced by electroosmotic flow and at least one parameter of the current signal pattern induced by electrophoresis; more preferably, it analyzes two parameters: the fluctuation range Amp.SD of the blocking current under the electrophoresis effect and the dwell time Dwell time of the blocking current under the electroosmotic flow effect.

9. The application of the detection method according to any one of claims 1-6 or the analytical apparatus according to claim 7 or 8 in the detection and identification of different proteins and their mutants.

10. The application as described in claim 9, characterized in that, The detection method or analytical device can distinguish proteins and one or more mutants, wherein at least one mutant is a frameshift mutant; more preferably, the analyte is H1.4 protein and its frameshift mutant.

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