A zebrafish protein-based nanopore and its application in single molecule detection

Abstract

The application belongs to the technical field of nanometer hole single molecule detection and biochemistry, and provides a nanometer hole based on zebra fish protein and application thereof in single molecule detection. The zebra fish protein is wild type JAC5 protein, and the nanometer hole based on the wild type JAC5 protein is applied to single molecule detection. Compared with other nanometer hole proteins, the wild type JAC5 protein has significant advantages in cloning expression and purification process, and is suitable for detecting nucleic acid, protein and pathogen, and has wide application scenarios. Compared with the wild type JAC5 protein, the mutant zebra fish protein after amino acid mutation of the wild type JAC5 protein can normally express, enhance structural stability and improve detection performance by eliminating the steric hindrance of the sugar binding region to enhance the affinity, or by introducing positive charges to enhance the electrostatic interaction between the transmembrane region and the phospholipid bilayer membrane.

Description

Technical Field

[0001] This invention belongs to the field of interdisciplinary technology of nanopore single-molecule detection and biochemistry, and particularly relates to a nanopore based on zebrafish protein and its application in single-molecule detection. Background Technology

[0002] Nanopore technology, with its advantages such as label-free and single-molecule resolution, has become a core tool for nucleic acid sequencing and protein analysis. However, existing mainstream nanopores (such as FraC, α-hemolysin, MSPA, and Aeromonas hydrolysin) have the following key drawbacks:

[0003] 1. Limited source leading to insufficient specificity: FraC (sea anemone), Aeromonas hydrolysin (bacteria), etc., are all from invertebrates / bacteria and lack the glycosylation molecular recognition sites specific to eukaryotes. They cannot specifically bind to highly mannose-modified nucleic acids / proteins (such as pathogen surface glycoproteins). For example, FraC nanopores rely on electroosmotic flow driven by the α-helical transmembrane region for detection. They can only capture positively charged folded proteins under acidic conditions of pH=4.5 and lack specific binding ability, making them susceptible to interference in complex clinical samples.

[0004] 2. Poor compatibility with eukaryotic samples: The optimal detection conditions for existing nanopores are mostly pH 7.5-8.0, suitable for bacteria / archaea (e.g., 600 mM KCl buffer at pH 8.0 for PHT nanopores), which do not match the physiologically compatible pH of 5.5-6.0 for eukaryotic macromolecules (such as folded proteins and high-mannose modified nucleic acids). For example, Aeromonas hydrolysin nanopores require pH 8.0 for protein phosphorylation detection, which leads to the easy precipitation of eukaryotic chymotrypsin (pI=8.75), failing to meet the requirements for detecting low-abundance eukaryotic proteins. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a zebrafish protein-based nanopore and its application in single-molecule detection, aiming to solve the problems mentioned in the background art.

[0006] In a first aspect, the present invention provides an application of a zebrafish protein-based nanopore in single-molecule detection, wherein the zebrafish protein is a wild-type JAC5 protein, and the amino acid sequence of the wild-type JAC5 protein is shown in SEQ ID NO:1.

[0007] The target analytes for single-molecule detection include peptides, proteins, or nucleotides.

[0008] Secondly, the present invention provides an application of nanopores based on mutant zebrafish protein in single-molecule detection, wherein the mutant zebrafish protein is obtained by mutation of wild-type JAC5 protein, and the amino acid sequence of wild-type JAC5 protein is shown in SEQ ID NO:1.

[0009] The mutant zebrafish protein specifically refers to the wild-type JAC5 protein with the following amino acid mutations: D135 is mutated to D135A, K171 to K171A, S190 to S190A, K192 to K192A, and N246 to N246A.

[0010] Furthermore, the mutant zebrafish protein undergoes further amino acid mutations, specifically: amino acid K186 is mutated to K186M, K186Q, K186C, K186R, K186H, K186W, K186A, K186F, or K186D.

[0011] Alternatively, the amino acid V187 may be mutated to V187N, V187I, V187T, V187G, V187P, V187L, V187D, V187Y, or V187S.

[0012] Alternatively, amino acid E188 may be E188N, E188K, E188S, E188Q, E188L, E188A, or E188T.

[0013] Furthermore, the amino acid mutation specifically refers to the mutation of amino acid T250 into T250N or T250G;

[0014] Alternatively, the amino acid T251 may mutate to T251S, T251I, or T251R;

[0015] Alternatively, the amino acid T252 may mutate to T252A, T252Y, or T252W.

