YaxAB nanopore, nanopore system containing it, and its applications

Abstract

This invention relates to YaxAB nanopores, systems containing the nanopores, and their applications. The YaxAB nanopores of this invention have a funnel-shaped three-dimensional structure, which maximizes the change in current interruption due to the behavior of the sample trapped inside the nanopore. This allows for very sensitive measurement of minute changes in physicochemical properties, including changes in the structure and dynamics of the sample. Furthermore, they possess high cation selectivity and can induce very strong electroosmosis, offering the advantage of capturing and analyzing various types of samples without being limited by the type of overall charge. In addition, by adjusting the amino acid sequence of the monomers constituting the nanopores, the purification yield of proteins can be improved and the oligomerization mechanism can be controlled, enabling the creation of YaxAB nanopores with pore sizes that cannot be formed with wild-type monomers. Moreover, the nanopores of this invention have the advantage of being able to secure YaxAB nanopores of various pore sizes in a single purification, and furthermore, of being able to selectively secure nanopores of a desired pore size. This overcomes the limitations of conventional biological nanopores, which have a fixed pore size. The YaxAB nanopore of the present invention has the advantage of being able to adjust the nanopore size according to the size of the sample. Furthermore, the YaxAB nanopore of the present invention has a very wide dynamic range of detection spectrum for samples ranging from small to large, due to its funnel-shaped three-dimensional structure with a large inlet and a narrow outlet. In addition, the YaxAB nanopore of the present invention has the largest nanopore structure of any biological nanopore to date, and based on its funnel-shaped structure with a large inlet, it enables the detection and analysis of single molecules in large samples that were impossible to detect with conventional biological nanopores. Moreover, the present invention establishes an optimized membrane composition in which the above-mentioned YaxAB nanopore can stably exist, and through a sensor realized based on this, various types of samples with a wide range of sizes and charges can be qualitatively and quantitatively analyzed at the single-molecule level with very high resolution and sensitivity.

Description

[Technical Field]

[0001] This application claims priority under Korean Patent Application No. 10-2022-0138724 dated October 25, 2022, and all content disclosed in the said Korean Patent Application is incorporated herein by reference.

[0002] The present invention relates to YaxAB nanopores and nanopore systems containing them, and to a novel nanopore sensing platform capable of precisely analyzing analytes of various sizes and charges at the single-molecule level, screening for drugs that bind to them, analyzing interactions with ligands, or detecting and quantitatively analyzing disease biomarkers. [Background technology]

[0003] Despite protein-protein interactions (PPIs) being efficient targets for disease treatment in drug discovery, commercialized methods for screening PPI inhibitors through large-scale screening are not yet available. While studies of structure-activity relationships (SARs) and modes of action (MOAs) to monitor protein-drug interactions (PDIs) in vitro are essential, and various methods exist for direct drug screening, such as nuclear magnetic resonance, surface plasma resonance, isothermal titration calorimeters, and fluorescence resonance energy transition, the high cost of equipment, inaccuracies due to labeling / immobilization, low sensitivity for detecting small molecule drugs, and limited solubility of target proteins and / or drugs highlight the urgent need for low-cost and efficient drug screening for protein-protein interactions.

[0004] Nanopores are novel, high-precision biosensors capable of detecting subtle morphological changes in biomolecules at the single-molecule level. Generally, nanopores act as the sole conduits, with each nanopore embedded in a thin insulating film, allowing ionic currents to flow between two liquid reservoirs. Nanopore experiments using the principle of a coulter counter relate to changes in ionic currents. When an analyte passes through a nanopore while the ionic current is electrophoretically induced in the presence of an external electric field, it induces a temporary interruption of the ionic current. This is measured by current amplitude, and based on such electrical signals, the length, size, charge, structure, and morphology of the biomolecule are determined. Nanopore biosensors are used in genome sequencing, detection of various individual biomolecules, and detection of biomolecular interactions. Cytolysin A (ClyA) is a pore-forming protein known to form 12- to 14-mer nanopores. ClyA nanopores have a constriction of 3.3 nm or more in diameter and are being studied for monitoring various biomolecules and protein-ligand interactions. Biomolecular analysis techniques using these nanopores have advantages such as single-molecule-level resolution, high sensitivity, label-free operation, and real-time measurement capabilities, and the development of nanopore-based biomolecular analysis techniques is actively underway. However, such nanopores are based on recognizing large structural changes induced by ligand binding, and have limitations when it comes to targets that cannot be targeted with drugs using conventional methods. Furthermore, since proteins basically have various surface charges, the principle of electrophoretic force (EPF), which is basically used as the driving force to attract the sample in nanopores, has limitations in capturing protein samples.

[0005] The inventors of this invention have diligently strived to develop a method for accurately and efficiently screening protein-protein interaction inhibitors even with minute amounts of sample. As a result, they discovered a novel biological nanopore that utilizes electroosmotic force (EOF), which is generated in a manner dependent on the surface charge inside the pore. By measuring electrical signals using a new nanopore sensing platform incorporating this nanopore, it is possible to detect single-molecule proteins or nucleic acids in response to changes in these signals. Furthermore, it is possible to efficiently screen for protein-drug interactions, protein-protein interactions, and small-molecule drugs that inhibit such protein-protein interactions. Moreover, it has been discovered that single-molecule-level "drug fingerprinting" based screening is possible, which can sensitively distinguish minute differences between other drugs bound to the same protein, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0006] One objective of the present invention is to provide a novel biological nanopore subunit. Another object of the present invention is to provide a nanopore comprising the nanopore subunit.

[0007] Another object of the present invention is to provide a nanopore-containing membrane comprising a membrane layer and a nanopore containing at least one nanopore subunit inserted into the membrane layer.

[0008] A further object of the present invention is to provide a nanopore system comprising a chamber and the nanopore-containing membrane, wherein the space within the chamber is divided into two compartments by the nanopore-containing membrane.

[0009] A further object of the present invention is to provide a method for single-molecule analysis of a single or multiple samples using the nanopore system described above. A further object of the present invention is to provide a method for analyzing the interaction between at least one sample and at least one ligand, or for screening ligands for a sample, using the nanopore system described above.

[0010] A further object of the present invention is to provide a method for analyzing or screening interaction inhibitors or promoters between interactable biomolecules using the nanopore system described above.

[0011] Another object of the present invention is to provide a method for identifying and quantitatively analyzing a sample in a sample using the nanopore system described above. Another object of the present invention is to provide a method for providing information for the diagnosis of biomarker-related diseases using the nanopore system described above. [Means for solving the problem]

[0012] To achieve the above objective, the present invention provides a YaxAB nanopore having a funnel shape and including a first opening, an intermediate region, and a second opening, wherein the outer diameter of the lumen of the first opening is 5 nm or more, the second opening includes a constricted portion with a diameter of 0.5 nm or more, the outer diameter of the lumen of the second opening is 1 nm or more, and the depth of the lumen is 5 nm or more.

[0013] Furthermore, the present invention includes a first monomer comprising an amino acid sequence having 70% or more homology to any one of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34, and an amino acid sequence having 70% or more homology to the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25. The present invention provides a YaxAB nanopore comprising a second monomer and at least one subunit containing a second monomer.

[0014] Furthermore, the present invention relates to a nanopore-containing membrane comprising a membrane layer and YaxAB nanopores inserted into the membrane layer, wherein the membrane layer comprises 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine 1,2-Dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG), 1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (1,2-dioleoyl-sn-gl (ycero-3-phosphoglycerol, DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and mycolic acid (mycolic At least one selected from the group consisting of (1-pal) acid, and campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (1-pal mitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine, The present invention provides a nanopore-containing membrane comprising at least one selected from the group consisting of (1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine, PChcPC), 1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, DChemsPC, 10,12-pentacosadiynoic acid, PCDA, 10,12-tricosadiynoic acid, TCDA, 5,7-hexadecadiynoic acid, HDDA, and 9,12-octadecadiynoic acid, ODDA.

[0015] Furthermore, the present invention provides a nanopore system comprising a chamber and a YaxAB nanopore-containing membrane, wherein the space within the chamber is divided into two compartments by the nanopore-containing membrane.

[0016] Furthermore, the present invention provides a method for single-molecule analysis of a single or multiple samples, comprising the steps of: placing at least one sample (analyte) in one compartment of the nanopore system; and measuring changes in electrical signals in the two compartments before and after the placement of the samples.

[0017] Furthermore, the present invention provides a method for analyzing the interaction between a sample and a ligand or screening a ligand for a sample, comprising the steps of: processing one or two compartments of the nanopore system with at least one sample (analyte) and at least one candidate ligand substance for the sample; and measuring changes in electrical signals in the two compartments before and after processing with the candidate substance.

[0018] Furthermore, the present invention provides a method for analyzing or screening for biomolecular interaction inhibitors or promoters, comprising the steps of: reacting a plurality of interactable biomolecules in one compartment of the nanopore system; treating one or two compartments with a candidate substance for interaction inhibitor or promoter; and measuring changes in electrical signals in the two compartments before and after treatment with the candidate substance.

[0019] Furthermore, the present invention provides a method for identifying and quantitatively analyzing samples in a sample, comprising the steps of: placing a sample in one compartment of the nanopore system; and comparing changes in electrical signals caused by multiple samples in the sample with a database of electrical signals for each substance.

[0020] Furthermore, the present invention provides a method for providing information for the diagnosis of biomarker-related diseases, comprising the steps of: placing a sample in one compartment of the nanopore system; and measuring a characteristic change in electrical signal caused by a biomarker for a specific disease among the changes in electrical signal caused by the sample. [Effects of the Invention]

[0021] The YaxAB nanopores of the present invention, under pH 7.5 conditions, have negatively charged amino acids constituting the inner wall of the pore. Because electroosmosis can be induced by cations predominantly flowing along the inner wall of the pore, samples can be trapped inside the nanopore regardless of the overall charge. Furthermore, the structure, including the diameter of the YaxAB nanopore, can be adjusted according to the number of heterodimers formed by the two monomers YaxA and YaxB, allowing for the production of YaxAB nanopores of various sizes in a single purification step. Therefore, it has the advantage that the target of analysis is not limited by the charge or size of the sample. Furthermore, because it has a funnel structure with a wide entrance and a relatively much narrower exit, even large samples can be captured inside the nanopore. In addition, due to the high ion density in the narrow region, the degree of current interruption originating from the sample is significantly different compared to conventional cylindrical nanopores, so even minute changes in the physicochemical properties of the sample can be detected with much greater sensitivity, and it is possible to analyze samples of various sizes.

[0022] Based on these unique physicochemical properties, the YaxAB nanopore of the present invention can detect a much wider range of samples with higher resolution compared to conventional nanopores. Furthermore, it can directly detect protein-small molecule complexes and sensitively distinguish protein-small molecule complexes bound to different small molecule compounds at the single-molecule level. Therefore, compared to conventional techniques, it can be used more efficiently in terms of time and cost for a wide range of applications, including sample-ligand interaction analysis, biomolecular structure and dynamics analysis, new drug screening, disease diagnosis, protein identification, post-translational modification (PTM) analysis, genome and proteome analysis, and protein and nucleic acid sequencing. [Brief explanation of the drawing]