[0016] Furthermore, the target analytes for the single-molecule detection include peptides, proteins, or nucleotides.

[0017] Furthermore, the nucleotide sequence encoding the wild-type JAC5 protein is shown in SEQ ID NO:2.

[0018] Furthermore, the method for preparing the mutant zebrafish protein includes the following steps:

[0019] Construct a vector for the mutant zebrafish protein;

[0020] Expression and purification of the mutant zebrafish protein.

[0021] Thirdly, the present invention provides a membrane layer embedded with the mutant zebrafish protein, wherein the membrane layer is a lipid bilayer.

[0022] Fourthly, the present invention provides a nanopore composed of the mutant zebrafish protein.

[0023] Fifthly, the present invention provides a product comprising the mutant zebrafish protein, said product being a composition, a complex, or a kit.

[0024] The present invention has the following beneficial effects:

[0025] (1) Wild-type JAC5 protein is a unique nanoporous protein that differs from FraC, Aeromonas hydrolysin, α-hemolysin, and MSPA. It has an octamer structure, with each subunit containing a mixed α-helix-β-sheet transmembrane region. The N-terminus is anchored to the cis compartment, and the C-terminus points to the trans compartment. The stem-loop precursor forms the core support of the channel. Nanopores based on wild-type JAC5 protein are used in single-molecule detection. Compared with other nanoporous proteins, the cloning, expression, and purification process of wild-type JAC5 protein has significant advantages: it does not require sphingomyelin liposome induction of FraC (direct membrane embedding), does not require high-salt buffer of Aeromonas hydrolysin (100mM NaCl low-salt adapted to eukaryotic samples), and its applicable detection range covers nucleic acids, proteins (folded / oligomeric proteins), and pathogens, making it suitable for a wide range of scenarios.

[0026] (2) The mutant zebrafish protein after the amino acid mutation of wild-type JAC5 protein enhances affinity by eliminating the steric hindrance of the sugar-binding region and introduces positive charge to enhance the electrostatic interaction between the transmembrane region and the phospholipid bilayer. Compared with wild-type JAC5 protein, mutant zebrafish protein can be expressed normally, enhance structural stability, thereby improving the quality of current signal and improving detection performance.

[0027] (3) Wild-type or mutant JAC5 protein, as a nanopore detection protein, is significantly superior to FraC, Aeromonas hydrolysin, PHT, γ-hemolysin and Aerolysin in terms of specificity, stability, detection range and clinical applicability. It provides a new tool for macromolecular analysis of eukaryotic organisms and diagnosis of pathogens and has important scientific research and clinical translation value. Attached Figure Description

[0028] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0029] Figure 1 This is a top view of the JAC5 protein channel ribbon structure model simulated by ALPHAFOLD, provided in an embodiment of the present invention.

[0030] Figure 2This is a side view of the JAC5 protein channel ribbon structure model simulated by ALPHAFOLD, provided in an embodiment of the present invention.

[0031] Figure 3 The mutation sites of the wild-type JAC5 protein provided in the embodiments of the present invention.

[0032] Figure 4 This is a purification electrophoresis diagram of Example 1 of the present invention. In the diagram, lane 1: protein marker; lane 2: wild-type JAC5 protein elution sample; lane 3: wild-type JAC5 protein elution sample; lane 4: JAC5-M1 protein elution sample; lane 5: JAC5-M1 protein elution sample.

[0033] Figure 5 This is the result of the detection of nanopore opening current of wild-type JAC5 protein in Example 2 of the present invention.

[0034] Figure 6 This is a diagram showing the results of detecting single-stranded DNA using wild-type JAC5 protein nanopores in Example 2 of this invention.

[0035] Figure 7 The results of the opening current detection of the JAC5-M1 protein nanopore in Example 3 of this invention are shown.

[0036] Figure 8 This is a diagram showing the results of single-stranded DNA detection using the JAC5-M1 protein nanopore in Example 4 of this invention.

[0037] Figure 9 This is a graph showing the current detection results during single-stranded DNA piercing in Example 5 of the present invention.

[0038] Figure 10 The image shows the current detection results during β2-microglobulin perforation in Example 6 of the present invention.

[0039] Figure 11 This is a graph showing the current detection results during short-chain RNA pore perforation in Example 7 of the present invention.