[0023] [Figure 1] These are the oligomer-specific bands obtained by electrophoresis of modified YaxAB_dN nanopores on a gradient blue native polyacrylamide gel. Modified YaxAB_dN nanopores are one type of modified YaxAB nanopore in which the amino acid sequence has been modified from wild-type YaxAB, and refer to the YaxAB oligomer formed from a first monomer consisting of SEQ ID NO: 1 (YaxA_(45-410)) and a second monomer consisting of SEQ ID NO: 2 (YaxB_(12_343)). The presence of modified YaxAB_dN-C8, -C9, and -C10 was confirmed by the three separated bands. [Figure 2]Figures 2a to 2c show the results of observing modified YaxAB_dN-C8, -C9, and -C10 nanopores via Negative-stain EM (negative staining electron microscopy), Figure 2d is a three-dimensional reconstruction based on the Negative-stain EM results, and Figure 2e shows the modified YaxAB_dN-C8 nanopore structure based on Cryo-EM (cryo-electron microscopy) results. [Figure 3] This image shows the structure of a modified YaxAB_dN nanopore in a longitudinal section. [Figure 4] Figure 4a shows the molecular dynamics (MD) simulation results illustrating the flow of potassium ions, chloride ions, and water molecules in a modified YaxAB_dN nanopore when 100 mV is applied, while Figures 4b and 4c show the molecular dynamics simulation results when the Bcl-xL protein is present at distances of 4 nm and 10 nm from the membrane, respectively. [Figure 5] Figure 5a shows a diagram of the sample trapping event when the Bcl-xL protein is trapped in a modified YaxAB_dN nanopore, along with the single-molecule sample signal. Figure 5b shows a diagram of the MD simulation when the sample is present at distances of 4 nm and 10 nm from the membrane, along with the sample's free energy as calculated by the MD simulation based on the distance (z) from the membrane. [Figure 6]The results show that modified YaxAB_dN nanopores are more stable when inserted into a DPhPC-sterol composite membrane than when inserted into a membrane consisting only of DPhPC. Figure 6a shows the change in conductivity when modified YaxAB_dN nanopores are inserted into a membrane consisting only of DPhPC, and the conductivity that flows after insertion, showing that membrane rupturing occurs. Figure 6b shows that modified YaxAB_dN-C8, -C9, and -C10 nanopores operate stably when inserted into a DPhPC-sterol composite membrane, where C represents a heterodimer subunit composed of a first monomer and a second monomer, and C8 represents a YaxAB nanopore composed of eight pairs of heterodimer subunits (hereinafter, YaxAB-C8). [Figure 7] This study demonstrates that when a voltage is applied to modified YaxAB_dN nanopores of various sizes, a constant current flows in response to the applied voltage. [Figure 8] The results of protein banding obtained by purification and electrophoresis, along with measurements of the conductivity of each nanopore, show the formation tendencies of modified YaxAB_dN nanopores (C8, C9, C10, C11, C12, C13, C14, and C15) for each band. [Figure 9] Figure 9a shows a schematic diagram of the electrical measurements of a modified YaxAB_dN-C8 nanopore inserted into a DPhPC-sterol composite membrane; Figure 9b shows the structure of the lumen connected by eight α-helics; Figure 9c shows the surface charge of the modified YaxAB_dN-C8 nanopore as a longitudinal section, with the negative charge increasing towards the transformer opening; and Figure 9d shows the reverse potential and cation selectivity (PK+ / PCl-) of the modified YaxAB_dN nanopore (C8, C9, C10). [Figure 10]This shows the results of single-molecule analysis of nine unlabeled proteins, including Hsp33, Bcl-xL, Holo-transferrin, FKBP12, MDM2, BSA, Aldolase, Ferritin, and Thyroglobulin, using modified YaxAB_dN nanopores. [Figure 11] This study demonstrates current cutoff and current noise analysis of protein-ligand interactions in modified YaxAB_dN nanopores. Figure 11a shows schematic diagrams of Bcl-xL, Bcl-xL+Bak-BH3 complexes, and Bcl-xL+ABT-737 complexes within the pore, along with the measured current cutoff signals. Figure 11b shows the residence time percentages for each current cutoff level (L1-L3). Figure 11c shows the power spectral densities of Bcl-xL, Bcl-xL+Bak-BH3 complexes, and Bcl-xL+ABT-737 complexes. Figures 11d-11f show the current noise (IN)-based analysis results. [Figure 12] This report concerns the results of analyzing the binding of Bax-BH3 peptide (KD=13μM), which has a low binding affinity to Bcl-xL protein, to a modified YaxAB_dN nanopore. Figure 12a shows the change in the ion current signal generated depending on whether or not Bax-BH3 peptide is bound to the Bcl-xL protein. Figure 12b shows the mean and standard deviation of the current noise (IN) generated depending on whether or not Bax-BH3 peptide is bound to the Bcl-xL protein. Figure 12c is a scatter plot showing the change in the pattern of the ion current trace generated depending on whether or not Bax-BH3 peptide is bound to the Bcl-xL protein. [Figure 13]This report concerns the results of analyzing the binding of a small molecule drug (Quercetin, KD=1.1μM) with low binding affinity to Bcl-xL protein using modified YaxAB_dN nanopores. Figure 13a shows the change in the ion current signal generated depending on whether or not Quercetin is bound to the Bcl-xL protein. Figure 13b shows the mean and standard deviation of the current noise (IN) generated depending on whether or not Quercetin is bound to the Bcl-xL protein. Figure 13c is a scatter plot showing the change in the pattern of the ion current trace generated depending on whether or not Quercetin is bound to the Bcl-xL protein. [Figure 14] This document shows the results of detecting GBP protein (Glucose / Galactose binding protein) using electroosmosis-based methods with modified YaxAB_dN nanopores. Figure 14a shows a schematic diagram of the detection of GBP protein on an electroosmotic base in a modified YaxAB_dN nanopore. Figure 14b shows the current cutoff signal for GBP protein and the current cutoff signal resulting from the structural change in GBP protein that occurs when it binds to galactose, an allosteric activator different from GBP protein. Figure 14c shows the current cutoff signal for lysozyme protein. Figure 14d shows the current cutoff signal for holo-myoglobin protein. Figure 14e shows the results of analyzing the current cutoff signal, standard deviation, and maintenance time factors for lysozyme protein using a two-dimensional scatter plot. [Figure 15] Figure 15a shows the bound fraction of the Bcl-xL+Bak-BH3 complex according to the concentration of the Bak-BH3 peptide, Figure 15b shows the bound fraction of the Bcl-xL+ABT-737 complex according to the concentration of ABT-737, and Figure 15c shows the bound fraction of the Bcl-xL+A-1331852 complex according to the concentration of A-1331852. [Figure 16]Figure 16a shows schematic diagrams of samples within a modified YaxAB_dN nanopore and the electrical signal results when only free Bcl-xL is present, when Bcl-xL is reacted with Bak-BH3, and when a small molecule compound (ABT-737) that inhibits the protein-protein interaction between Bcl-xL and Bak-BH3 is treated after Bcl-xL has been pre-reacted with Bak-BH3. Figure 16b shows the frequency measurements of signals for three types of samples (Bcl-xL, Bcl-xL+Bak-BH3, Bcl-xL+ABT-737) when the small molecule compound (ABT-737) was treated at different concentrations after Bcl-xL and Bak-BH3 had been pre-reacted. Figure 16c shows current traces of the signals for the three types of samples. [Figure 17] The interaction between Bcl-xL protein and small molecule compounds was detected using modified YaxAB_dN nanopores. Figure 17a shows the ion current traces when Bcl-xL protein was individually treated with non-binding agents (LCL-161, GDC-0152) and binding agents (ABT-737, A-1331852), respectively. Figure 17b shows the 2D density contour plot of current cutoff (ΔI / Io)-current noise (IN) when Bcl-xL protein was individually treated with small molecule compounds (LCL-161, GDC-0152, A-1331852, ABT-737). Figure 17c shows the 2D density contour plot of current interruption (ΔI / Io)-current noise (IN) and its real-time monitoring results when Bcl-xL protein was simultaneously treated with multiple small molecule compounds (LCL-161, GDC-0152, A-1331852, ABT-737). [Figure 18]This report concerns the results of analyzing the competitive binding of drugs to Bcl-xL protein using modified YaxAB_dN nanopores. Figure 18a shows a schematic diagram of the competitive binding of drugs to Bcl-xL protein, Figure 18b shows the ion current trace when ABT-737 was titrated on the Bcl-xL / Bak-BH3 complex, and Figures 18c and 18d are 2D density contour plots and violin plots, respectively, showing the competitive binding of Bak-BH3 and ABT-737 to Bcl-xL protein in real time. Figure 18e shows the ion current trace when A-1331852 was titrated on the Bcl-xL+ABT-737 complex, while Figures 18f and 18g are 2D density contour plots and violin plots, respectively, showing the competitive binding of ABT-737 and A-1331852 to the Bcl-xL protein in real time. [Figure 19] Figure 19a shows the results of detecting peptides that bind to Bcl-xL protein using modified YaxAB_dN nanopores and the resulting peptide drug screening. Figure 19a shows the Bcl-xL protein and the Bcl-xL+ peptide drug complex that appear after reacting Bcl-xL protein with three Bak_BH3 mutant peptides that bind to Bcl-xL protein (Bak_BH3_D84A, Bak_BH3_I85A, Bak_BH3_L78A) and wild-type Bak_BH3 peptide. Figure 19b shows a 2D scatter plot of current noise (SD)-current interruption (ΔI / Io) for current interruption signals derived from Bcl-xL protein and Bcl-xL+ peptide drug conjugates, and Figure 19c shows a scatter plot of the current interruption signals that appeared after reacting a solution containing Bcl-xL protein, three mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, Bak_BH3_L78A), and wild-type Bak_BH3. [Figure 20]Figures 20a to 20e show the current cutoff signal patterns and 2D scatter plots of current noise (SD)-current cutoff (ΔI / Io) for each protein. Figure 20f shows the current cutoff signal and the results of the analysis after reacting a solution containing equal amounts of Bcl-2 proteins (Bcl-xL, Bcl-w, Bcl-2, Mcl-1) and the negative control group mTOR protein. Figure 20g shows the current cutoff signal and the results of the analysis after adding an equal amount of A1155463, a small molecule drug targeting Bcl-xL, to the mixed solution. [Figure 21]The following shows the detection results of ternary complexes (protein complexes containing three different molecules bound together) using modified YaxAB_dN nanopores. Figure 21a is a schematic diagram showing that the mTOR-FRB domain and the FKBP12 protein can interact via rapamycin. Figures 21b and 21c show the current cutoff signals for mTOR and FKBP12 proteins and the resulting scatter plots, respectively. Figures 21d and 21e show the current cutoff signals for the binary complex of mTOR and rapamycin and the binary complex of FKBP12 and rapamycin and the resulting scatter plots, respectively. Figure 21f shows the current cutoff signals and the resulting scatter plots when both mTOR and FKBP12 are added. Figure 21g shows the current cutoff signals and the resulting scatter plots when mTOR, FKBP12, and rapamycin are added together. Figure 21h shows the current cutoff signals and scatter plot results obtained when mTOR and FKBP12 are added together with eight negative small molecule compounds that do not bind to the mTOR-FRB domain (Astemizole, Mibefradil, Terfenadine, Pranlukast, Capecitabine, Dimantine, Sulfanilamide, and Monobenzone). Figure 21i shows the current cutoff signals and scatter plot results obtained when mTOR and FKBP12 are added together with the eight negative small molecule compounds and rapamycin. [Figure 22]Figures 22a and 22b show the results of protein-post-translational modification (PTM) peptide interaction analysis and protein-protein interaction inhibitor analysis of modified YaxAB_dN nanopores. Figures 22a and 22b show schematic diagrams of the binding of the 12-amino acid H4 peptide (H4_1-12) and the acetylated peptide (H4_1-12 K5acK8ac) at residues 5 and 8 to the target protein BRD4-BD1 in the histone protein. Figure 22c shows the current cutoff signal measured after adding the H4 peptide (H4_1-12) or the acetylated peptide (H4_1-12 K5acK8ac) at residues 5 and 8 to the target protein BRD4-BD1. Figure 22d shows the change in nanopore current cutoff signal generated by the interaction between the BRD4-BD1 protein and the acetylated peptide (H4_1-12 K5acK8ac), expressed as current noise (IN) value including PSD (Power Spectrum Density) or standard deviation (SD) analysis of ion current, and the binding affinity (KD) of the acetylated peptide (H4_1-12 K5acK8ac) to the BRD4-BD1 protein, measured by quantitative analysis. Figure 22e shows the results of measuring the nanopore current cutoff signal after reacting four small molecule compounds that bind to the BRD4-BD1 protein (GSK778, Y06026, XMD8-92, PLX51107) with the BRD4-BD1 protein. Figure 22f shows the relationship between the BRD4-BD1 target protein and the acetylated peptide (H4_1-12 This shows the results of analyzing the residence time ratio for each current cutoff level (L1-L3) after reacting K5ac and K8ac, and their respective low molecular weight compounds, and then capturing the samples in a nanopore and measuring the change in minute current. [Figure 23]This shows the selective detection results for cancer biomarkers in the presence of serum using modified YaxAB_dN nanopores. Figure 23a shows a two-dimensional scatter plot of the nanopore current cutoff signal and current noise (SD)-current cutoff (ΔI / Io) for the cancer biomarker Bcl-xL protein, and Figure 23b shows the current cutoff signal in depeleted FBS (Fetal Bovine Serum) alone. Figure 23c shows the result after adding 10 nM of Bcl-xL protein in the presence of depleted FBS, and Figure 23d shows the result after measuring the nanopore current cutoff signal after adding 20 nM of Bcl-xL protein in the presence of depleted FBS. [Figure 24] The results of detecting and analyzing Bcl-xL wild-type protein and Bcl-xL(E31K, E36K) and Bcl-xL(R100E, R103E) mutant proteins using modified YaxAB_dN nanopores are shown, demonstrating that the nanopore electrical signals unique to the three proteins appear distinctly from each other. Figure 24a shows the structural characteristics, Figure 24b shows the current signals, and Figure 24c shows the scatter plot results of the analysis of the current interruption signals. [Figure 25] This shows the results of electroosmotic detection of mTOR proteins using modified YaxAB_dN nanopores, and the capture or pass-through signals generated in a voltage-dependent manner are shown. [Figure 26] This shows the results of electrophoresis-based detection of EGF protein using modified YaxAB_dN nanopores. [Figure 27] This shows the detection results for the intrinsically disordered proteins (IDPs) p53TAD1 peptide (1.8 kDa, residues 15-29) and p53TAD protein (8.3 kDa, residues 1-73) using electrophoresis with modified YaxAB_dN nanopores. [Figure 28]This shows the detection results of double-stranded and single-stranded nucleic acids using electrophoresis with modified YaxAB_dN nanopores. [Figure 29] This study compares the protein yields of wild-type YaxA_FL (SEQ ID NO: 3), wild-type YaxB_FL (SEQ ID NO: 4), YaxA_(45-410) (SEQ ID NO: 1), and YaxB_(12-343) (SEQ ID NO: 2) purified under identical conditions. [Figure 30] This study concerns the oligomer-specific bands obtained by electrophoresis of modified YaxAB_dN nanopores on a gradient blue native polyacrylamide gel, demonstrating that wild-type YaxAB (YaxAB_FL) and modified YaxAB_dN have different oligomer formation tendencies. In Figure 30, wild-type YaxAB refers to the oligomer formed from YaxA_FL (the first monomer consisting of SEQ ID NO: 3) and YaxB_FL (the second monomer consisting of SEQ ID NO: 4). Figure 30a shows the results of oligomerization and nanopore formation under low-concentration protein conditions, and Figure 30b shows the results under high-concentration protein conditions. [Figure 31] This shows a comparison of the formation tendency of YaxAB nanopores from each protein band obtained by electrophoresis of wild-type nanopores and modified YaxAB_dN nanopores, as well as the measured conductivity of each nanopore. [Figure 32] This study concerns the oligomer bands obtained by electrophoresis of YaxAB nanopores formed using first and second monomers with adjusted sequence lengths on a gradient blue native polyacrylamide gel. The results show that the formation tendency of YaxAB oligomers differs depending on the sequence of YaxA, obtained by reacting the first monomer, YaxA_(45-410) consisting of SEQ ID NO: 1, and YaxA_(X-411) with adjusted sequence length, with the second monomer, YaxB_(12-343) consisting of SEQ ID NO: 2. [Figure 33]Figure 33a shows the YaxAB oligomer bands formed after electrophoresis of YaxAB nanopores on a gradient blue native polyacrylamide gel. The first monomer, YaxA_(45-410), consisting of wild-type YaxA_FL or SEQ ID NO: 1, reacted with the second monomer, YaxB_(12-343), consisting of SEQ ID NO: 2. Figure 33b shows the YaxAB oligomer bands formed after electrophoresis of YaxAB nanopores on a gradient blue native polyacrylamide gel. The results show the YaxAB oligomer tendencies formed after individually reacting YaxA_(45-410), YaxA_(3-410), YaxA_(5-410), YaxA_(8-410), or YaxA_(9-410) with the second monomer, YaxB_(12-343), consisting of SEQ ID NO: 2. [Figure 34] This report concerns the results of electrically verifying the Bcl-xL detection ability of a nanopore system by applying YaxAB oligomers to a nanopore-containing membrane. Figure 34a shows the results of electrically verifying the detection ability of a YaxAB nanopore system for Bcl-xL protein using YaxAB oligomers formed by the binding of YaxA_(11-411), YaxA_(21-411), YaxA_(31-411), YaxA_(41-411), and YaxB_(12-343), a second monomer consisting of the amino acid sequence of SEQ ID NO: 2, by electrical measurement experiments. Figure 34b shows the results of an electrometric measurement experiment verifying the detection ability of the YaxAB nanopore system for Bcl-xL protein, which utilizes the YaxAB oligomer formed by the binding of YaxA_(3-410) and YaxB_(12-343), a second monomer consisting of the amino acid sequence of SEQ ID NO: 2. [Figure 35]This shows the results of electrophoresis on a gradient blue native polyacrylamide gel, revealing the oligomer-specific bands for four types of YaxAB oligomers, each consisting of a nanopore subunit formed by the binding of YaxA_(57-398) or YaxA_(52-383), which is the minimum sequence capable of forming a YaxAB oligomer, and YaxAB_dN oligomer, which consists of a nanopore subunit formed by the binding of YaxA_(45-410) and YaxB_(12-343). [Figure 36] This paper concerns YaxAB nanopores fabricated with the smallest possible sequence for the formation of YaxAB oligomers. Four types of YaxAB oligomers, formed by the binding of YaxA_(57-398) or YaxA_(52-383) to YaxB_(26-342) or YaxB_(12-316), were applied to a nanopore-containing membrane, and the Bcl-xL detection capability of the nanopore system was verified by electrical measurement experiments. [Figure 37] This result shows the minimum YaxA and YaxB sequences capable of forming YaxAB oligomers. [Figure 38] This is a histogram showing the changes in current traces and the relative signal generation frequency for different current cutoff ratios (ΔI / Io) when FK506 is added to the electrically neutral FKBP12 protein in the YaxAB nanopore system. [Figure 39] This is a histogram showing the changes in current traces and the relative signal generation frequency for each current cutoff ratio (ΔI / Io) when holo-transferrin protein is added to the YaxAB nanopore system, both in its standalone state and when oxaliplatin is added to holo-transferrin. [Figure 40] This shows the results of comparing the signals of the sample-compound complex under low and high concentrations of ABT-737 compound in the YaxAB nanopore system. [Figure 41]This result shows whether or not a nonspecific signal is generated when a non-target compound is added to the YaxAB nanopore system depending on the presence or absence of a sample. [Figure 42] This shows the results of comparing the signals of sample-compound complexes under various DMSO concentrations in the YaxAB nanopore system. [Modes for carrying out the invention]

[0024] The present invention will be described in detail below. 1. YaxAB nanopore subunit and YaxAB nanopore containing the same One aspect of the present invention provides YaxAB nanopore subunits and YaxAB nanopores.

[0025] YaxAB is a heterodimer formed by the binding of YaxA and YaxB proteins, and is one of the pore-forming toxins that allows Yersinia enterocolitic, an intestinal pathogen that causes a variety of diseases ranging from mild diarrhea to severe systemic bacteremia, to bind to and invade host cells. In Yersinia enterocolitic, RovA regulates the transcription of toxic genes, and among them, YE1984 and YE1985 have been confirmed to be upregulated by RovA. Because they are homologous to the cytotoxin genes xaxA and xaxB of Xenorhabdus nematophila, they were named yaxA and yaxB, respectively. Both the YaxA and YaxB proteins have a membrane-active moiety, but only the YaxA protein can bind to the membrane on its own. The YaxB protein binds to the membrane-embedded YaxA protein, inducing the heterodimer to form a soluble membrane-penetrating helical structure. YaxAB can then form YaxAB nanopores in the host cell membrane through additional oligomerization. Wild-type YaxAB nanopores primarily have a decamer structure composed of 10 YaxAB heterodimers. In YaxAB nanopores, the YaxB protein is positioned to face the lumen of the pore, while the YaxA protein surrounds the YaxB protein. In other words, YaxAB-mediated toxicity can induce osmotic lysis by forming pores in the host cell membrane.

[0026] In one embodiment, the YaxAB nanopore of the present invention may be formed by including at least one subunit that includes a membrane-active moiety or a site that forms a membrane-penetrating α-helices structure as part of wild-type YaxAB, which is a pore-forming toxin. In particular, the subunit constituting the YaxAB nanopore of the present invention described above may include a first monomer and a second monomer. In this case, the first monomer may include an amino acid sequence having 70% or more homology to any one of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34, and the second monomer may include an amino acid sequence having 70% or more homology to the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25.

[0027] In the present invention, "YaxAB nanopore" includes all types of proteins called YaxAB proteins, and may specifically include all isoform forms of YaxAB proteins, as well as all homolog, ortholog, and paralog proteins. Furthermore, the YaxAB nanopore of the present invention may be a non-naturally occurring protein. A non-naturally occurring protein means a protein composed of an amino acid sequence that does not exist in nature, is not normally expressed by cells or organisms, and is artificially produced.