[0040] Figure 12 This is a graph showing the current detection results during protein fragment perforation in Example 8 of the present invention. Detailed Implementation

[0041] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0043] This invention provides an application of zebrafish protein-based nanopores in single-molecule detection, wherein the zebrafish protein is wild-type JAC5 protein, and the amino acid sequence of wild-type JAC5 protein is shown in SEQ ID NO:1.

[0044] Single-molecule detection targets include peptides, proteins, or nucleotides.

[0045] In some embodiments, the mutation sites of the wild-type JAC5 protein are as follows: Figure 3 As shown, this invention provides an application of nanopores based on mutant zebrafish protein in single-molecule detection. The mutant zebrafish protein is obtained by mutation of wild-type JAC5 protein, and the amino acid sequence of wild-type JAC5 protein is shown in SEQ ID NO:1.

[0046] The mutant zebrafish protein is specifically: the amino acid D135 of the wild-type JAC5 protein is mutated to D135A, K171 is mutated to K171A, S190 is mutated to S190A, K192 is mutated to K192A, and N246 is mutated to N246A.

[0047] In some embodiments, the mutant zebrafish protein is further mutated to amino acids, specifically by mutating amino acid K186 to K186M, K186Q, K186C, K186R, K186H, K186W, K186A, K186F, or K186D.

[0048] Alternatively, the amino acid V187 may be mutated to V187N, V187I, V187T, V187G, V187P, V187L, V187D, V187Y, or V187S.

[0049] Alternatively, amino acid E188 may be E188N, E188K, E188S, E188Q, E188L, E188A, or E188T.

[0050] In some embodiments, the amino acid mutation is further specifically: the amino acid T250 is mutated to T250N or T250G;

[0051] Alternatively, the amino acid T251 may mutate to T251S, T251I, or T251R;

[0052] Alternatively, the amino acid T252 may mutate to T252A, T252Y, or T252W.

[0053] In some embodiments, the target analytes for single-molecule detection include peptides, proteins, or nucleotides.

[0054] In some embodiments, the nucleotide sequence encoding the wild-type JAC5 protein is shown in SEQ ID NO:2.

[0055] In some embodiments, the method for preparing mutant zebrafish protein includes the following steps:

[0056] Constructing a vector for mutant zebrafish protein;

[0057] Expression and purification of mutant zebrafish protein.

[0058] In some embodiments, the present invention provides a membrane layer incorporating mutant zebrafish protein, the membrane layer being a lipid bilayer.

[0059] In some embodiments, the present invention provides a nanopore composed of mutant zebrafish protein.

[0060] In some embodiments, the present invention provides a product comprising mutant zebrafish protein, the product being a composition, a complex, or a kit.

[0061] Example 1: Standardized preparation process of JAC5 protein and its mutants

[0062] 1. Carrier construction:

[0063] (1) Using pET28a as the base vector, the coding genes of JAC5 (wild type) and JAC5-M1 proteins were inserted between the Nde I / Xho I restriction sites, and the N-terminus was fused with a His6 tag; the JAC5-M1 protein is specifically: the amino acid D135 of the wild type JAC5 protein is mutated to D135A, K171 is mutated to K171A, S190 is mutated to S190A, K192 is mutated to K192A and N246 is mutated to N246A;

[0064] (2) Transformed into Escherichia coli DH5α competent cells, single clones were selected and sequenced to verify that the mutation sites were unbiased, and the recombinant vectors were named pET28a-JAC5, pET28a-JAC5-M1, and pET28a-JAC5-M2.

[0065] 2. Induced expression:

[0066] (1) JAC5 protein: pET28a-JAC5 was transformed into BL21(DE3), inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C until OD. 600 =0.6-0.8, add 0.5mM IPTG, induce at 25℃ and 200rpm for 12h;

[0067] (2) JAC5-M1 mutant: pET28a-JAC5-M1 was transformed into BL21(DE3), inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C until OD. 600 =0.6-0.8, add 0.5mM IPTG, induce at 25℃ and 200rpm for 12h.

[0068] 2. Bacterial cell collection

[0069] (1) After induction, centrifuge at 4℃ and 8000rpm for 30min, discard the supernatant and collect the bacterial pellet; resuspend the bacterial pellet in pre-cooled PBS buffer (pH=7.4, containing 1mM PMSF protease inhibitor), centrifuge again (4℃, 8000rpm, 10min), wash twice to remove residual culture medium components, and finally store the bacterial pellet in a -80℃ freezer (short-term storage, not exceeding 1 week).

[0070] (2) Two-step purification:

[0071] Step 1: Ni-NTA affinity chromatography (capture of target protein)

[0072] ① Cell disruption and supernatant preparation

[0073] Lysis buffer preparation: 50 mM Tris-HCl (pH=8.0), 500 mM NaCl, 10 mM imidazole, 1 mM PMSF, 0.1% Triton X-100.

[0074] Ultrasonic disruption: Resuspend the bacterial precipitate in lysis buffer at a ratio of 1:10 (w / v), and sonicate under ice bath conditions (power 300 W, working time 3s, interval 5s, total time 20min) until the bacterial solution is clear (no obvious turbidity); centrifuge at 4℃ and 18000rpm for 30min, collect the supernatant and discard the precipitate.

[0075] ②Ni-NTA resin pretreatment and sample loading

[0076] Take 5 mL of Ni-NTA resin (GE Healthcare), equilibrate it with 5 column volumes of lysis buffer, and slowly load the supernatant into the resin column.

[0077] ③ Washing and Collection

[0078] Washing procedure: Elute contaminating proteins with 10 column volumes of washing buffer (50 mM Tris-HCl, pH=8.0; 500 mM NaCl, 20 mM imidazole), and analyze the eluent by SDS-PAGE until no obvious contaminating protein bands are visible.

[0079] Target protein elution: JAC5 protein was eluted with 5 column volumes of elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 300 mM imidazole). Elution fractions were collected at 1 mL / tube and analyzed by SDS-PAGE. Fractions containing the target protein were combined. The purified electrophoresis image of JAC5 nanoporous protein is shown below. Figure 4 As shown.

[0080] Step 2: Gel filtration chromatography (to remove impurities and polymers)

[0081] Chromatography column selection and operation

[0082] Chromatography column: Superdex 200 10 / 300GL (GE Healthcare) was used. Elution was performed with gel filtration buffer at a flow rate of 0.5 mL / min. Components were collected at 0.5 mL / tube. The target peak position was determined by SDS-PAGE and UV monitoring (280 nm), and the target peak was collected.

[0083] 3. Optimization of the oligomerization process for JAC5 nanopores

[0084] (1) Liposome formulation: DOPC:DOPE:DOPS=9:9:2 (weight ratio), the acidic phospholipid head of DOPS interacts with the positively charged residues on the surface of JAC5 protein;

[0085] (2) Oligopolymerization conditions: Incubate at 37°C for 1 h in MES buffer (50 mM MES, 100 mM NaCl) at pH 5.5-6.0;

[0086] A top view of the JAC5 protein channel ribbon structure model simulated by ALPHAFOLD is shown below. Figure 1 As shown; a side view of the JAC5 protein channel ribbon structure model simulated by ALPHAFOLD. Figure 2 As shown.

[0087] The results showed that the JAC5 protein consists of eight identical subunits that assemble symmetrically around a central axis forming a transmembrane channel. The octamer has a central hollow structure with an inlet diameter of 38±2 Å and a bottom diameter of 26±2 Å. The transmembrane β-barrel is formed by the recombination of stem-loop precursors from the eight JAC5 protein subunits, with each subunit contributing two β-chains. The inner diameter of the transmembrane β-barrel is 2.8±0.2 nm.

[0088] The amino acid sequence of the wild-type JAC5 protein is shown in SEQ ID NO:1, and the nucleotide sequence encoding the wild-type JAC5 protein is shown in SEQ ID NO:2.

[0089] amino acid sequence:

[0090] SEQ ID NO:1:

[0091] MTYPTNLEIIGGQGGSSFSFTGENNGASLEKIWVWVGGWQIKAVRAWLSDGRDETFGVPSGSHQEYVFTPGECFTSLSLWGNGAGTRLGAIKFKTNKGGEFFAHMTSWGLKTEYPMDVGSGYCLGIVGRGGSDIDCMGFMFLNAVQSTVLTNVNYPTINQLIPKVATEEIKSVSFENKTSVKQEQKVETSKKVIKTSSWSMTKSFSSTFSVEVSAGIPEIAEVSTGFSISFGVESTHSLEQTDEKNETLTTTVEVPPKKKVDVHITIGRASFDLPYTGTVKITCKNGSVLQYETKGQYKGVAYTDIKVNTVEKDL;

[0092] Nucleotide sequence:

[0093] SEQ ID NO:2:

[0094] ATGACCTATCCTACTAACCTGGAAATTATCGGTGGTCAGGGTGGTTCCTCTTTCTCCTTTACCGGTGAAAACAACGGTGCTTCTCTGGAGAAAATCTGGGTTTGGGTTGGCGGCTGGCAGATCAAAGCTGTGCGCGCCTGGCTGAGCGACGGTCGTGACGAAACCTTCGGCGTTCCATCCGGCTCCCACCAGGAATATGTGTTCACTCCAGGCGAATGCTTCACCAGCCTGTCCCTGTGGGGCAACGGTGCTGGCACCCGTCTGGGTGCCATCAAATTCAAAACCAACAAAGGTGGTGAATTCTTCGCTCACATGACCTCCTGGGGTCTGAAAACCGAATACCCAATGGACGTTGGTTCTGGCTATTGCCTGGGTATCGTTGGTCGTGGTGGCTCTGACATTGACTGTATGGGCTTCATGTTTCTGAACGCTGTTCAATCCACTGTTCTGACCAACGTTAACTACCCAACTATCAACCAGCTGATCCCAAAAGTCGCTACCGAAGAAATCAAATCCGTGAGCTTCGAAAACAAAACCTCTGTGAAACAGGAACAGAAAGTTGAAACCTCTAAGAAAGTTATCAAGACTTCCTCTTGGAGCATGACTAAATCTTTCTCTTCCACCTTTAGCGTTGAAGTGAGCGCTGGTATTCCAGAAATCGCTGAAGTTAGCACCGGCTTTTCTATTAGCTTTGGTGTGGAATCTACTCACAGCCTGGAGCAGACCGATGAGAAGAATGAGACCCTGACCACTACTGTGGAGGTGCCACCAAAGAAAAAAGTCGATGTTCATATCACCATTGGCCGCGCTTCTTTCGATCTGCCATACACCGGCACCGTTAAGATTACTTGTAAAAACGGCTCTGTCCTGCAGTACGAAACCAAAGGCCAATACAAAGGTGTTGCTTACACCGATATCAAAGTTAATACCGTTGAGAAAGATCTGTAA。

[0095] Example 2: Transmembrane experiment of wild-type JAC5 protein

[0096] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 100 mV was applied through the nanofilm patch clamp device, and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0097] (2) Nanopore embedding: 5 μg of oligomeric wild-type JAC5 protein was added to the cis-detection cell, and a voltage of 200 mV was applied to promote protein embedding. The current change was monitored in real time using an Axopatch 200B amplifier. After successful embedding, the cis-detection cell was rinsed with detection buffer to remove unembedded proteins, thus completing the nanopore construction.

[0098] (3) The analog signal is low-pass filtered at 100kHz using a 4-pole Bessel filter and digitized at 500kHz. Data acquisition is controlled by Origin.

[0099] (4) Single-molecule detection of single-stranded DNA: setting nanopatch clamp parameters: sampling frequency 10kHz, filtering frequency 2kHz, applying initial voltage 100mV, adding 50μL of 100nM single-stranded DNA (SEQ ID NO:3: 5'-TTTTTTTTTT-3') solution to the cis compartment, and acquiring current signals using Clampex 10.4 software.

[0100] The results of nanopore opening current detection of wild-type JAC5 protein are as follows: Figure 5 As shown, the results indicate that the pore current signal of wild-type JAC5 protein is unstable and has many current spikes.

[0101] Current detection results during single-stranded DNA pore drilling are as follows Figure 6 As shown, the results indicate that the pore current signal of wild-type JAC5 protein is unstable and has many current spikes.

[0102] Example 3: Transmembrane experiment of JAC5-M1 protein with amino acid mutation

[0103] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 100 mV was applied through the nanofilm patch clamp device, and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0104] (2) Nanopore embedding: 5 μg of oligomerized JAC5-M1 protein (wild-type JAC5 protein with amino acid mutations D135 to D135A, K171 to K171A, S190 to S190A, K192 to K192A, and N246 to N246A) was added to the cis-detection cell, and a voltage of 200 mV was applied to promote protein embedding. The current change was monitored in real time using an Axopatch 200B amplifier. After successful embedding, the cis-detection cell was rinsed with detection buffer to remove unembedded protein, thus completing the nanopore construction.

[0105] (3) The analog signal is low-pass filtered at 100kHz using a 4-pole Bessel filter and digitized at 500kHz. Data acquisition is controlled by Origin.

[0106] The results of the nanopore opening current detection of JAC5-M1 protein are as follows: Figure 7 As shown, the results indicate that the JAC5-M1 protein has a stable pore-opening current signal but many current spikes.