[0028] For example, in the present invention, the first monomer contained in YaxAB nanopore is one amino acid sequence of SEQ ID NO. 7, SEQ ID NO. 24, or SEQ ID NO. 34, or NCBI accession NO. WP_005169901.1 (SEQ ID NO. 3), WP_011816273.1 (SEQ ID NO. 35), HDL8508204.1, HDL8476175.1, HDL7646675.1, PNM17258.1, WP_050336257.1, HDL8287337.1, HDL7822654.1, HDL7748605.1, HDL6737510.1, HEB4799346.1, WP_221871123.1, EKN3734298.1、EKN3569010.1、HDL7089514.1、WP_050160085.1、WP_221876226.1、EKN4747040.1 、WP_050916836.1、HDM8435746.1、HDL8238855. 1、WP_019079472.1、EKN3341278.1、WP_050163370.1、HDL7317648.1、HDM8081034.1、WP_0508708 97.1、EKN3737775.1、HDL7467110.1、HDL7471348.1、WP_046694975.1、WP_301211860.1、HDL7726 911.1、WP_268215747.1、EKN5089609.1、WP_057620566.1、EKN6360258.1、WP_019083154.1、EKN5 159302.1、WP_050131624.1、ELI8015017.1、EKN 4802793.1、EKN6165952.1、ELI8293390.1、ELI8097018.1、WP_050157801.1、EKN4708697.1、EKN 5102380.1、EKN5913011.1、WP_050164534.1、WP_242365000.1、HDL6759266.1、WP_050327956.1、 EKN3969341.1、HDL8127360.1、EKN3724092.1、WP_050334096.1、HDL7761807.1、HDL6872781.1、 EKN3573207.1、EKN3562735.1、EKN5949803.1、HDL6624424.1、WP_219649234.1、EKN3957447.1、 EKN3611203.1、EKN5162868.1、WP_172654801.1 、HDL6886351.1、WP_263797395.1、EKN3989601. 1、HDL7855057.1、HDW8038128.1、EKN3870380.1、WP_057631219.1、EKN5074149.1、WP_219650267.1、HDL6522072.1、EKN4058214.1、EKN4068486.1、HDL6961480.1、WP_075338710.1、EKN5112876 .1、HDL8368473.1、HDL7423034.1、HDM8447363.1、EKN6363360.1、EKN3564743.1、HDL7684594.1、HDL7735658.1、HDL7339639.1、HDL7966642.1、CNH41000.1、HDL7909665.1、WP_219648645.1、W P_219643575.1、CQD54886.1、CNG18579.1、EKN4 119565.1、WP_145592082.1、WP_038639531.1、AJJ34365.1、WP_050114520.1、WP_271298628.1、WP_004390824.1、WP_050095452.1、WP_145562021. 1、WP_159678334.1、WP_309477958.1、WP_145510 697.1、WP_219646505.1、WP_145556850.1、WP_14 5537515.1、WP_219655358.1、CNG40075.1、EKN3 837153.1、EKN3315583.1、WP_301178346.1、HDL 8028676.1、HDL8052082.1、HDL7585724.1、HDL8104119.1、WP_005164820.1、WP_050127938.1、WP_271306546.1、CBX71227.1、KGA73448.1、HDL82 79144.1, HDL8024634.1, WP_115240285.1, WP_254461835.1, HDL8320198.1, WP_050124432.1, WP _050326508.1, HDL8083732.1, EKN3713082.1, HDL8750155.1, CNK45388.1, WP_221864008.1, SUP The protein may include, but is not limited to, an amino acid sequence such as 62773.1 or a variant thereof. Specifically, it may be a protein consisting of the aforementioned amino acid sequence or a variant thereof. The variant of the amino acid sequence may include a variant peptide having a different sequence due to deletion, insertion, substitution, or combination thereof of amino acid residues, to the extent that it does not affect the function of the amino acid sequence, or it may be in the form of a protein fragment having the same function. Amino acid modifications at the protein and peptide levels that do not alter the activity of the aforementioned amino acid sequence are known in the art of the present invention and may include phosphorylation, sulfation, acrylation, glycosylation, methylation, etc. The amino acids may be modified by thylation, farnesylation, etc. Therefore, variants of the amino acid sequence may have an amino acid sequence that is "substantially" identical to the amino acid sequence. The peptides having substantially the same amino acid sequence may have amino acid sequences with homology of 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more, respectively, but are not limited thereto. Any protein having homology of 70% or more of the amino acid sequence to the aforementioned amino acid sequence and possessing activity capable of binding to a lipid membrane is included within the scope of the present invention.

[0029] Furthermore, in the present invention, the second monomer contained in YaxAB nanopore is the amino acid sequence of SEQ ID NO. 18 or SEQ ID NO. 25, or NCBI accession NO. WP_0 11816274.1 (Sequence ID 4), WP_050336255.1 (Sequence ID 36) , WP_005169898.1, PNM17259.1, WP_050137918.1, HDL7748604.1, HDL6508072.1, WP_050137583. 1, WP_050322893.1, HDL7822655.1, HDL7646676 .1, WP_050321988.1, WP_221871120.1, HDL6961479.1, WP_050298175.1, CNG06681.1, EKN591960 1.1, EKN5074150.1, WP_075338711.1, HDL70579 06.1, WP_219656641.1, WP_050157802.1, ELI8015018.1, EKN4068485.1, HDL7533782.1, WP_0576 31218.1, WP_050916837.1, EKN6259816.1, EKN3 396085.1, HDL7423033.1, EKN4822249.1, WP_05 0880005.1, WP_050875182.1, HDL7335482.1, WP_050163371.1, EKN3734297.1, HDL6886350.1, HEB4799167.1, WP_263700684.1, EKN3341279.1, H DL7478311.1, EKN3957448.1, WP_050327958.1, HDL7339638.1, HDL6946689.1, WP_019079471.1 , HDV7159892.1, HDL7467111.1, WP_172654802. 1、WP_050870895.1、EKN3469507.1、WP_019083153.1、WP_076706633.1、HDW8038129.1、CQG99659 .1、WP_050945964.1、HDL6872782.1、WP_046694976.1、WP_263699488.1、WP_263697703.1、EKN3724091.1、HDL7089513.1、CQH55963.1、HDL7666636.1、EKN5102379.1、WP_046050899.1、HDL69594 93.1、EKN5159301.1、HDL8096476.1、EKN3683754.1、EKN3973834.1、WP_219650266.1、EKN63657 51.1、HDL8258876.1、EKN4119566.1、HDM8089399.1、WP_263696663.1、EKN6178815.1、ELI80970 17.1、HDL7909664.1、WP_219643580.1、WP_050149278.1、CNH43347.1、HDL8000813.1、WP_020283 191.1、HDL8788876.1、HDL7394360.1、HDL7984656.1、WP_005164817.1、HDL8028561.1、CBY2714 9.1、HDL8032249.1、HDL8024436.1、HDL8004719.1、HDL6519422.1、HDL8048091.1、WP_01626618 5.1、CCV61752.1、WP_049526748.1、WP_145535945.1、WP_050114521.1、WP_309477957.1、WP_2898 16262.1、WP_050291733.1、WP_145589360.1、WP_ 038633022.1、WP_265526189.1、WP_145537514.1、WP_050095454.1、WP_057634251.1、WP_0502896 82.1、WP_145531133.1、WP_145586506.1、WP_100 286507.1、WP_219655343.1、WP_049557371.1、WP_087795913.1、WP_145510696.1、WP_271304184. 1、WP_004390823.1、WP_145556851.1、WP_050120 The protein may include, but is not limited to, amino acid sequences such as 114.1, WP_219648204.1, and WP_159678331.1 or their variants. Specifically, it may be a protein consisting of the aforementioned amino acid sequences or their variants. The variant of the amino acid sequence may, like the first monomer, include a variant peptide having a different sequence due to deletion, insertion, substitution, or combination thereof of amino acid residues, to the extent that it does not affect the function of the amino acid sequence, or it may be in the form of a protein fragment having the same function. Amino acid modifications at the protein and peptide levels that do not alter the activity of the amino acid sequence are known in the art of the present invention and may, in some cases, be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the variant of the amino acid sequence may have an amino acid sequence that is "substantially" identical to the aforementioned amino acid sequence. The peptides having substantially the same amino acid sequence may have amino acid sequences with homology of 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more, respectively, but are not limited thereto. Any protein having homology of 70% or more of the amino acid sequence to the aforementioned amino acid sequence and possessing activity capable of binding to a lipid membrane is included within the scope of the present invention.

[0030] Specifically, in the present invention, the subunit may include, but is not limited to, a first monomer comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 30, 31, 32, 33, and 34, and a second monomer comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 25, 26, 27, 28, and 29.

[0031] In another embodiment, the YaxAB nanopore of the present invention includes a first opening, an intermediate region, and a second opening, and has a funnel shape, wherein the outer diameter of the lumen of the first opening is 5 nm or more, the second opening includes a constriction, the diameter of the constriction is 0.5 nm or more, the outer diameter of the lumen of the second opening is 1 nm or more, and the depth of the lumen is 5 nm or more.

[0032] Specifically, the YaxAB nanopore includes a first opening, an intermediate region, and a second opening. The first opening is a portion that is exposed without being inserted into the membrane and serves as an entrance, and the outer diameter of the lumen located at the first opening may be 5 nm or more, for example, about 5-30 nm, 6-25 nm, 7-20 nm, 8-18 nm, or 10-17 nm, specifically YaxAB C8-C 10 In a nanopore, the outer diameter of the lumen of the first opening is approximately 1 The wavelength may be 0 to 17 nm. The second opening is inserted into the film and serves as an exit point. The length is min, and the outer diameter of the lumen of the second opening may be 1 nm or more, for example, about 1 to 20 nm, 1.1 to 15 nm, 1.2 to 10 nm, 1.3 to 5 nm, or 1.4 to 3 nm, specifically YaxAB C8-C 10 In the case of nanopores, the second opening is located The diameter of the internal lumen may be approximately 1.9 to 3.1 nm. The intermediate region is a portion connecting the first opening and the second opening, and the intermediate region may have a shape in which the diameter of the internal lumen gradually decreases from the first opening to the second opening. In the YaxAB nanopore, the depth of the internal lumen extending from the first opening through the intermediate region to the second opening may be 5 nm or more, for example, approximately 5 to 20 nm, 6 to 18 nm, 7 to 15 nm, or 10 to 13 nm. Therefore, the YaxAB nanopore may be funnel-shaped, with a wide entrance and a relatively much narrower exit. Furthermore, the internal lumen of the nanopore may be funnel-shaped or coffee dripper-shaped, with a deep depth and a narrow width.

[0033] The second opening includes a constricted portion. The constricted portion is a region in the lumen where the width is narrowed, and the diameter of the constricted portion may be 0.5 nm or more, for example, about 0.5 to 18 nm, 1 to 15 nm, 1.5 to 10 nm, 1.7 to 5 nm, or 1.9 to 3.1 nm. Specifically, YaxAB nanopore C8-C 10 In this case, a diameter of approximately 1.9 to 3.1 nm is also acceptable.

[0034] The constricted portion may be electronegative or electropositive. If the constricted portion is electronegative, it is characterized by having a net negative surface charge in the lumen of the constricted portion. Specifically, in the YaxAB nanopore of the present invention, if the subunit is arranged such that the second monomer faces the lumen and the surface charge of the second monomer is negative, the constricted portion may have a negative surface charge at pH 7.5. Therefore, the electronegative constricted portion can be conferred with selectivity for substances and / or cations that are positively charged from the first opening direction to the second opening direction or from the second opening direction to the first opening direction. In this case, the YaxAB nanopore may have a cation selectivity for anions of 1.1 times or more, 1.4 times or more, 1.7 times or more, or 2.3 times or more. In a specific embodiment of the present invention, calculations of the ion selectivity of the YaxAB nanopore described above confirmed that the YaxAB nanopore exhibits a cation selectivity of 1.4 times or more for anions under pH 7.5 conditions (see Figure 9).

[0035] When the constricted portion is electropositive, it is characterized by having a net positive surface charge in the lumen of the constricted portion. Specifically, in the YaxAB nanopore of the present invention, the subunit is arranged such that the second monomer faces the lumen, and when the surface charge of the second monomer is positive, the constricted portion may have a positive surface charge at pH 7.5. Therefore, the electropositive constricted portion can be conducive to substances and / or anions that are negatively charged from the first opening direction to the second opening direction or from the second opening direction to the first opening direction. In this case, the YaxAB nanopore may have an anion selectivity of 1.1 times or more, 1.4 times or more, 1.7 times or more, or 2.3 times or more for cations. Therefore, the YaxAB nanopore of the present invention may have an ion selectivity of 1.1 times or more.

[0036] On the other hand, the first monomer is one of the amino acid sequences of SEQ ID NO. 7, SEQ ID NO. 24, or SEQ ID NO. 34, or NCBI accession NO. WP_00516 9901.1 (Sequence ID 3), WP_011816273.1 (Sequence ID 35), HDL 8508204.1, HDL8476175.1, HDL7646675.1, PNM17258.1, WP_050336257.1, HDL8287337.1, HDL78 22654.1, HDL7748605.1, HDL6737510.1, HEB4799346.1, WP_221871123.1, EKN3734298.1, EKN35 69010.1, HDL7089514.1, WP_050160085.1, WP_221876226.1, EKN4747040.1, WP_050916836.1, HD M8435746.1, HDL8238855.1, WP_019079472.1, E KN3341278.1, WP_050163370.1, HDL7317648.1, HDM8081034.1, WP_050870897.1, EKN3737775.1 , HDL7467110.1, HDL7471348.1, WP_046694975. 1, WP_301211860.1, HDL7726911.1, WP_268215747.1, EKN5089609.1, WP_057620566.1, EKN63602 58.1, WP_019083154.1, EKN5159302.1, WP_050131624.1, ELI8015017.1, EKN4802793.1, EKN6165952.1, ELI8293390.1, ELI8097018.1, WP_05015 7801.1, EKN4708697.1, EKN5102380.1, EKN5913011.1, WP_050164534.1, WP_242365000.1, HDL6759266.1, WP_050327956.1, EKN3969341.1, HDL8 127360.1, EKN3724092.1, WP_050334096.1, HDL 7761807.1、HDL6872781.1、EKN3573207.1、EKN3562735.1、EKN5949803.1、HDL6624424.1、WP_21 9649234.1、EKN3957447.1、EKN3611203.1、EKN5162868.1、WP_172654801.1、HDL6886351.1、WP_263797395.1、EKN3989601.1、HDL7855057.1、HDW8038128.1、EKN3870380.1、WP_057631219.1、EK N5074149.1、WP_219650267.1、HDL6522072.1、E KN4058214.1、EKN4068486.1、HDL6961480.1、WP_075338710.1、EKN5112876.1、HDL8368473.1、H DL7423034.1、HDM8447363.1、EKN6363360.1、EKN3564743.1、HDL7684594.1、HDL7735658.1、HDL7339639.1、HDL7966642.1、CNH41000.1、HDL7909665.1、WP_219648645.1、WP_219643575.1、CQD54886.1、CNG18579.1、EKN4119565.1、WP_145592 082.1、WP_038639531.1、AJJ34365.1、WP_050114520.1、WP_271298628.1、WP_004390824.1、WP_05 0095452.1、WP_145562021.1、WP_159678334.1、WP_309477958.1、WP_145510697.1、WP_219646505 .1、WP_145556850.1、WP_145537515.1、WP_21965 5358.1、CNG40075.1、EKN3837153.1、EKN3315583.1、WP_301178346.1、HDL8028676.1、HDL80520 82.1, HDL7585724.1, HDL8104119.1, WP_005164 Includes amino acid sequences such as 820.1, WP_050127938.1, WP_271306546.1, CBX71227.1, KGA73448.1, HDL8279144.1, HDL8024634.1, WP_115240285.1, WP_254461835.1, HDL8320198.1, WP_050124432.1, WP_050326508.1, HDL8083732.1, EKN3713082.1, HDL8750155.1, CNK45388.1, WP_221864008.1, SUP62773.1, etc. The first monomer may be expressed from a nucleic acid sequence or variant thereof that codes for a protein. For example, the first monomer may be a protein containing the amino acid sequence of SEQ ID NO: 7, a protein containing the amino acid sequence of SEQ ID NO: 24, a protein containing the amino acid sequence of SEQ ID NO: 1, a protein containing the amino acid sequence of SEQ ID NO: 5, a protein containing the amino acid sequence of SEQ ID NO: 6, a protein containing the amino acid sequence of SEQ ID NO: 8, a protein containing the amino acid sequence of SEQ ID NO: 9, a protein containing the amino acid sequence of SEQ ID NO: 10, a protein containing the amino acid sequence of SEQ ID NO: 11, a protein containing the amino acid sequence of SEQ ID NO: 12, a protein containing the amino acid sequence of SEQ ID NO: 13 It may be expressed from nucleic acid sequences encoding a protein, a protein containing the amino acid sequence of SEQ ID NO: 14, a protein containing the amino acid sequence of SEQ ID NO: 15, a protein containing the amino acid sequence of SEQ ID NO: 16, a protein containing the amino acid sequence of SEQ ID NO: 17, a protein containing the amino acid sequence of SEQ ID NO: 19, a protein containing the amino acid sequence of SEQ ID NO: 20, a protein containing the amino acid sequence of SEQ ID NO: 21, a protein containing the amino acid sequence of SEQ ID NO: 22, a protein containing the amino acid sequence of SEQ ID NO: 23, a protein containing the amino acid sequence of SEQ ID NO: 30, a protein containing the amino acid sequence of SEQ ID NO: 31, a protein containing the amino acid sequence of SEQ ID NO: 32, a protein containing the amino acid sequence of SEQ ID NO: 33, or a protein containing the amino acid sequence of SEQ ID NO: 34. Furthermore, as described above, the nucleic acid sequence encoding the protein may be a codon optimized for expression in a suitable host, such as Escherichia coli, or the chromosomal DNA encoding the wild-type first monomer extracted from a YaxAB nanopore-producing organism, such as Yersinia enterocolitic, may be extracted from a nanopore-producing organism, such as Yersinia enterocolitic. The extracted or synthesized nucleic acid sequence may be amplified using PCR with specific primers, and mutants may be produced by inducing site-directed mutagenesis using the amplified sequence. Suitable methods for site-directed mutagenesis are known in the relevant art, and for example, a method using polymerase chain reaction may be used.The variant of the nucleic acid sequence may be a nucleic acid sequence that exhibits at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology, respectively, to the nucleic acid encoding the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34, but is not limited thereto.