[0107] Example 4: JAC5-M1 protein detection of single-stranded DNA

[0108] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 100 mV was applied through the nanofilm patch clamp device, and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0109] (2) Nanopore embedding: 5 μg of oligomeric JAC5-M1 protein was added to the cis-detection cell, and a voltage of 200 mV was applied to promote protein embedding. The current change was monitored in real time by an Axopatch 200B amplifier. After successful embedding, the cis-detection cell was rinsed with detection buffer to remove unembedded proteins, thus completing the nanopore construction.

[0110] (3) The analog signal is low-pass filtered at 100kHz using a 4-pole Bessel filter and digitized at 500kHz. Data acquisition is controlled by Origin.

[0111] (4) Single-molecule detection of single-stranded DNA: setting nanopatch clamp parameters: sampling frequency 10kHz, filtering frequency 2kHz, applying initial voltage 100mV, adding 50μL of 100nM single-stranded DNA (SEQ ID NO:3: 5'-TTTTTTTTTT-3') solution to the cis compartment, and acquiring current signals using Clampex 10.4 software.

[0112] The current detection results during single-stranded DNA pore drilling of mutant JAC5 protein (JAC5-M1 protein with amino acid K186 mutated to K186A) are as follows: Figure 8 As shown, the results indicate that compared to the wild-type JAC5 protein, the mutant JAC5 protein exhibits more stable current characteristics, a narrower current signal width, and fewer spikes when detecting single-stranded DNA. The principle behind the improved current signal quality is as follows: selecting five side chain residues (Asp135, Lys171, Ser190, Lys192, and Asn246) and mutating them to alanine results in stronger hydrophobic interactions across the membrane β-barrel, making it easier to form pores. After membrane insertion, it is less likely to detach, leading to a more stable signal.

[0113] Example 5: Detection of high-mannose-modified single-stranded DNA based on nanopores of mutant JAC5 protein (amino acid K186 of JAC5-M1 protein)

[0114] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 30 mV was applied through the nanofilm patch clamp device and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0115] (2) Nanopore embedding: 5 μg of oligomerized mutant JAC5 protein (the amino acid K186 of JAC5-M1 protein is mutated to K186M, K186Q, K186C, K186R, K186H, K186W, K186A, K186F or K186D) is added to the cis-detection cell, and a voltage of 200mV is applied to promote protein embedding. The current change is monitored in real time by an Axopatch 200B amplifier. After successful embedding, the cis-detection cell is rinsed with detection buffer to remove unembedded protein, thus completing the nanopore construction.

[0116] (3) Single-molecule detection of single-stranded DNA: setting nanopatch clamp parameters: sampling frequency 10kHz, filtering frequency 2kHz, applying initial voltage 100mV, adding 50μL of 100nM single-stranded DNA (SEQ ID NO:4: 5'-Man-α1-2Man-TTTTTTTTTTTTTTTTTTTT-3') solution to the cis compartment, and acquiring current signals using Clampex 10.4 software.

[0117] The current detection results during single-stranded DNA pore drilling of mutant JAC5 protein (JAC5-M1 protein with amino acid K186 mutated to K186A) are as follows: Figure 9 As shown, the results indicate that compared to JAC5-M1 protein and wild-type JAC5 protein, mutant JAC5 protein (the amino acid K186 of JAC5-M1 protein is mutated to K186A) has stable current properties, narrower current signal width, and fewer spikes when detecting single-stranded DNA. The principle behind the improved current signal quality is that after the lysine with a positively charged side chain is mutated to alanine with a methyl side chain, the positive charge repulsion is eliminated and the steric hindrance is reduced.

[0118] The current detection results of single-stranded DNA pores for other mutant JAC5 proteins (amino acid K186 of JAC5-M1 protein) are shown in Table 1.

[0119] Table 1. Current detection results during single-stranded DNA pore drilling of other mutant JAC5 proteins.

[0120]

[0121] Example 6: Single-molecule detection of β2-microglobulin based on mutant JAC5 protein (amino acid E188 of JAC5-M1 protein)

[0122] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 30 mV was applied through the nanofilm patch clamp device and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0123] (2) Nanopore embedding: 5 μg of oligomerized mutant JAC5 protein (the amino acid E188 of JAC5-M1 protein is E188N, E188K, E188S, E188Q, E188L, E188A or E188T) was added to the cis-detection cell, and a voltage of 200mV was applied to promote protein embedding. The current change was monitored in real time by an Axopatch 200B amplifier. After successful embedding, the cis-detection cell was rinsed with detection buffer to remove unembedded protein, thus completing the nanopore construction.

[0124] (3) Setting nanopatch clamp parameters for single molecule detection of β2-microglobulin: sampling frequency 10kHz, filtering frequency 2kHz, initial voltage 30mV, adding 50μL of 20nM β2-MG solution to the cis compartment, and acquiring current signal using Clampex 10.4 software.

[0125] The current detection results of β2-microglobulin perforation of mutant JAC5 protein (JAC5-M1 protein with amino acid E188 mutated to E188A) are as follows: Figure 10 As shown, the results indicate that compared to JAC5-M1 protein and wild-type JAC5 protein, mutant JAC5 protein (the amino acid E188 of JAC5-M1 protein is mutated to E188A) has stable current properties, narrower current signal width, and fewer spikes when detecting the current generated by β2-microglobulin. The principle behind the improved current signal quality is that after glutamic acid is mutated to alanine, the nonpolar side chain of alanine replaces the carboxyl group of glutamic acid, eliminating negative charge repulsion. Furthermore, the methyl side chain does not change the pore size of the contraction region and does not affect molecular resolution.

[0126] The current detection results of β2-microglobulin perforation of other mutant JAC5 proteins (amino acid E188 of JAC5-M1 protein) are shown in Table 2.

[0127] Table 2. Current detection results of β2-microglobulin perforation of other mutant JAC5 proteins.

[0128]

[0129] Example 7: Detection of short RNA based on mutant JAC5 protein (amino acid V187 of JAC5-M1 protein)

[0130] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 30 mV was applied through the nanofilm patch clamp device and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0131] (2) Nanopore embedding: 5 μg of oligomerized mutant JAC5 protein (the amino acid V187 of JAC5-M1 protein is mutated to V187N, V187I, V187T, V187G, V187P, V187L, V187D, V187Y or V187S) is added to the cis-detection cell, and a voltage of 200mV is applied to promote protein embedding. The current change is monitored in real time by an Axopatch 200B amplifier. After successful embedding, the cis-detection cell is rinsed with detection buffer to remove unembedded protein, thus completing the nanopore construction.

[0132] (3) Setting nanopatch clamp parameters for single-molecule detection of short-chain RNA: sampling frequency 10kHz, filtering frequency 2kHz, initial voltage 100 mV applied, 50μL of 100nM short-chain RNA (SEQ ID NO:5: 5'-Man-α1-2Man-AUUGGAGCUCUUGUUUACCGGGUUUCAU-3') solution added to the cis compartment, and current signal acquired using Clampex 10.4 software.

[0133] The current detection results during short-chain RNA pore perforation of mutant JAC5 protein (JAC5-M1 protein with amino acid V187 mutated to V187G) are as follows: Figure 11 As shown, the results indicate that compared to JAC5-M1 protein and wild-type JAC5 protein, mutant JAC5 protein (the amino acid V187 of JAC5-M1 protein is mutated to V187G) has stable current properties, narrower current signal width, and fewer spikes when detecting short-chain RNA nucleic acids. The principle behind the improved current signal quality is that after valine is mutated to glycine, glycine has no side chain, no polarity interference, does not affect the overall structural stability of the β-barrel, and eliminates steric hindrance.

[0134] The current detection results of short-chain RNA pores of other mutant JAC5 proteins (amino acid V187 of JAC5-M1 protein) are shown in Table 3.

[0135] Table 3. Current detection results during short-chain RNA pore perforation of other mutant JAC5 proteins.

[0136]

[0137] Example 8: Protein fragment detection based on mutant JAC5 protein (amino acids T250, T251, and T252 of JAC5-M1 protein)

[0138] (1) Preparation of phospholipid bilayer membrane: 5 μL of pentane solution containing 10 mg / mL DPhPC was dropped onto the surface of the polytetrafluoroethylene membrane (diameter 100±10 μm) of the nanobilayer membrane support system. The solution was left at room temperature for 2 min to allow the pentane to evaporate and form a uniform phospholipid monolayer membrane. 500 μL of detection buffer (50 mM MES, 100 mM NaCl, 0.01% DDM, pH=5.5) was added to the cis detection cell and trans detection cell of the electrolytic cell, respectively. The liquid level was slowly adjusted to allow the phospholipid monolayer membrane to fuse into a bilayer membrane. A voltage of 30 mV was applied through the nanofilm patch clamp device and the current was confirmed to be 0 pA, which proved that the phospholipid bilayer membrane was intact.