[0037] Furthermore, the first monomer is one of the amino acid sequences of SEQ ID NO. 7, SEQ ID NO. 24, or SEQ ID NO. 34, or NCBI accession NO. WP_00516 9901.1 (Sequence ID 3), WP_011816273.1 (Sequence ID 35), HDL 8508204.1, HDL8476175.1, HDL7646675.1, PNM17258.1, WP_050336257.1, HDL8287337.1, HDL78 22654.1, HDL7748605.1, HDL6737510.1, HEB4799346.1, WP_221871123.1, EKN3734298.1, EKN35 69010.1, HDL7089514.1, WP_050160085.1, WP_221876226.1, EKN4747040.1, WP_050916836.1, HD M8435746.1, HDL8238855.1, WP_019079472.1, E KN3341278.1, WP_050163370.1, HDL7317648.1, HDM8081034.1, WP_050870897.1, EKN3737775.1 、HDL7467110.1、HDL7471348.1、WP_046694975. 1、WP_301211860.1、HDL7726911.1、WP_268215747.1、EKN5089609.1、WP_057620566.1、EKN63602 58.1、WP_019083154.1、EKN5159302.1、WP_050131624.1、ELI8015017.1 、EKN4802793.1、EKN6165952.1、ELI8293390.1、ELI8097018.1、WP_05015 7801.1、EKN4708697.1、EKN5102380.1、EKN5913011.1、WP_050164534.1 、WP_242365000.1、HDL6759266.1、WP_050327956.1、EKN3969341.1、HDL8 127360.1, EKN3724092.1, WP_050334096.1, HDL 7761807.1、HDL6872781.1、EKN3573207.1、EKN3562735.1、EKN5949803.1、HDL6624424.1、WP_21 9649234.1、EKN3957447.1、EKN3611203.1、EKN5162868.1、WP_172654801.1、HDL6886351.1、WP_ 263797395.1、EKN3989601.1、HDL7855057.1、HDW8038128.1、EKN3870380.1、WP_057631219.1、EK N5074149.1、WP_219650267.1、HDL6522072.1、E KN4058214.1、EKN4068486.1、HDL6961480.1、WP_075338710.1、EKN5112876.1、HDL8368473.1、H DL7423034.1, HDM8447363.1, EKN6363360.1, EKN3564743.1, HDL7684594.1, HDL7735658.1, HDL7339639.1, HDL7966642 .1, CNH41000.1, HDL7909665.1, WP_219648645.1, WP_219643575.1, CQD54886.1, CNG18579.1, EKN4119565.1, WP_145592 082.1, WP_038639531.1, AJJ34365.1, WP_050114520.1, WP_271298628.1, WP_004390824.1, WP_05 0095452.1, WP_145562021.1, WP_159678334.1, WP_309477958.1, WP_145510697.1, WP_219646505 .1, WP_145556850.1, WP_145537515.1, WP_21965 5358.1, CNG40075.1, EKN3837153.1, EKN3315583.1, WP_301178346.1, HDL8028676.1, HDL80520 82.1, HDL7585724.1, HDL8104119.1, WP_005164 Includes amino acid sequences such as 820.1, WP_050127938.1, WP_271306546.1, CBX71227.1, KGA73448.1, HDL8279144.1, HDL8024634.1, WP_115240285.1, WP_254461835.1, HDL8320198.1, WP_050124432.1, WP_050326508.1, HDL8083732.1, EKN3713082.1, HDL8750155.1, CNK45388.1, WP_221864008.1, SUP62773.1, etc. The nucleic acid sequence encoding the protein may be expressed from a host cell into which the nucleic acid sequence has been introduced. For example, the first monomer may be expressed from a vector containing a nucleic acid sequence encoding a protein including the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34, and from a host cell containing the vector. The vector may incorporate the nucleic acid sequence encoding the protein including the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34 into a recombinant replicatable vector, such as a cloning vector or an expression vector. The nucleic acid sequence encoding the protein including the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34 may be operably linked to a promoter contained within the vector. The nucleic acid sequence can be replicated and expressed in a host cell using the vector. Therefore, the first monomer can be obtained by introducing the nucleic acid sequence into a replicatable vector, introducing such a vector into a compatible host cell, and growing the host cell under conditions that induce replication of the vector, and the vector can be recovered from the host cell. Host cells known in the relevant art may be used as host cells suitable for cloning or expressing the nucleic acid sequence.

[0038] On the other hand, the second monomer is the amino acid sequence of SEQ ID NO. 18 or SEQ ID NO. 25, or NCBI accession NO. WP_011816274.1 (SEQ ID NO. 4), WP_050336255.1 (Sequence ID 36), WP_005169898.1, PNM17259.1, WP_050137918.1, HDL7748604.1, HDL6 508072.1, WP_050137583.1, WP_050322893.1, HDL7822655.1, HDL7646676.1, WP_050321988.1, W P_221871120.1、HDL6961479.1、WP_050298175.1、CNG06681.1、EKN5919601.1、EKN5074150.1、WP_075338711.1、HDL7057906.1、WP_219656641.1、WP_050157802.1、ELI8015018.1、EKN4068485.1 、HDL7533782.1、WP_057631218.1、WP_050916837.1、EKN6259816.1、EKN3396085.1、HDL7423033.1、EKN4822249.1、WP_050880005.1、WP_050875182.1、HDL7335482.1、WP_050163371.1、EKN37342 97.1、HDL6886350.1、HEB4799167.1、WP_263700 684.1、EKN3341279.1、HDL7478311.1、EKN3957448.1、WP_050327958.1、HDL7339638.1、HDL6946 689.1、WP_019079471.1、HDV7159892.1、HDL746 7111.1、WP_172654802.1、WP_050870895.1、EKN3469507.1、WP_019083153.1、WP_076706633.1、HDW8038129.1、CQG99659.1、WP_050945964.1、HDL 6872782.1、WP_046694976.1、WP_263699488.1、WP_263697703.1、EKN3724091.1、HDL7089513.1、 CQH55963.1、HDL7666636.1、EKN5102379.1、WP_ 046050899.1、HDL6959493.1、EKN5159301.1、HDL8096476.1、EKN3683754.1、EKN3973834.1、WP_ 219650266.1, EKN6365751.1, HDL8258876.1, EKN4119566.1, HDM8089399.1, WP_263696663.1, E KN6178815.1, ELI8097017.1, HDL7909664.1, WP_219643580.1, WP_050149278.1, CNH43347.1, HDL8000813.1, WP_020283191.1, HDL8788876.1, H DL7394360.1, HDL7984656.1, WP_005164817.1, HDL8028561.1, CBY27149.1, HDL8032249.1, HDL8024436.1, HDL8004719.1, HDL6519422.1, HDL8048091.1, WP_016266185 .1, CCV61752.1, WP_049526748.1, WP_145535945.1, WP_050114521.1, WP_309477957.1, WP_289816262.1, WP_050291733. 1, WP_145589360.1, WP_038633022.1, WP_265526 189.1, WP_145537514.1, WP_050095454.1, WP_05 7634251.1, WP_050289682.1, WP_145531133.1, WP_145586506.1, WP_100286507.1, WP_219655343 .1, WP_049557371.1, WP_087795913.1, WP_14551 0696.1, WP_271304184.1, WP_004390823.1, WP_1 Nucleic acid codes encoding proteins, including amino acid sequences such as 45556851.1, WP_050120114.1, WP_219648204.1, and WP_159678331.1. The second monomer may be expressed from the sequence or a variant thereof. The second monomer may be expressed from a nucleic acid sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 2, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29. Furthermore, the nucleic acid sequence encoding the protein as described above may be a codon optimized for expression in a suitable host, such as E. coli, or the chromosomal DNA encoding the wild-type second monomer extracted from a YaxAB nanopore-producing organism, such as Yersinia enterocolitic, may be extracted from a nanopore-producing organism, such as Yersinia enterocolitic. The extracted or synthesized nucleic acid sequence may be amplified using PCR with specific primers, and the amplified sequence may be used to induce site-directed mutagenesis to produce mutants. Suitable methods for site-directed mutagenesis are known in the relevant art, and for example, a method using polymerase chain reaction may be used. The variant of the nucleic acid sequence may be a nucleic acid sequence that exhibits at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology, respectively, to the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25, but is not limited thereto. It includes any nucleic acid sequence that encodes a protein containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25. Furthermore, the second monomer may be expressed using a vector and a host cell containing the vector, as described above in the description of the first monomer.

[0039] Furthermore, the second monomer is the amino acid sequence of SEQ ID NO. 18 or SEQ ID NO. 25, or NCBI accession NO. WP_011816274.1 (SEQ ID NO. 4), WP_050336255.1 (Sequence ID 36), WP_005169898.1, PNM17259.1, WP_050137918.1, HDL7748604.1, HDL6 508072.1, WP_050137583.1, WP_050322893.1, HDL7822655.1, HDL7646676.1, WP_050321988.1, W P_221871120.1、HDL6961479.1、WP_050298175.1、CNG06681.1、EKN5919601.1、EKN5074150.1、WP_075338711.1、HDL7057906.1、WP_219656641.1、WP_050157802.1、ELI8015018.1、EKN4068485.1 、HDL7533782.1、WP_057631218.1、WP_050916837.1、EKN6259816.1、EKN3396085.1、HDL7423033.1、EKN4822249.1、WP_050880005.1、WP_050875182.1、HDL7335482.1、WP_050163371.1、EKN37342 97.1、HDL6886350.1、HEB4799167.1、WP_263700 684.1、EKN3341279.1、HDL7478311.1、EKN3957448.1、WP_050327958.1、HDL7339638.1、HDL6946 689.1、WP_019079471.1、HDV7159892.1、HDL746 7111.1、WP_172654802.1、WP_050870895.1、EKN3469507.1、WP_019083153.1、WP_076706633.1、HDW8038129.1、CQG99659.1、WP_050945964.1、HDL 6872782.1、WP_046694976.1、WP_263699488.1、WP_263697703.1、EKN3724091.1、HDL7089513.1、 CQH55963.1、HDL7666636.1、EKN5102379.1、WP_ 046050899.1、HDL6959493.1、EKN5159301.1、HDL8096476.1、EKN3683754.1、EKN3973834.1、WP_ 219650266.1, EKN6365751.1, HDL8258876.1, EKN4119566.1, HDM8089399.1, WP_263696663.1, E KN6178815.1, ELI8097017.1, HDL7909664.1, WP_219643580.1, WP_050149278.1, CNH43347.1, HDL8000813.1, WP_020283191.1, HDL8788876.1, H DL7394360.1, HDL7984656.1, WP_005164817.1, HDL8028561.1, CBY27149.1, HDL8032249.1, HDL8024436.1, HDL8004719.1, HDL6519422.1, HDL8048091.1, WP_016266185.1, CCV61752.1, WP_049 526748.1, WP_145535945.1, WP_050114521.1, WP_309477957.1, WP_289816262.1, WP_050291733. 1, WP_145589360.1, WP_038633022.1, WP_265526 189.1, WP_145537514.1, WP_050095454.1, WP_05 7634251.1, WP_050289682.1, WP_145531133.1, WP_145586506.1, WP_100286507.1, WP_219655343 .1, WP_049557371.1, WP_087795913.1, WP_14551 0696.1, WP_271304184.1, WP_004390823.1, WP_1 Nucleic acid codes encoding proteins, including amino acid sequences such as 45556851.1, WP_050120114.1, WP_219648204.1, and WP_159678331.1. The sequence may be expressed from a host cell into which it has been introduced. For example, the first monomer may be expressed from a vector containing a nucleic acid sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25, and from a host cell containing the vector. The vector may incorporate the nucleic acid sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25 into a recombinant replicabeast vector, such as a cloning vector or an expression vector. The nucleic acid sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25 may be operably linked to a promoter contained within the vector. The vector can be used to replicate and express the nucleic acid sequence in a host cell. Thus, the second monomer can be obtained by introducing the nucleic acid sequence into a replicabeast vector, introducing such a vector into a compatible host cell, and growing the host cell under conditions that induce replication of the vector, and the vector can be recovered from the host cell. Host cells known in the relevant art may be used as host cells suitable for cloning or expressing the nucleic acid sequence.

[0040] On the other hand, the first monomer and the second monomer may combine with each other to form a heterodimer. The heterodimer may then be used as a YaxAB nanopore subunit, and these subunits may come together to form a YaxAB nanopore. Therefore, the subunit is a protomer that constitutes the YaxAB nanopore of the present invention as described above.

[0041] Furthermore, the nanopore-forming ability of the first monomer and the second monomer can be improved by adjusting their amino acid sequences. In the present invention, the term "nanopore-forming ability" refers to the ability of subunits containing the first monomer and the second monomer to be repeatedly linked to one another, forming nanometers (nm, 10⁻¹⁰⁻¹ -9This involves the formation of pores of a certain size, which means oligomerization of the subunits. Therefore, excellent nanopore-forming ability means that the subunits stably form pores, or that nanopores can be formed even when the subunits are present at low concentrations.

[0042] In a specific embodiment of the present invention, the wild-type YaxAB nanopore of the present invention does not exhibit oligomer bands under low-concentration protein conditions, whereas the modified YaxAB_dN nanopore, composed of a YaxAB subunit consisting of the first monomer of SEQ ID NO: 1 (YaxA_(45-410)) and the second monomer of SEQ ID NO: 2 (YaxB_(12-343)), exhibits low-concentration We confirmed that oligomeric bands clearly appeared even under these protein conditions, and that nanopores were formed more stably compared to wild-type YaxAB (see Figure 30).

[0043] Furthermore, in a specific embodiment of the present invention, in order to confirm the minimum amino acid sequence capable of forming the YaxAB nanopore of the present invention, various sequences were prepared and their oligomerization tendencies were analyzed. As a result, it was found that a nanopore containing a first monomer comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 24 and a second monomer comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25 was formed. In the case of the asabuunit, it was confirmed that it stably forms a YaxAB oligomer, and that the oligomer can successfully realize the nanopore-containing membrane of the present invention, thereby enabling the successful capture of the sample protein (Bcl-xL) using the nanopore system (see Figures 35 and 36).

[0044] In another specific embodiment of the present invention, it was confirmed that a nanopore subunit comprising a first monomer consisting of at least one of the amino acid sequences composed of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, and a second monomer containing the amino acid sequence of SEQ ID NO: 2, stably forms a YaxAB oligomer (see Figure 32).

[0045] The YaxAB nanopore of the present invention described above may be formed by assembling two or more subunits, for example, three or more, five or more, or seven or more subunits, specifically four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more, specifically four to twenty subunits. Depending on the number of subunits, the diameter of the lumen of the YaxAB nanopore can be changed, specifically, the larger the number of subunits, the larger the diameter of the lumen of the YaxAB nanopore of the present invention can be.

[0046] The YaxAB nanopore of the present invention described above allows for easy adjustment of the number of subunits constituting the nanopore by adjusting the amino acid sequence lengths of the first and second monomers. In other words, by changing the amino acid sequences of the first and second monomers, the number of subunits forming the nanopore can also be changed. In a specific embodiment of the present invention, a nanopore can be formed using wild-type YaxAB subunits and modified YaxAB_dN subunits. We confirmed that there are differences in the types of gradient blue native polyacrylamide gel electrophoresis bands that appear when pores are formed, and that the number of subunits constituting the nanopores can be adjusted by changing the amino acid sequence of the subunits (see Figure 30). As a result, we were able to form novel YaxAB-C8 pores that could not be formed with wild-type YaxAB.

[0047] Furthermore, in a specific embodiment of the present invention, the modified YaxAB_dN subunit The results of electrophoresis analysis of the gradient blue native polyacrylamide gel that appeared when nanopores were formed revealed YaxAB-C8, YaxAB-C9, and YaxAB-C 10 The bands that correspond to this (C8, C9, and C 10 Bands are the main type of band that occurs, but in addition to these, C10 bands (C located above the band 11 、C 12 、C 13 、C 14 、and C 15 bands) also appeared in a well-separated state, and it was confirmed that the number of subunits constituting the nanopore could be adjusted by varying the amino acid sequences of the subunits (see Figure 8). As a result, novel nanopores of various sizes that could not be formed with the wild-type YaxAB sequence were able to be formed. Therefore, the modified YaxAB_dN nano pores of the present invention have a significant advantage over conventional biological nanopores with a fixed pore size in that various sizes of nanopores can be secured by a single purification and nanopores having a desired size can be selectively secured.

[0048] In addition, the first monomer and the second monomer can enhance the purification efficiency of the first monomer and the second monomer by adjusting the amino acid sequence length. In particular, when the first monomer consists of the amino acid sequence of SEQ ID NO: 1, the protein purification efficiency can be 2-fold or more, 3-fold or more, or 4-fold or more higher than that of the wild-type YaxA protein. Also, when the second monomer consists of the amino acid sequence of SEQ ID NO: 2, the protein purification efficiency can be 1 .1-fold or more higher. The protein purification process may be performed by a method known as a normal protein purification method. When the purification efficiency of each monomer is high, nanopores can be formed more stably than with the wild-type YaxAB subunits.

[0049] In a specific embodiment of the present invention, it was confirmed that the first monomer having the amino acid sequence of SEQ ID NO: 1 of the present invention had a 4.7-fold higher purified protein yield compared to wild-type YaxA, and the second monomer having the amino acid sequence of SEQ ID NO: 2 had a 1.1-fold higher purified protein yield compared to wild-type YaxB, and it was confirmed that the purification efficiency was excellent under the same protein purification conditions (see Figure 29).

[0050] 2. Nanopore-containing membrane Another aspect of the present invention provides a nanopore-containing membrane comprising a membrane layer and YaxAB nanopores inserted into the membrane layer.

[0051] The membrane serves as a support capable of fixing the nanopores, and is not limited to any membrane that can divide the space or chamber containing the nanopores into two compartments. The membrane serves to block the passage of fluids or substances contained in fluids without additional means. The membrane layer may contain phospholipids and amphiphilic substances.

[0052] The phospholipids, as amphiphilic molecules containing at least one phosphorus group, serve as support structures constituting the membrane. The phospholipid membrane refers to a structure in which the hydrophobic heads of the phospholipids are aligned in one direction and the hydrophilic tails are aligned in the opposite direction. The phospholipids are 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (1,2-dipalmitoyl-s n-glycero-3-phosphorylethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dihexa 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (D MPC), 1,2-Dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG), 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG),2-Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (D, It may also be OPE, 1,2-Dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and mycolic acid, etc.

[0053] The aforementioned amphiphilic substance is a substance that has both water-soluble and lipid-soluble properties, and refers to a substance that maintains a constant fluidity of the membrane containing the phospholipid and stabilizes the membrane. When the amphiphilic substance is included, the stability of the membrane can be increased, the number of nanopores formed can be affected, and the lifespan of the nanopores can be adjusted by changing the nanopore disjoining force through an effect on the elasticity of the membrane.

[0054] The aforementioned amphiphilic substances include, for example, campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), and 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine. 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), 1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), 10,12-pentacosadiic acid (10,12-Pentacosadiic acid) This may include, but is not limited to, amphiphilic substances that increase the stability of the membrane, such as acid (PCDA), 10,12-tricosadiynoic acid (TCDA), 5,7-hexadecadiynoic acid (HDDA), and 9,12-octadecadiynoic acid (ODDA).

[0055] The membrane may contain phospholipids and amphiphilic substances in proportions of 50-95 mol% and 5-50 mol%, respectively, or 55-95 mol% and 5-45 mol%, or 60-95 mol% and 5-40 mol%, respectively. When the proportions are within these ranges, the elasticity of the lipid membrane can be reduced as the proportion of amphiphilic substances in the membrane increases, thereby reducing the disjoining force for nanopore formation and increasing the nanopore lifespan, which in turn increases the nanopore formation frequency. If the proportions of phospholipids and amphiphilic substances in the membrane deviate from the above proportions, the YaxAB nanopores of the present invention cannot be stably inserted into the membrane, the membrane may rupture, or a constant electrical conductivity cannot be measured when a voltage is applied, and no ionic current flows according to Ohm's law.

[0056] In the present invention, the YaxAB nanopore is the same as that described above in the section "YaxAB Nanopore Subunit and YaxAB Nanopore Containing the Same," and therefore, the above description is used by reference and will not be repeated.

[0057] The YaxAB nanopores may be inserted into the membrane by known methods. For example, the nanopores may be inserted into the membrane by a change in ionic current. In the nanopore-containing membrane, the stability of the YaxAB nanopores of the present invention is improved. The YaxAB nanopores of the present invention are efficiently inserted into the membrane without rupturing, the nanopore formation frequency is high, and the number of stably inserted nanopores can be increased. Furthermore, when using the nanopore-containing membrane, even when a voltage is applied, a stable electrical conductivity value and current flow according to Ohm's law can be observed through the YaxAB nanopores.

[0058] In a specific embodiment of the present invention, it was confirmed that the YaxAB nanopore of the present invention is stably inserted into a DPhPC-sterol composite membrane compared to an artificial membrane composed solely of DPhPC (see Figure 6).

[0059] 3. Nanopore System Another aspect of the present invention provides a nanopore system using the YaxAB nanopore. The nanopore system of the present invention described above comprises a chamber and a YaxAB nanopore-containing membrane.

[0060] The chamber refers to a structure having an internal space capable of containing a certain volume of fluid. The space within the chamber is a fluid-filled compartment, and the fluid may be an electrolyte solution. The chamber may also include electrodes that generate an electrical potential difference across the membrane to facilitate the flow of electrolytes through nanopores between chambers. The internal space of the chamber is divided into two compartments by the nanopore-containing membrane, for example, a first and second compartment, or a cis and a trans compartment. When a sample is added to the compartment facing the first opening, the first opening of the YaxAB nanopore is a cis opening, and the second opening is a trans opening. The YaxAB nanopore and the nanopore-containing membrane described above will be described by reference to those described above and will not be repeated.

[0061] In this invention, the term "nanopore system" refers to a novel system that can be used as a sensor system or the like because it can detect or analyze changes in physicochemical properties, including changes in the fine structure and dynamics of biomolecules at the single-molecule level. In a nanopore system, the nanopore is generally inserted into an insulating film and acts as the sole conduit that allows ionic current to flow between two compartments separated by the insulating film. The nanopore system operates by detecting minute fluctuations in the ionic current when a charged biomolecule passes through the nanopore or is trapped in the lumen of the nanopore in the presence of an external electric field, and by determining the length, size, structure, dynamics, charge, and morphology of the biomolecule.

[0062] The nanopore system may contain a liquid medium, which may be an aqueous solution containing a salt. An ionic current can flow through the nanopore as the salt passes between two compartments separated by an insulating film. When the salt is an ion, it may be a metal salt, a halide salt, such as an alkali metal chloride salt. Specifically, in the salt, the cation is NH 4+ , K + na + Li + Mg 2+ Ca 2+ Gdm + It may also be, and the anion is F - SO4 2- HPO4 2- C2H3O 2- Cl - , Br - NO 3- , 3- , I - , 4- SCN - It could be something like this, for example, The salt may be sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl), calcium chloride (CaCl2), cesium chloride (CsCl), guanidine chloride (GdmCl), potassium ferrocyanide, potassium ferricyanide, etc. The salt may also be an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. However, it is not limited to these, and any electrically charged salt may be included without limitation.

[0063] Furthermore, the concentration of the salt may be 10 mM to 5 M, 20 mM to 4.8 M, 30 mM to 4.5 M, 40 mM to 4.2 M, or 50 mM to 4 M. Also, the concentrations of the salt present in the two compartments may be asymmetrical.

[0064] Furthermore, the two compartments within the nanopore system may include electrodes. By applying one or more electrical potentials to the electrodes, an ion flow can be generated across the nanopore-containing membrane from one compartment to the other.

[0065] The nanopore system of the present invention can operate by generating electroosmotic or electrophoretic forces, causing a sample to pass through or be captured through the pore, when a gradient due to a difference in salt concentration is formed between two compartments separated by the nanopore-containing membrane, or when a voltage is applied, or both. The nanopore system may operate by generating electroosmosis or electrophoresis, or by generating both electrophoresis and electroosmosis, or by a balance between electrophoresis and electroosmosis.

[0066] In the present invention, the term "electroosmosis" refers to the phenomenon in which, when electrodes are attached to both sides of a porous membrane, such as the nanopore-containing membrane, and a DC voltage is applied, a liquid moves toward one electrode through the nanopores contained in the membrane. In this invention, the term "electrophoresis" refers to the phenomenon in which molecules move in an electric field according to their charge and size.

[0067] In the aforementioned nanopore system, when the working electrode is located in the compartment where the cis opening of the YaxAB nanopore exists, the potential difference (voltage) between the two compartments is -200mV, -180mV, -160mV, -140mV, -120mV, -100mV, -90mV, -85mV, -80mV, -75mV, -70mV, -65mV, -60mV, -55mV, -50mV, -45mV, -40mV, -35mV, - 30mV, -25mV, -20mV, -15mV, -10mV, -5mV, 0mV, +5mV, +10mV, +15mV, +20mV, +25mV, +30mV, +35mV, +40mV, +45mV, +50mV, +55mV, +60mV, +90mV, +100mV, +120mV, +130mV, +140mV, +200mV, +300mV, +400mV~500mV are also acceptable. In contrast, when the working electrode is located in the compartment where the transform opening of the YaxAB nanopore exists, the potential difference (voltage) between the two compartments is +200mV, +180mV, +160mV, +140mV, +120mV, +100mV, +90mV, +85mV, +80mV, +75mV, +70mV, +65mV, +60mV, +55mV, +50mV, +45mV, +40mV, +35mV The applied potential may be +30mV, +25mV, +20mV, +15mV, +10mV, +5mV, 0mV, -5mV, -10mV, -15mV, -20mV, -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -90mV, -100mV, -120mV, -130mV, -140mV, -200mV, -300mV, -400mV to -500mV. The applied potential is the working electrode. This refers to the potential of the electrode, and the cis aperture of the YaxAB nanopore. When a positive potential is applied to the section containing the part, a stable open-circuit fundamental current can be observed. That is, in the nanopore system, when a working electrode is present at the cis opening of the pore in the two sections separated by the nanopore-containing membrane, and a voltage in the range of -100mV to +200mV or -200mV to +500mV is applied to the YaxAB nanopore, when a working electrode is present at the trans opening of the pore, and a voltage in the range of +100mV to -200mV or +200mV to -500mV is applied, the YaxAB nanopore is stably maintained in an open state, and charged ions can move across the nanopore between the two sections, allowing an ionic current to flow.

[0068] The analyte is translocated through the nanopores of the nanopore system of the present invention, trapped in the lumen, or operated in such a way that the nanopores are opened and closed (gating). For example, when a voltage is applied to the nanopore system to form an electric field, the analyte can be translocated, trapped, or gated. In this case, minute fluctuations caused by the interruption of the flow of ionic current through the nanopore by the analyte appear as a current signal, which can be used to detect the analyte or to analyze its properties. The properties of the analyte may be electrical, chemical, or physical properties of the analyte, and specifically, at least one selected from the group consisting of the surface charge of the analyte, the isoelectric point of the analyte, the stability of the analyte, isomers, the length, size, molecular weight, structure, dynamics, intensity and orientation of the dipole moment, orientation, flexibility, conformational heterogeneity, shape, quantity, and changes therein.

[0069] The YaxAB nanopore has a funnel shape with a wide entrance (first opening), allowing it to detect samples of various sizes and molecular weights. The sample may be a peptide, protein, lipid, carbohydrate, inorganic substance, low molecular weight compound, nucleic acid, or inorganic particle (particle) composed of nanomaterials. For example, the sample may be a receptor, antibody, or aptamer. If the sample is a protein, its molecular weight may be 1-1200 kDa, 1-1000 kDa, or 1-800 kDa. In a specific embodiment of the present invention, it was confirmed that the nanopore system of the present invention can detect proteins with molecular weights of 1-670 kDa (see Figures 10, 18, and 19). The nanopore system allows the sample to be trapped in the lumen of the nanopore regardless of its surface charge, because the lumen of the YaxAB nanopore has an electronegative surface charge, and this property can induce a greater electroosmotic force than electrophoretic force.

[0070] Furthermore, because the YaxAB nanopore has a funnel shape, the ion current density increases as you move towards the narrower part of the nanopore's lumen, maximizing the degree of ion current blockage by the sample. At the same time, the electroosmotic and electrophoretic forces are in equilibrium, causing the sample to remain within the nanopore's lumen while exhibiting its own unique movement, resulting in a signal with higher resolution compared to conventional nanopore systems.

[0071] In a specific embodiment of the present invention, when a positive voltage is applied to the nanopore system of the present invention by molecular dynamics simulation, it was confirmed that cations and water molecules predominantly flow from the first opening to the second opening of the YaxAB nanopore, and that electroosmosis occurs (see Figure 4). Furthermore, the YaxAB nanopore can detect proteins with various surface charges having isoelectric points of 2-12, 3-11, and 4-10, not only by electrophoresis but also by electroosmosis. In a specific embodiment of the present invention, proteins with various surface charges (neutral, negative, positive) in the isoelectric point range of 4.1-9.6 can also be detected within the lumen. By confirming that the protein is captured, we confirmed that it can be detected regardless of its overall charge (see Figures 10, 18, and 19).

[0072] In another specific embodiment of the present invention, the nanopore system of the present invention was observed to exhibit a greater change than the ion current interruption change caused by conventional nanopore systems, and to show distinctly different electrical signal patterns depending on the type of sample, confirming that it has excellent resolution (see Figures 11 and 14).

[0073] Furthermore, in another specific embodiment of the present invention, as shown in Figure 10, the detection range of the YaxAB-C8 nanopore system of the present invention is 1-77 kDa; YaxAB-C 11 The system is ~158kDa; and YaxAB-C 12 The system exhibited a detection range of ~669 kDa, confirming that it has a significantly wider sample detection range compared to the sample detection range of conventional nanopore systems: ~25 kDa (FraC nanopore); 25-42 kDa (ClyA nanopore); and ~64.5 kDa (PlyAB nanopore). In other words, the YaxAB nanopore system of the present invention not only allows for the creation of pores of various sizes in a single purification step, but also enables adjustment of the pore size. Therefore, it was confirmed that the YaxAB nanopore system of the present invention has a superior sample detection range compared to conventional nanopores.

[0074] 4. Utilization of Nanopore Systems Furthermore, yet another aspect of the present invention provides a method for single-molecule analysis of one or more samples, comprising the steps of: placing at least one sample (analyte) in one compartment of the nanopore system; and measuring changes in electrical signals in the two compartments before and after the placement of the samples.

[0075] The sample may be a peptide, protein, lipid, carbohydrate, inorganic substance, small molecule compound, nucleic acid, or inorganic particle (particle) composed of nanomaterials. For example, the sample may be a receptor, antibody, or aptamer. More specifically, the sample may be a biomolecule, specifically a single compound such as nucleic acid, amino acid, or monosaccharide, or a polymer such as polynucleotide, peptide, protein, polysaccharide, carbohydrate, or lipid, or a molecule such as a natural or synthetic organic substance, inorganic substance, or a complex thereof. For example, the nucleic acid may be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA / RNA hybrid, or a combination thereof. The protein may be a fibrous protein whose structure has been modified using a surfactant such as SDS, and a peptide, but is not limited thereto.

[0076] The monomolecule analysis method for a single or multiple samples may include the step of placing at least one sample in one of the compartments separated by the nanopore-containing membrane, for example, the step of placing the sample in any one of the compartments, particularly the compartment to which the first opening of the nanopore faces. The processing of the sample may also be carried out in a situation in which the movement of the sample to other compartments via the nanopore is induced, for example, in a situation in which a gradient due to a difference in salt concentration is formed between the two compartments separated by the membrane, or a voltage is applied, or both.

[0077] The procedure also includes measuring changes in electrical signals in the two compartments before and after the placement of the sample. The aforementioned "electrical signal" refers to a signal generated when ions and / or charged low-molecular-weight substances pass from one compartment separated by a membrane to the other, for example, from the first compartment to the second compartment. Specifically, this includes the current pattern of the ion current and the open-pore current (I). o , I open ), magnitude of current drop (ΔI) , degree of current interruption, event duration, frequency of nanopore signal phases per unit time, current noise (I N ) and other such methods may be used, but are not limited thereto. The frequency per unit time of the nanopore signaling phase may include, but are not limited to, the event frequency and the inter-event interval. The basic current in the open state may mean the current in the nanopore-only state. The magnitude of the current drop (ΔI) may mean the magnitude by which the amount of electrolyte, e.g., ions and / or charged low-molecular-weight substances passing through decreases as a sample is trapped inside the nanopore or as the sample passes through the nanopore. The degree of current blockade is the basic current in the open pore state (ΔI). o The ratio of ΔI, which is the magnitude of the current drop due to the sample, to (ΔI / I) o) may also be. The event duration may be the duration of the event when the sample event appears as the magnitude of the current drop (ΔI). The current noise may include power spectral density (PSD) or standard deviation (SD) of ion current, the power spectral density is the noise power generated by the flow of ion current shown on the frequency spectrum, and shows the power density distribution according to the signal frequency. The standard deviation is the standard deviation value of the current cutoff signal itself that is generated by the decrease in ion current. N ) represents the square root of the integral value of the power spectrum of the ion current, or the standard deviation of the ion current when the current decreases, and may represent a quantification of the electrical noise characteristics of the nanopore, and may include the current noise obtained by amplifying the difference in current noise by signal filtering in the low-oscillation section. Furthermore, the electrical signal is the current interruption (ΔI / I o ) and current noise (I N The analysis method for a single or multiple samples may include a density contour plot, a two-dimensional scatter plot, or a violin plot showing the sample in two dimensions. For example, the analysis may include the presence or absence of the sample, the type of sample, the structure of the sample, its dynamics and deformation, and the quantity of the sample.

[0078] In one embodiment of the present invention, when using the nanopore system, it was confirmed that clearly different ion current change patterns were observed for samples of various sizes and with various surface charges, enabling the analysis of single-molecule samples (see Figure 10).

[0079] In another embodiment of the present invention, it was confirmed that the nanopore system allows for the detection and analysis of intrinsically disordered proteins and peptide samples by passing EGF, a small monomolecule compound negatively charged at pH 7.5, or the p53TAD1 peptide, an intrinsically disordered protein (IDP) with a small molecular weight of 1.8 kDa, and the p53TAD protein, an intrinsically disordered protein with a molecular weight of 8.3 kDa, through the nanopore system using electrophoretic force (see Figures 26 and 27).