[0139] (2) Nanopore embedding: Add 5 μg of oligomerized mutant JAC5 protein (the amino acid T250 of JAC5-M1 protein is mutated to T250N, T250G, or T251 is mutated to T251S, T251I, T251R, or T252 is mutated to T252A, T252Y, T252W) to the cis detection cell, and apply a voltage of 200mV to promote protein embedding; monitor the current change in real time with an Axopatch 200B amplifier. After successful embedding, rinse the cis detection cell with detection buffer to remove unembedded protein and complete the nanopore construction.

[0140] (3) Setting nanopatch clamp parameters for single-molecule detection of protein fragments: sampling frequency 10kHz, filtering frequency 2kHz, initial voltage 100mV applied, 50μL of 100nM protein fragment (SEQ ID NO:6: N-Man-α1-2Man-YLPTQGFQLLIS-C) solution added to the cis compartment, and current signal acquired by Clampex 10.4 software.

[0141] The current detection results during pore puncture of a mutant JAC5 protein (the amino acid T252 of the JAC5-M1 protein is mutated to T252A) are as follows: Figure 12As shown, the results indicate that compared to JAC5-M1 protein and wild-type JAC5 protein, the mutant JAC5 protein (the amino acid T252 of JAC5-M1 protein is mutated to T252A) produces a stable current signal with a narrower current signal width and fewer spikes. The principle behind the improved current signal quality is that after threonine is mutated to alanine, the steric hindrance of the methyl side chain of alanine is minimal. Eliminating steric hindrance enhances the flexibility of the β chain, allowing for greater deformation. Furthermore, alanine lacks a hydroxyl group, avoiding the formation of nonspecific hydrogen bonds with water molecules.

[0142] The current detection results of protein fragment pores of other mutant JAC5 proteins (amino acids T250, T251, and T252 of JAC5-M1 protein) are shown in Table 4.

[0143] Table 4. Current detection results during pore perforation of protein fragments of other mutant JAC5 proteins.

[0144]

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of nanopores based on mutant zebrafish protein in single-molecule detection, characterized in that: The mutant zebrafish protein is obtained by mutation of wild-type JAC5 protein, and the amino acid sequence of wild-type JAC5 protein is shown in SEQ ID NO:1; The mutant zebrafish protein specifically refers to the wild-type JAC5 protein with the following amino acid mutations: D135 is mutated to D135A, K171 to K171A, S190 to S190A, K192 to K192A, and N246 to N246A.

2. The application as described in claim 1, characterized in that: The mutant zebrafish protein is then subjected to amino acid mutations, specifically: amino acid K186 is mutated to K186M, K186Q, K186C, K186R, K186H, K186W, K186A, K186F, or K186D. Alternatively, the amino acid V187 may be mutated to V187N, V187I, V187T, V187G, V187P, V187L, V187D, V187Y, or V187S. Alternatively, the amino acid E188 may mutate to E188N, E188K, E188S, E188Q, E188L, E188A, or E188T.

3. The application as described in claim 2, characterized in that: The amino acid mutation specifically refers to the mutation of amino acid T250 into T250N or T250G. Alternatively, the amino acid T251 may mutate to T251S, T251I, or T251R; Alternatively, the amino acid T252 may mutate to T252A, T252Y, or T252W.

4. The application as described in any one of claims 1-3, wherein the target analyte for single-molecule detection includes peptides, proteins, or nucleotides.

5. The application as described in claim 4, characterized in that: The nucleotide sequence encoding the wild-type JAC5 protein is shown in SEQ ID NO:

2.

6. The application as described in claim 5, characterized in that: The method for preparing the mutant zebrafish protein includes the following steps: Construct a vector for the mutant zebrafish protein; Expression and purification of the mutant zebrafish protein.

7. A membrane based on mutant zebrafish protein, characterized in that: The membrane is a lipid bilayer, and the membrane is embedded in the mutant zebrafish protein as described in any one of claims 1-3.

8. A nanopore based on mutant zebrafish protein, characterized in that: The nanopores are composed of mutant zebrafish proteins as described in any one of claims 1-3.

9. A product comprising mutant zebrafish protein, characterized in that: The product comprises the mutant zebrafish protein as described in any one of claims 1-3, and the product is a composition, complex, or kit.

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