[0080] In yet another embodiment of the present invention, Lambda DNA, single-stranded nucleic acid (50 mer), and double-stranded nucleic acid (23 bp) were independently added to the nanopore system and their passage was observed. The results showed that an ion current blockage phenomenon was observed before and after the addition of each nucleic acid, confirming that the nanopore system of the present invention allows nucleic acids to pass through not only by electroosmosis but also by electrophoresis, thus enabling the detection and analysis of nucleic acids through the nanopore system (see Figure 28).

[0081] In another embodiment of the present invention, the nanopore system was used to perform protein profiling of Bcl-2 system proteins (Bcl-xL, Bcl-w, Bcl-2, Mcl-1) and drug identification based on the affinity of the target protein. As a result, Bcl We observed that the nanopore current cutoff signals for the -2 proteins Bcl-xL, Bcl-w, Bcl-2, and Mcl-1 all appeared in distinct, unique patterns. Even when equal amounts of Bcl-2 proteins were mixed, unique current cutoff signals originating from each protein were observed. Furthermore, when an equal amount of A1155463, a small molecule drug targeting Bcl-xL, was added, protein-specific current cutoff signals appeared for Bcl-w, Bcl-2, Mcl-1, and mTOR, while the unique current cutoff signal for the target protein Bcl-xL disappeared, and a new current cutoff signal appeared. This confirmed that profiling of Bcl-2 proteins is possible via the nanopore system, and that identification of drugs with selectivity for target proteins is also possible (see Figure 20).

[0082] The aforementioned analytical methods for single or multiple samples can be used to measure and analyze the presence, type, behavior, charge, size, structure, dynamics, orientation, flexibility, structural heterogeneity, or quantity of single or multiple samples. They can be used for biosensors of various samples such as proteins and nucleic acids, for detection and structural and dynamic analysis of samples at the single-molecule level, for biomolecular interaction analysis, and for sequence analysis of nucleic acids and proteins. Furthermore, the aforementioned analytical methods for single or multiple samples can be utilized in new drug screening, disease diagnosis, and proteomic analysis, including protein identification and post-translational modification. In addition, the aforementioned analytical methods for single or multiple samples can be used for biomolecule detection, identification, quantitative analysis, and screening by detecting characteristic electrical signals of target substances and performing fingerprinting analysis in the presence of multiple samples.

[0083] For example, if the sample is nucleic acid, when the nucleic acid passes through the nanopore, different ion current change patterns appear depending on the type of nucleotide. By analyzing these patterns, nucleic acids can be identified, which can then be used for nucleic acid sequence analysis. Furthermore, by analyzing these patterns, the nucleotides constituting the nucleic acid can be identified, enabling accurate and rapid analysis of the nucleic acid sequence. The nucleotides constituting the nucleic acids include a base, a sugar, and at least one phosphate group, and include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and deoxygenated nucleotides. It may also be adenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), teoccytymidine monophosphate (dTMP), teoccytymidine diphosphate (dTDP), teoccytymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), or deoxycytidine triphosphate (dCTP).

[0084] A further aspect of the present invention provides a method for analyzing the interaction between a sample and a ligand or screening a ligand for a sample, comprising the steps of: processing one or two compartments of a nanopore system with at least one sample (analyte) and at least one candidate ligand substance for the sample; and measuring changes in electrical signals in the two compartments before and after processing with the candidate substance.

[0085] Method for analyzing the interaction between the sample and the ligand, or for screening the ligand for the sample. The procedure includes the step of treating at least one sample with at least one ligand candidate substance for the sample in one or two compartments of the nanopore system.

[0086] The aforementioned sample means a target object for detecting, analyzing interactions with, or screening a ligand by the ligand interaction analysis or ligand screening method for the sample. The aforementioned sample may, but is not limited to, peptides, polypeptides, proteins, inorganic substances, small molecules, or nucleic acids. In particular, the term "small molecule" refers to organic compounds with a small molecular weight, for example, 1000 Da or less, in the fields of molecular biology and pharmacology. Small molecules often act as modulators or drugs in biological processes. -9 Having a small size of approximately m, in the field of pharmaceuticals, small molecules refer to molecules or drugs that bind to biomacromolecules such as proteins and nucleic acids and regulate the function of these biomacromolecules. Small molecule compounds have various biological functions as cellular signaling substances. Small molecule compounds may be of natural origin (secondary metabolites) or artificially synthesized. The nucleic acids may be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA / RNA hybrids, or combinations of the above nucleic acids.

[0087] The ligand candidate substance refers to a substance expected to interact with the sample. In particular, when the ligand interacts with the free sample, the oscillation, rotational motion, and translocation patterns of the sample when it is trapped in the lumen of the nanopore may be altered for physical or chemical reasons. For example, when the ligand interacts with the free sample to form a complex, the proportion of the sample present in the free state decreases. Therefore, not only is the electrical signal exhibited by the free sample significantly reduced, but the new electrical signal exhibited by the sample-ligand complex significantly increased, allowing for direct detection of the sample-ligand conjugate.

[0088] The present invention's method for analyzing the interaction between a sample and a ligand, or for screening ligands for a sample, can be used as a drug screening method. Specifically, when the sample is a drug target and the candidate ligand is a candidate drug, interaction analysis or screening between the drug target and the candidate drug is possible, thereby effectively screening candidate drug substances.

[0089] In one embodiment of the present invention, the nanopore system was used to analyze the interaction between a target protein and a peptide that binds to the target protein, thereby screening peptide drugs. Bcl-xL protein and three Bak-BH3 mutant peptides that bind to Bcl-xL protein (Bak_BH3_D84A, Bak_BH3_I85A, Bak_BH3_L78A) were reacted with wild-type Bak_BH3 peptide, and at this time, Observation and analysis of the current interruption signals derived from the Bcl-xL protein and the Bcl-xL+ peptide drug complex confirmed that the scatter plot results obtained by measuring and analyzing the Bcl-xL protein with four mixed peptides matched the scatter plot trends observed in individual experiments. This confirmed that the signal frequency of the Bcl-xL-peptide complex matched the order of binding affinity between the Bcl-xL protein and each peptide. Thus, the YaxAB nanopore of the present invention enables binding affinity-based screening for peptide drugs that bind to proteins, and can effectively select peptide drugs with high binding affinity to target proteins even in peptide mixed solutions, thus proving its usefulness as a drug screening platform (see Figure 19). The explanation of the electrical signals is the same as described above in the explanation of the method of analyzing the sample, so it will be used again without repetition.

[0090] Method for analyzing the interaction between the sample and the ligand, or for screening the ligand for the sample. This may include the step of simultaneously processing at least one sample and at least one ligand candidate substance for the sample in one or two compartments of the nanopore system.

[0091] The step of simultaneously processing at least one sample and at least one candidate ligand substance for the sample in one or two compartments of the nanopore system may involve first placing at least one sample in either one or two compartments, and then simultaneously processing multiple ligands for the sample in the same compartment, for example, two or more, four or more, eight or more, ten or more, twenty or more, fifty or more, or 100 or more.

[0092] The present invention provides a method for analyzing the interaction between a sample and a ligand, or for screening a ligand for a sample, which includes measuring changes in the electrical signal of the sample before and after processing with the candidate substance. By measuring the electrical signals shown by the nanopore vibrational motion phase, rotational motion phase, or nanopore passage phase of the sample when the sample is free and trapped in the lumen of a nanopore, it is possible to determine whether the sample has interacted with the candidate substance, for example, whether the sample and the candidate substance have bound to form a complex.

[0093] Furthermore, the method for analyzing the interaction between the sample and the ligand, or for screening ligands for the sample, may further include the step of determining the candidate substance to be a ligand that interacts with the sample if the change is significant. If the change is significant, this may include the appearance of a new signal that was not present before or after processing with the candidate ligand substance.

[0094] In one embodiment of the present invention, when the YaxAB nanopore of the present invention is used to treat Bak-BH3, a peptide that binds to Bcl-xL protein, or a small molecule compound that binds to protein (ABT-737), a clear change in the ion current pattern is observed compared to the negative control group (see Figure 11). It is also confirmed that a change in the ion current pattern is observed when treating Bak-BH3 peptide or a small molecule compound that is a weak binder with low binding affinity to Bcl-xL protein (see Figures 12-13).

[0095] In another embodiment of the present invention, using the YaxAB nanopore of the present invention, we measured the current cutoff signal generated when glucose / galactose binding protein (GBP), which undergoes an alloteric motion (transformation of its three-dimensional structure upon binding to glucose or galactose), is bound to galactose, a small molecule compound that targets GBP. The results showed that when galactose is added, the frequency of gating generated from the GBP-derived current cutoff signal increases significantly, confirming that the GBP-galactose interaction can be effectively analyzed depending on the presence or absence of galactose (see Figure 14).

[0096] In yet another embodiment of the present invention, when the target protein Bcl-xL is treated individually with a non-binding agent (LCL-161, GDC-0152) and a binding agent (ABT-737, A-1331852), the current noise (I) of the non-binding agent (LCL-161, GDC-0152) is measured using the YaxAB nanopore system. N The signal remains unchanged, and the current noise (I) from the binder (ABT-737, A-1331852) is not present. N We confirmed that only the signal (I) was specifically elevated. This allowed us to determine the current noise (I) of the YaxAB nanopore. N Signal analysis confirmed that the interaction of small molecule compounds that bind to the Bcl-xL protein can be specifically detected (see Figure 17a).

[0097] In yet another embodiment of the present invention, a small molecule compound (LCL-161, GDC-0152, ABT-737, A-133) that binds to the target protein Bcl-xL is used. The ratio of the combined effect with 1852) is controlled in real time to interrupt the current (ΔI / I o ) and current noise (I NWe confirmed that high-resolution single-molecule fingerprinting analysis of protein-drug interactions is possible using a 2D density contour plot, which shows the interaction in two dimensions. This confirmed that the signal of the Bcl-xL + small molecule compound complex changes depending on the type of small molecule compound that binds to Bcl-xL, and that various small molecule compounds that bind to the Bcl-xL protein can be clearly distinguished (see Figure 17b). This confirmed that the interaction of the Bcl-xL protein sample with various ligands can be analyzed.

[0098] In another specific embodiment of the present invention, when the Bcl-xL+Bak-BH3 complex was simultaneously treated with four low-molecular-weight compounds that bind to Bcl-xL (LCL-161, GDC-0152, ABT-737, and A-1331852), it was confirmed that the proportion of the Bcl-xL+ABT-737 complex gradually decreased over time, while the proportion of the Bcl-xL+A-1331852 complex steadily increased. This confirmed that the YaxAB nanopore system of the present invention can be used to simultaneously analyze the binding activity between a sample and multiple low-molecular-weight compounds in real time (see Figure 17c).

[0099] In yet another specific embodiment of the present invention, when a ligand (Bak-BH3) that binds to the target protein Bcl-xL is treated with a small molecule compound (ABT-737, A-1331852), the ratio of individual complexes of competitively binding ligands and small molecule compounds is controlled in real time by current interruption (ΔI / I). o )-current noise (I NThe 2D density contour plots and violin plots of the 2D density contour plots confirmed that analysis could be performed at the single-molecule fingerprinting level with high resolution. This confirmed that after adding ABT-737 to the Bcl-xL+Bak-BH3 complex, the signal of the Bcl-xL+Bak-BH3 complex decreased and the signal of the Bcl-xL+ABT-737 complex increased in real time. Similarly, after adding A-1331852 to the Bcl-xL+ABT-737 complex, the signal of the Bcl-xL+ABT-737 complex decreased and the signal of the Bcl-xL+A-1331852 complex increased in real time. This confirmed that ABT-737 and A-1331852 competitively bind to the same binding site as Bak-BH3 on the Bcl-xL protein (see Figure 18).

[0100] The molecular weight ratio of the sample to the ligand may be 1:1 or higher, 1:2 or higher, 1:5 or higher, 1:10 or higher, 1:20 or higher, 1:20 or higher, 1:50 or higher, 1:150 or higher, 1:170 or higher, 1:190 or higher, 1:250 or higher, 1:500 or higher, or 1:1000 or higher. In a specific embodiment of the present invention, it was confirmed that the nanopore system of the present invention can clearly distinguish the ion current patterns of the sample and the sample-ligand complex even when the molecular weight ratio of the sample to the ligand differs by 16 or 194 times, and therefore can sensitively analyze whether or not there is interaction between the sample and the ligand (see Figures 38 and 39).

[0101] The concentration ratio of the sample to the ligand may be 1:2 or higher, 1:4 or higher, 1:10 or higher, 1:50 or higher, 1:60 or higher, 1:80 or higher, 1:90 or higher, 1:100 or higher, 1:120 or higher, 1:150 or higher, 1:200 or higher, 1:300 or higher, 1:500 or higher, or 1:1000 or higher. In a specific embodiment of the present invention, it was confirmed that even with a large concentration difference, such as a ratio of 1:2 or 1:100 between the sample and the ligand that binds to the sample, the difference in ion current patterns was clearly distinguishable from when the sample was captured alone, and the presence or absence of interaction between the sample and the ligand could be sensitively analyzed (see Figures 40 and 41).

[0102] Furthermore, the method for analyzing the interaction between the sample and the ligand, or for screening ligands for the sample, may be a qualitative and / or quantitative analysis of the interaction between the sample and the ligand. For example, the presence or absence of ligand candidate substance binding to the sample, ligand The type and quantity of the substance may also be analyzed in real time. Depending on the change in the electrical signal, it can be seen that the degree and frequency of interaction between the sample and the ligand are stronger or higher. Furthermore, the degree and frequency of such protein-protein interactions can be expressed, for example, by the binding affinity (binding dissociation constant (K)). D It can be quantified using )).

[0103] In a specific embodiment of the present invention, current-noise-capacitance-dependent quantitative analysis is performed on the target protein Bcl-xL and the ligand Bak-BH3 peptide or the small molecule drugs ABT-737 and A-1331852 using the YaxAB nanopore, thereby determining the binding affinity (K) to Bcl-xL. D ) was calculated (see Figure 15).

[0104] The aforementioned method for analyzing the interaction between a sample and a ligand, or for screening ligands for a sample, can be used for studying protein-protein interactions, protein-ligand interactions, and drug screening. When multiple candidate substances are processed simultaneously, it is possible to analyze competitive binding or structure-activity relationships (SARs) between the sample and candidate ligands. Therefore, it can be used for drug screening of protein-protein interaction inhibitors or enhancers, small molecule compounds, and for discovering and designing new drugs through binding site mapping.

[0105] A further aspect of the present invention provides a method for analyzing or screening biomolecular interaction inhibitors or promoters, comprising the steps of: reacting a plurality of interactable biomolecules in one compartment of the nanopore system; treating one or two compartments with a candidate substance for interaction inhibitor or promoter; and measuring changes in electrical signals in the two compartments before and after treatment with the candidate substance.

[0106] The present invention provides a method for analyzing or screening interaction inhibitors or promoters, which first includes the step of treating a free biomolecule in one of two compartments of the nanopore system. In one embodiment of the present invention, the biomolecule may be placed in one of the compartments first and reacted, and then the candidate substance may be treated in the reactant or in the opposite compartment. In other words, the candidate substance may be treated after the free biomolecule has been placed in one of the compartments. Alternatively, the free biomolecule and the candidate substance may be treated simultaneously in one of the compartments. Furthermore, the treatment of the candidate substance may be carried out in a situation in which the biomolecule is induced to move to the other compartment via the YaxAB nanopore contained in the nanopore system, for example, in a situation in which a gradient due to a difference in salt concentration is formed between the two compartments separated by the separation membrane, or a voltage is applied, or both.

[0107] Furthermore, the biomolecules can be used without limitation as long as they can interact with each other, for example, by binding to form a complex. For example, they may be peptides, proteins, lipids, carbohydrates, small molecule compounds, nucleic acids, or inorganic particles (particles) made of nanomaterials. For example, the biomolecules may be receptors, antigens, antibodies, or aptamers.

[0108] The candidate ligand substance refers to any type of molecule expected to inhibit or promote the interaction between the biomolecules, such as a single compound such as nucleic acids, amino acids, or monosaccharides, or a polymer such as a polynucleotide, peptide, protein, or polysaccharide, or a molecule such as a natural or synthetic organic, inorganic, or complex thereof. For example, the candidate ligand substance may be a receptor, antigen, antibody, or aptamer.

[0109] Among the candidate ligands, those that inhibit the interaction between biomolecules, particularly those that competitively bind to a portion of the biomolecules and inhibit the interaction between biomolecules, are referred to as "inhibitors." These inhibitors may reduce the interaction between biomolecules through such competitive binding. In other words, the inhibitor can bind to a portion of the biomolecules, increasing the proportion of the biomolecules that exist in a free state without interacting with each other, and increasing the proportion of complexes in which the biomolecules and the inhibitor interact. As described above, an increase in the proportion of biomolecules that exist in a state without interacting with each other reduces the electrical signals caused by the interaction between biomolecules, and a new electrical signal pattern exhibited by complexes in which the biomolecules and the inhibitor interact can appear, allowing for the direct detection or analysis of inhibitors that inhibit the interaction between biomolecules.

[0110] Furthermore, among the candidate ligand substances, those that promote the interaction between biomolecules are referred to as "promoters," and these promoters may include molecular glue and proteolytic agents (PROTACs) that induce or increase the interaction between biomolecules. For example, the promoter can act as a molecular glue, binding to the biomolecules and inducing or increasing the interaction between them. As described above, an increase in the proportion of biomolecules interacting with each other increases the electrical signals resulting from these interactions, and a new electrical signal pattern can appear in the complex formed by the interaction of biomolecules and promoters, allowing for the direct detection or analysis of promoters that enhance the interaction between biomolecules.

[0111] Therefore, the method for analyzing or screening for the interaction inhibitor or promoter between biomolecules may further include the step of determining that the candidate substance is an inhibitor that inhibits the interaction between biomolecules or an interaction promoter that promotes the interaction if the change in the electrical signal is significant.

[0112] If the aforementioned change is significant, it may include the appearance of new signals that were not present before or after treatment with the candidate interaction inhibitor or enhancer, or an increase or decrease in electrical signals resulting from interactions between biomolecules. In particular, current interruption (ΔI / I o ) and current noise (I N This may include cases where the pattern appearing in the two-dimensional density contour plot changes.

[0113] The aforementioned methods for analyzing or screening inhibitors or enhancers of biomolecular interactions can be used for drug screening, such as for protein-protein interaction inhibitors or enhancers, and for discovering and designing new drugs through protein binding site analysis.

[0114] In a specific embodiment of the present invention, the YaxAB nanopore system was used to analyze the activity of a protein-protein inhibitor targeting the Bcl-xL+Bak-BH3 complex. When treated with a small molecule compound (ABT-737) that binds to the Bcl-xL protein, it was confirmed that the signal for the Bcl-xL+Bak-BH3 complex decreased and the signal for the Bcl-xL+ABT-737 complex increased, confirming that the binding activity of the inhibitor of interaction between the sample and ligand can be analyzed in real time (see Figure 16).

[0115] In another embodiment of the present invention, the YaxAB nanopore system is used to connect the BRD4-BD1 target protein with the acetylated peptide (H4_1-12 K5acK8ac). We attempted to analyze the activity of interaction inhibitors between BRD4-BD1 target protein and acetylated peptide (H4_1-12 K5acK8ac), and BRD4-BD1 When four small molecule compounds that bind to proteins (GSK778, Y06026, XMD8-92, PLX51107) are reacted, a current cutoff signal unique to the target protein-small molecule compound complex can be observed. The signals from the four target protein-small molecule compound complexes are significantly different from the signals from the target protein-acetylated peptide complex. We confirmed that differences exist even among protein-small molecule compound complexes, enabling us to analyze the presence or absence of activity of interaction inhibitors that inhibit target protein-acetylated peptide interactions, and further confirming that we can distinguish between small molecule compounds that bind to target proteins (see Figure 22).

[0116] In yet another embodiment of the present invention, the YaxAB nanopore system was used to analyze the promotion of protein-protein interactions by molecular adhesives. When mTOR protein, FKBP12 protein, and rapamycin, which acts as a molecular adhesive, were added together, a novel current-cutting signal appeared along with the current-cutting signals corresponding to the mTOR protein and FKBP12 protein. It was confirmed that this signal was not a binary complex formed by the binding between the mTOR or FKBP12 protein and the molecular adhesive, but rather a current-cutting signal from a ternary complex in which mTOR, FKBP12, and the molecular adhesive are all bound together. This confirmed that it is possible to distinguish between proteins and ternary complexes, and thus the promotion of protein-protein interactions by molecular adhesives can be analyzed (see Figure 21).

[0117] The aforementioned analysis may be a qualitative or quantitative analysis of the interaction inhibitor between the sample and the ligand. For example, it may involve determining the presence or absence of the interaction inhibitor, the amount of the inhibitor, whether or not there is competitive binding between the sample and the ligand, identifying the site where the inhibitor binds to the target substance (binding site mapping), and determining the binding affinity (binding / dissociation constant, K) between the sample and the inhibitor. D ) is also acceptable, but is not limited to this.

[0118] Furthermore, the analysis may be a qualitative or quantitative analysis of an interaction enhancer between at least one sample and at least one ligand. For example, it may be, but is not limited to, the presence or absence of the interaction enhancer, the amount of the enhancer, the presence or absence of a binary complex, or the presence or absence of a ternary complex.

[0119] A further aspect of the present invention provides a method for identifying and quantitatively analyzing samples in a sample, comprising the steps of: placing a sample in one compartment of the nanopore system; and comparing characteristic changes in electrical signals caused by multiple samples in the sample with a substance-specific electrical signal database.

[0120] Furthermore, yet another aspect of the present invention provides a method for providing information for the diagnosis of biomarker-related diseases, comprising the steps of: placing a sample in one compartment of the nanopore system; and measuring a characteristic change in electrical signal caused by a biomarker for a specific disease among the changes in electrical signal caused by the sample.

[0121] The term "sample" means any sample that can contain the aforementioned specimen or biomarker, and may include, for example, one or more samples selected from the group consisting of cell samples, tissue samples, blood samples, urine samples, saliva samples, lymph samples, cerebrospinal fluid samples, amniotic fluid samples, pleural fluid samples, pericardial fluid samples, ascites samples, aqueous humor samples, bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, forensic samples, archaeological samples, paleontological samples, infectious samples, organic samples, plant samples, microbial samples, viral samples, soil samples, marine samples, and freshwater samples.

[0122] The tissue sample may be derived from one or more tissues selected from the group consisting of the epididymis, eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchi, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, bladder, cecum, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonads, testes, ovaries, uterus, fallopian tubes, thymus, pituitary gland, thyroid gland, adrenal gland, and parathyroid gland. Furthermore, the tissue sample may be derived from a human or other organism. It may originate from any variety of organs.

[0123] The cell sample may be derived from animal cells, plant cells, fungal cells, bacterial cells, or protozoan cells, and more specifically, it may be derived from animal cells, and more specifically, it may be derived from human cells.

[0124] For example, the cell sample may include germ cells (egg cells, sperm, etc.), ovarian epithelial cells, ovarian fibroblasts, immune cells, B cells, T cells, NK cells, dendritic cells, cancer cells, eukaryotic cells, stem cells, blood cells, muscle cells, adipocytes, skin cells, nerve cells, osteocytes, pancreatic cells, endothelial cells, pancreatic epithelial cells, pancreatic α cells, pancreatic β cells, pancreatic endothelial cells, bone marrow lymphoblasts, bone marrow B lymphoblasts, bone marrow macrophages, bone marrow erythroblasts, bone marrow dendritic cells, bone marrow adipocytes, bone marrow osteocytes, bone marrow chondrocytes, preosteoblasts, bone marrow megakaryocytes, brain B lymphocytes, brain glial cells, neurons, brain astrocytes, neuroectoderm cells, brain macrophages, brain microglia, brain epithelial cells, cortical neurons, brain fibroblasts, mammary epithelial cells, colonic epithelial cells, colonic B lymphocytes, mammary myoepithelial cells, mammary fibroblasts, colonic cells, cervical epithelial cells, and mammary duct cells. The cells may be derived from one or more cells selected from the group consisting of cutaneous cells, tongue epithelial cells, tonsillar dendritic cells, tonsillar B lymphocytes, peripheral blood lymphoblasts, peripheral blood T lymphoblasts, peripheral blood cutaneous T lymphocytes, peripheral blood NK cells, peripheral blood B lymphoblasts, peripheral blood mononuclear cells, peripheral blood myeloblasts, peripheral blood monoblasts, peripheral blood preosteoblasts, peripheral blood macrophages, peripheral blood basophils, liver endothelial cells, liver mast cells, liver epithelial cells, liver B lymphocytes, spleen endothelial cells, spleen epithelial cells, spleen B lymphocytes, hepatocytes, liver fibroblasts, lung epithelial cells, bronchial epithelial cells, lung fibroblasts, lung B lymphocytes, lung Schwann cells, lung squamous cells, lung macrophages, lung osteoblasts, neuroendocrine cells, alveoli, gastric epithelial cells, gastric fibroblasts, stem cells, fetal cells, tumor cells, suspected cancer cells, cancer cells, and cells that have undergone gene editing procedures.

[0125] In the method for identifying and quantitatively analyzing a sample in a sample according to the present invention, the sample means the object to be analyzed for its presence and amount in the sample by the method, and the sample may, but is not limited to, peptides, polypeptides, proteins, inorganic substances, low molecular weight compounds, or nucleic acids.

[0126] The present invention provides a method for identifying and quantitatively analyzing samples within a sample. This method involves placing the sample in one compartment of the nanopore system, measuring characteristic changes in electrical signals caused by multiple samples within the sample, and comparing these changes with a database of electrical signals specific to each substance. This allows for the identification and quantitative analysis of the samples within the sample. The database of electrical signals specific to each substance can be constructed by measuring characteristic electrical signals for each substance via the nanopore system of the present invention.

[0127] The present invention provides a method for identifying and quantitatively analyzing samples within a sample. Even when a variety of samples contain numerous types of samples, this method accurately measures the presence and quantity of a specific sample by detecting characteristic changes in the electrical signal of the sample to be identified from the overall changes in the electrical signal of the sample. Therefore, it can be effectively used for sample identification and quantitative analysis, as well as drug screening.

[0128] Furthermore, in the present invention, the term "biomarker" means an anatomical, physiological, biochemical, or molecular parameter associated with the presence of a specific physiological state or progression. The biomarker may include organic biomolecules such as polypeptides, proteins, or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, or sugars (e.g., monosaccharides, disaccharides, oligosaccharides) that show an increase or decrease in an individual or in a specific sample derived from an individual.

[0129] The present invention provides information for the diagnosis of biomarker-related diseases by placing a sample in one compartment of the nanopore system and measuring characteristic changes in electrical signals caused by biomarkers known to be associated with specific diseases. This allows for accurate measurement of the presence and amount of biomarkers in the sample, making it effective for the diagnosis of biomarker-related diseases and even for predicting disease prognosis.

[0130] The aforementioned diseases are not limited to those in which an association with a specific biomarker has been clarified, and may be infectious or non-infectious diseases. The aforementioned infectious diseases are diseases caused by microorganisms such as bacteria and viruses, and include, for example, sepsis, septic shock, SARS-CoV infection, Middle East respiratory syndrome (MERS), salmonellosis, food poisoning, typhoid fever, paratyphoid fever, systemic inflammatory response syndrome (SIRS), and multiple organ dysfunction syndrome. Syndrome (MODS), pneumonia, pulmonary tuberculosis, tuberculosis, common cold, influenza, respiratory tract infection, rhinitis, nasopharyngitis, otitis media, bronchitis, lymphadenitis, mumps, lymphadenitis, angular cheilitis, stomatitis, arthritis, myositis, dermatitis, vasculitis, gingivitis, periodontitis, keratitis, conjunctivitis, wound infection, peritonitis, hepatitis, osteomyelitis, cellulitis, meningitis, encephalitis, brain abscess, encephalomyelitis, meningitis, osteomyelitis, nephritis, carditis, endocarditis, enteritis, gastritis, esophagitis, duodenitis, colitis, urinary tractitis, cystitis, vaginitis, cervicitis, salpingitis, infectious erythema, bacterial dysentery, abscesses and ulcers, bacteremia, Diarrhea, dysentery, gastroenteritis, gastrointestinal inflammation, urogenital abscess, open wound or wound infection, purulent inflammation, abscess, swelling, pyoderma, impetigo, folliculitis, cellulitis, postoperative wound infection, laceration syndrome, burn syndrome, thrombotic thrombocytopenia, hemolytic uremic syndrome, renal failure, pyelonephritis, glomerulonephritis, nervous system abscess, otitis media, sinusitis, pharyngitis, tonsillitis, mastoiditis, cellulitis, odontogenic infection, dacryocystitis, pleurisy, abdominal abscess, liver abscess, cholecystitis, splenic abscess, pericarditis, myocarditis, placenta, amniotic fluid, mastitis, mastitis, puerperal fever, toxic shock syndrome This may also include shock syndrome, Lyme disease, gas gangrene, atherosclerosis, mycobacterium avium syndrome (MAC), enterohemorrhagic Escherichia coli (EHEC) infection, enteropathogenic Escherichia coli (EPEC) infection, enteroinvasive Escherichia coli (EIEC) infection, methicillin-resistant Staphylococcus aureus (MRSA) infection, vancomycin-resistant Staphylococcus aureus (VRSA) infection, or listeriosis.

[0131] The aforementioned non-infectious diseases may include cancer, autoimmune diseases, non-infectious inflammatory diseases, neurodegenerative diseases, heart diseases, strokes, diabetes, metabolic diseases, chronic kidney diseases, or chronic respiratory diseases. The aforementioned cancers include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, kidney cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, classical Hodgkin lymphoma (CHL), mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, and ep These may include Stein-Barr virus (EBV)-positive and EBV-negative post-transplant lymphoproliferative disorders (PTLD), EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, external NK / T-cell lymphoma, nasopharyngeal carcinoma, human herpesvirus 8 (HHV8)-associated primary exudative lymphoma, other hematological malignancies including Hodgkin lymphoma, primary central nervous system (CNS) lymphoma, spinal tumors, and brainstem gliomas. The aforementioned autoimmune diseases include lupus, systemic lupus erythematosus, Sjögren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, and pilonidal disease. I disease), peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barr syndrome, atopic dermatitis, autoimmune hepatitis, fibrotic alveolitis, Graves' disease, IgA kidney disease, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis The neurodegenerative diseases may include granulomatosis, autoimmune diseases, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, heart disease including ischemic diseases such as myocardial infarction and atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria, infertility related to lack of fetal-maternal tolerance, vitiligo, myasthenia gravis, and systemic sclerosis. The neurodegenerative diseases may also include cognitive impairment, brain tumors, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis (ALS). This may include lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain, and memory impairment.

[0132] In a specific embodiment of the present invention, we confirmed whether selective detection and quantitative analysis of specific biomarker proteins present in serum samples is possible. We checked for the presence or absence of characteristic electrical signals specific to Bcl-xL at concentrations of 10 nM and 20 nM in the presence of FBS (Fetal Bovine Serum) similar to human serum. When a voltage of 80 mV was applied in the presence of FBS from which some proteins had been removed (depleted FBS), in addition to the various nanopore electrical signals measured in the depleted FBS, a characteristic electrical signal specific to the 10 nM Bcl-xL protein was observed, and we confirmed a signal distribution that matched the Bcl-xL protein specific electrical signal among the various dispersed signals in the depleted FBS. Furthermore, electrical measurements performed under 20 nM Bcl-xL protein conditions in the presence of depleted FBS showed that more Bcl-xL-specific electrical signals were detected compared to 10 nM Bcl-xL protein conditions. This was confirmed by the two-dimensional scatter plot, which clearly showed a concentration-dependent clustering of Bcl-xL protein signals. This confirmed that the nanopore system of the present invention can accurately measure the presence and amount of sample proteins in mixed samples containing many different types of substances (see Figure 23).

[0133] The present invention will be described in detail below with reference to examples. However, the following embodiments are merely illustrative examples of the present invention, and the content of the present invention is not limited to these embodiments.

[0134] [Example 1] Fabrication of YaxAB nanopore sensors [1-1] Sample preparation The compounds used in the nanopore measurement buffer were Sigma-Aldrich (St. Louise, USA), lipids and surfactants were purchased from Avanti Polar Lipids (Alabaster, AL, USA), and Bak-BH3 peptide and Bax-BH3 peptide were purchased from Peptron Inc. (Daejeon, South Korea). The low molecular weight compounds ABT-737 (Cayman Chemical), LCL-161 (Selleckchem), GDC-0152 (Selleckchem), A-1331852 (Selleckchem), and Quercetin (Sigma-Aldrich) were dissolved in dimethyl sulfoxide-d6 (Sigma-Aldrich). I'm ready.

[0135] [1-2] Purification of the first monomer (YaxA) and the second monomer (YaxB) The first monomer (YaxA) and second monomer (YaxB) used in the experiment were synthesized by Cosmogenetech. The first monomer (YaxA) consisted of the amino acid sequence of SEQ ID NO: 1 (YaxA_(45-410)) and the wild-type amino acid sequence of SEQ ID NO: 3. The no-acid sequence (YaxA_FL) is used as the second monomer (YaxB) in sequence number 2. The amino acid sequence (YaxB_(12-343)) and the amino acid sequence of the wild type, sequence number 4 ( YaxB_FL was synthesized and purified.

[0136] Specifically, YaxA and YaxB were introduced into pET15b vectors containing thrombin cleavage sites, respectively, along with N-terminal 6xHis-SUMO and 6xHis tags. YaxA was expressed in E. coli BL21(DE3)pLysS cells induced with 0.5 mM IPTG and then cultured overnight in 2xYT medium at 18°C. YaxB was expressed under the same conditions as YaxA. To purify the expressed YaxA protein, the cell pellet was resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 1 mM PMSF, 1 g / ml DNase I, and 0.2 mg / ml lysozyme) and degraded using a microfluidizer. The YaxA protein was lysed in 3% DDM at 4°C for 2 hours. 5 mL of the purified lysate was equilibrated with buffer A (50 mM Tris pH 8.0, 300 mM NaCl, 0.05% Simul-6, and 10 mM imidazole). The Ni-NTA affinity column was loaded, and the bound protein was eluted in a linear gradient of 10 mM-1 M imidazole. In the presence of thrombin protease, buffer B (50 mM) was used. The cells were dialyzed at 4°C against Tris (pH 8.0, 300 mM NaCl). YaxA was further purified in a secondary Ni-NTA step to remove degraded tags and proteases. For YaxB protein, the cell pellet was resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 1 mM PMSF, 1 g / mL DNase I, 0.2 mg / mL lysozyme) and degraded using an ultrasonic disruptor. The purified lysate was loaded onto a 5 mL Ni-NTA affinity column equilibrated with buffer C (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole), and bound proteins were eluted within a linear gradient from 10 mM to 1 M imidazole. The cells were dialyzed at 4°C against buffer E (20 mM HEPES pH 7.0, 25 mM NaCl) in the presence of thrombin protease. Next, the samples were coupled to a 5 mL HiTrap Q column equilibrated with buffer E and eluted within a linear salt gradient from 0 to 1 M NaCl. The peak fractions were further purified using a Superdex 75 16 / 600 gel filtration column (GE Healthcare) with buffer F (25 mM HEPES pH 7.0, 150 mM NaCl).

[0137] [1-3] Fabrication of YaxAB nanopores The first monomer of SEQ ID NO: 1 (YaxA_(45-410) purified in Examples 1-2 )) and the second monomer of sequence number 2 (YaxB_(12-343)) are used to modify Ya xAB_dN nanopores were fabricated using a similar method. Wild-type first monomer (SEQ ID NO: 3, Ya We fabricated wild-type YaxAB nanopores consisting of xA_FL and the wild-type second monomer (SEQ ID NO: 4, YaxB_FL).

[0138] Specifically, 6 mg each of the first and second monomers were cultured at 25°C for 30 minutes to bind them. Cymal-6 (1.5% w / v) was added to the mixture of YaxA and YaxB, and the mixture was incubated at 4°C for 30 minutes. After incubation, the mixture was injected into a Superose 6 column in gel filtration buffer (25 mM HEPES pH 7.0, 150 mM NaCl, 0.05% w / v Cymal-6). The peak fraction was analyzed by 4-16% blue native polyacrylamide gel electrophoresis (BN- By PAGE (Thermo Fisher Scientific Korea, Invitrogen), YaxAB oligomeric proteins of different sizes and with varying numbers of YaxAB nanopore subunits were separated (see Figure 1). The bands corresponding to YaxAB oligomeric proteins were excised from the gel and placed in gel filtration buffer, and YaxAB oligomeric proteins were extracted from each band at 4°C. Wild-type YaxAB nanopores were extracted in the same manner, and the modified YaxAB_dN nanopores or wild-type YaxAB nanopores were separated. The supernatant was used in the experiment.

[0139] [1-4] Fabrication of nanopore systems and measurement methods for nanopore experimental data The first monomer of sequence number 1 prepared in Examples 1-3 (YaxA_(45-410 Ya, composed of )) and the second monomer of sequence number 2 (YaxB_(12-343)) A nanopore sensor was fabricated using xAB-C8 nanopores.

[0140] Specifically, two compartments were assembled by positioning a Teflon film with 100 μm pores between two Teflon chambers, and 0.8 mL of a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 M KCl was placed in each compartment. In addition to Tris-HCl, buffers such as HEPES, phosphate buffer, PBS, and sodium acetate can be used in the solution composition. The nanopores were pre-coated with a lipid bilayer before assembly.

[0141] For electrical measurements, the Ag / AgCl electrode was configured such that the ground electrode was connected to the transformer side and the working electrode to the cis side. Purified YaxAB-C8~YaxAB-C was placed in the cis compartment so that a single YaxAB nanopore was inserted into the lipid membrane. 13 After adding the substance, the nanopore current signal was measured by applying a voltage of +100mV to the cis compartment. Following insertion of the YaxAB nanopore, various proteins (Bcl-xL, Hsp33, FKBP12, holo-transferrin, MDM2, BSA, Aldolase, Ferritin, Thyroglobulin) or Bcl-2 system proteins were added, followed by the addition of one or more various peptide ligands (Bak-BH3, Bak-BH3-I83A, Bak-BH3-D84A, Bak-BH3-L78A) or one or more small molecule drugs (ABT-737, LCL-161, GDC-0152, A-1331852, Quercetin, A1155463, FK506, Oxaliplatin) to the cis compartment. Electrical signals were recorded using a patch-clamp amplifier (Axopatch 200B, Molecular Devices Inc., Sunnyvale, CA, USA) with a bias voltage of 40-120mV. The collected data was analyzed using the single-channel search function of pClamp 11 software (Molecular Devices Inc.). Ion signals recorded at a sampling rate of 100kHz were analyzed using a built-in 8-pole at 10kHz. The samples were filtered using a low-pass Bessel filter. The sampling rate and filter could be varied under different conditions for signal optimization, and all nanopore experiments were performed at 25°C.

[0142] [Example 2] Analysis of YaxAB nanopore formation and electrical properties [2-1] Formation of YaxAB nanopores To determine whether the YaxAB nanopore proteins prepared in Examples 1-3 could be used as nanopore sensors, the structures of modified YaxAB_dN nanopores were analyzed using Negative-stain EM and Cryo-EM. The results are shown in Figures 2a-2. As shown in c, the modified YaxAB_dN nanopore is composed of 8 to 10 subunits. It was confirmed to be a simulated α-helical pore complex.

[0143] Based on the aforementioned EM data, the modeling structure shown in Figure 2d and the three-dimensional structure of YaxAB-C8 were derived as shown in Figure 2e. As a result, it was confirmed that the YaxAB nanopore has a clear symmetrical structure, with a wide first opening, a narrow second opening, and an intermediate region connecting the first and second openings. It was also confirmed that the lumen of the nanopore has a constriction where the diameter narrows. In particular, in the case of the YaxAB-C8 nanopore, the depth of the lumen is 17 nm, the diameter of the lumen of the wide first opening is 10 nm, the outer diameter of the lumen of the second opening is 2.7 nm, and the diameter of the constriction of the lumen located at the second opening is 1.9 nm. It has a funnel structure with a wide entrance and a narrow exit, and it was confirmed that the lumen of the nanopore is coffee dripper-shaped with a constriction and has a wavy shape due to the α-helix. Based on this, the structure of the YaxAB nanopore is shown in Figure 3.

[0144] [2-2] Molecular dynamics simulation analysis Prior to the experiment, the properties of the YaxAB nanopore were analyzed using molecular dynamics (MD) simulations. A YaxAB-C8 simulation setup was configured with a 25*25 nm 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and sterol composite membrane inserted. Molecular dynamics simulations were performed using the GROMACS 2020.2 package under conditions of 303 K with no surface tension in the presence of a 1 M KCl solution.

[0145] To measure the free energy of the Bcl-xL protein via a YaxAB-C8 nanopore at 100mV, Bcl-xL was added to a distance of z=10nm from the nanopore-containing membrane. MD simulations were then performed by adjusting the distance (z) from the membrane in 1nm increments within the range of 2nm-13nm. The Bcl-xL protein moved freely in the x and y directions, and the free energy was calculated by integrating the average forces at each set z value.

[0146] As a result, as shown in Figure 4a, we were able to confirm the local distribution of water and ion molecules inside the nanopore, and we were able to confirm that the flow velocity of potassium ions and water increased as they approached the constriction of the nanopore when a positive voltage was applied. Flow velocity of potassium ions in the constriction of the lumen (0.16 nm -2 ns -1 ) is a chloride ion (0.04 nm -2 ns -1 This is faster than the flow velocity of cis, which means that when a positive voltage is applied, an electroosmotic force (EOF) acts from cis to transformer.

[0147] Furthermore, MD simulations revealed that the free energy was doubly minimum when the Bcl-xL protein was located at z=4, 10 nm, as shown in Figure 5. This suggests that the temporary residence of the Bcl-xL protein at the minimum free energy level induces oscillations between multiple residence sites (R1-R3) while inducing various current cutoff levels (L1-L3). The minimum free energy value of Bcl-xL observed at z=4 nm indicates that once the protein is trapped within the YaxAB nanopore, it preferentially occupies that position. MD simulations predicted that when the Bcl-xL protein is trapped at z=4 nm, the flow of potassium ions and water in the constricted region is significantly reduced compared to an empty YaxAB nanopore (Figure 4b). Therefore, due to the weakened EOF, the Bcl-xL protein moved to the cis side (z=10 nm), and MD simulations showed that when the Bcl-xL protein was trapped at a distance of z=10 nm, the potassium ion and water flow was even greater than when it was trapped at a distance of z=4 nm (Figure 4c). This suggests that, upon recovery of the EOF, the Bcl-xL protein moved again to the trans side (z=4 nm).

[0148] [2-3] Improvements to enhance the stability of nanopore-containing membranes In the case of the YaxAB nanopore, it was confirmed that it is not well inserted into DPhPC membranes, which are artificial biomembrane materials that have been widely used in the past, or even if it is inserted, it has low stability and the membrane is prone to rupture (see Figure 6a).

[0149] To complement this, DPhPC is combined with 5-50 mol% sterol molecules to form a DPhPC-sterol composite membrane, and in the case of such a composite membrane, the YaxAB nanopores of the present invention are C6, C7, C8, C9, C 10 , C 11 , C 12 , and C 13Not only were the nanopores successfully inserted, but it was also confirmed that a constant amount of current flowed very stably according to Ohm's law even after insertion into the membrane, as shown in Table 1 below (see Figures 6b and 7).

[0150] [Table 1]

[0151] Furthermore, the conductivity results measured when a voltage of 100 mV was applied to the cis compartment were matched with those of each modified YaxAB_dN nanopore. YaxAB-C4 showed a conductivity of 1.0 ± 0.1 nS; YaxAB-C5 is 1.8±0.1nS; YaxAB-C6 is 3.0±0.1nS; YaxAB-C7 is 4.1±0.1nS; YaxAB-C8 is 5.0±0.1nS; YaxAB-C9 is 7.8±0.2nS; YaxAB-C 10 is 10.9±0.2nS; YaxAB-C 11 is 14.6±0.2nS; YaxAB-C 12 is 18.5 nS; YaxAB-C 13 A constant conductivity of 24.4 nS was observed (see Figures 7 and 8). The above experimental values ​​are current values ​​that were theoretically derived due to the structure of the YaxAB nanopores (YaxAB-C8: 5.4 nS; YaxAB-C9: 8.2 nS; YaxAB-C 10 Since it was confirmed that the values ​​(11.7 nS) matched each other, it was found that the YaxAB nanopore of the present invention exists very stably on the DPhPC-sterol composite membrane according to Ohm's law. Experiments were then conducted after fabricating a nanopore sensor using a DPhPC-sterol composite membrane into which YaxAB-C8 (hereinafter, YaxAB nanopore) was inserted.

[0152] [2-4] Measurement of cation selectivity of YaxAB nanopores To measure the ion transfer between the pores of the YaxAB nanopore of the present invention, various voltages were applied and the ion current was measured. Using the Goldman-Hodgkin-Katz equation of the following formula 1, the ion selectivity (P K + / P Cl - ) which is the passing ratio of potassium ions / chloride ions was calculated.

[0153]

Equation

[0154] Here, [α K+ / Cl- cis / trans means the activity of potassium ions in the cis chamber or the activity of chloride ions in the trans chamber, R means the ideal gas constant, F means the Faraday constant, and Vr means the reversal potential measured under the conditions of an asymmetric salt.

[0155] The reversal potential (Vr) of the YaxAB nanopore was measured using KCl solutions with different concentrations in a 10 mM Tris HCl buffer solution with pH 7.5 in both compartments of the nanopore (trans / cis: 2 M KCl / 0.5 M KCl). Specifically, after putting 800 μL of 1 M KCl solution in both the cis and trans compartments, the YaxAB nanopore was inserted into the membrane. After adjusting the offset and balancing electrodes and discarding 400 μL of 1 M KCl solution from both compartments, 400 μL of 3 M KCl was added to the trans compartment, and the same volume of salt-free buffer was added to the cis compartment to induce a salt gradient. The cation selectivity was calculated using the value obtained by multiplying the molar concentration of the given ion by the mean ionic activity coefficient (2 M KCl: 0.573, 0.5 M KCl: 0.649).

[0156] As a result, as shown in Fig. 9, it was confirmed that all three types of the modified YaxAB_dN nanopores (C8, C9 and C 10 nanopores) have asymmetric I-V curves. YaxAB-C8 has a cation selectivity of 2.34; YaxAB-C9 has a cation selectivity of 1.72; YaxAB-C 10 has a cation selectivity of 1.47 (P​​+ / P Cl - ) was confirmed to have, and such high cation selectivity of the YaxAB nanopore of the present invention can cause an electroosmotic force that predominantly acts in the opposite direction to the electrophoretic force. When a positive voltage is applied under the condition of pH 7.5, since the amino acids constituting the inner wall of the pore are negatively charged, strong electroosmosis can be caused by cations that predominantly flow from cis to trans along the inner wall of the pore (due to excellent cation selectivity).

[0157] Thus, since the YaxAB nanopore of the present invention can cause electroosmosis, the analyte can be captured inside the nanopore regardless of the overall charge. In addition to the wide entrance, the diameter of the YaxAB nanopore can be adjusted by adjusting the number of heterodimers constituted by a part of two monomeric YaxA and YaxB, so that large analytes can also be captured inside the nanopore, and there is an advantage that the analysis target is not limited by the charge and size of the analyte. Furthermore, since it has a funnel structure in which the entrance is wide while the exit is relatively much narrower, compared with the conventional cylindrical nanopore, when the analyte is captured inside the nanopore, the degree of current interruption by the analyte can change more drastically, and as a result, there is an advantage that the difference in current interruption by the analyte can be maximized and even small changes in the analyte can be detected much more sensitively.

[0158] [Example 3] Detection of Analytes at the Single-Molecule Level via the YaxAB Nanopore Sensor In order to confirm whether the YaxAB nanopore sensor of the present invention can detect proteins at the single-molecule level, experiments were conducted using the YaxAB-C8 nanopore sensor fabricated in Examples 1-4 above.

[0159] First, in order to confirm the principle by which the ion current changes depending on the sample in the YaxAB nanopore of the present invention, a Bcl-xL protein (3.4 × 5.1 nm) that is larger than 2 nm, cannot pass through the YaxAB nanopore of the present invention, and has a negative overall charge was selected as a representative sample. The selected Bcl-xL protein was placed in the cis compartment, and voltages of (+) and (-) 100 mV were applied to the cis and trans compartments, respectively.

[0160] In the case of Bcl-xL protein, the inside of the pore becomes negatively charged at pH 7.5, and therefore, although the electrophoretic force (EPF) caused by the voltage applied to both compartments prevents inflow into the nanopore, the electroosmotic force (EOF) generated by the YaxAB nanopore of the present invention causes inflow into the pore (see Capture (I) in Figure 5A), and at this time, a fundamental current (I) is added to the ionic current. open A current interruption of 38.4 ± 4.7% was caused by this (see L1 in Figure 5A). After flowing into the pore, the Bcl-xL protein undergoes oscillation movement due to the balance between the forces of electroosmosis and electrophoresis (see Trap(II) in Figure 5A), and this oscillation movement causes a fundamental current (I) to be added to the ionic current. open Current interruption was triggered in the range of 68.4±3.6% to 79.0±1.8% (see L2 and L3 in Figure 5A). Then, when the Bcl-xL protein, which was in the oscillating motion as described above, leached out of the nanopore (see Escape(III) in Figure 5), no further current interruption was triggered, and the ionic current became the basic current in the open state (I open It was restored to the ) level.

[0161] As a result, the Bcl-xL protein captured by the YaxAB nanopore exhibited three types of ion current patterns (L1, L2, and L3), and it was confirmed that the degree of current interruption between L1 and L2 / L3 was at the level of 30.1-41.0%, which is greater than that of conventional nanopores (e.g., ClyA, PlyAB, MspA, NeoTrap, etc.).

[0162] The current cutoff levels of conventional ClyA nanopores were confirmed to be SBP1 1.3%, SBP2 1.7%, BtuF 8.3%, GBP 3.4%, SiaP 4.8%, TbpA 2.5%, LBP 6.2%, SpuD 3.6%, and MBP 4.1% (Zernia et al., “Current Blockades of Proteins inside Nanopores for Real-Time Metabolome In "Analysis," ACS Nano, 14(2):2296-2307 (2020), the current cutoff levels of conventional PlyAB nanopores were confirmed to be 23.2% for HbA and 21.5% for HbS (Huang et al., "Detection of single amino acid differences in haemoglobin from blood samples using a nanopore," Analytical Chemistry (2021)).

[0163] Furthermore, while specific current cutoff levels have not been separately provided for conventional MspA nanopores, it has been confirmed that lysozyme levels are within 15% and holo-myoglobin levels are within 18% (S. Huang et al., "Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap", JACS (2022)).

[0164] Furthermore, although specific current cutoff levels have not been separately provided for NeoTrap nanopores, it has been confirmed that the current cutoff level is within 20% for Hsp90 and Avidin, confirming that the YaxAB nanopores of the present invention have a relatively high resolution. (Schmid et al., “Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations”, Nat. Nanotechnol. (2021)).

[0165] On the other hand, among conventional nanopores, the degree of current interruption was relatively high for holo-myoglobin (within 18%) in MspA nanopores and HbA (within 23.2%) in PlyAB nanopores, but this was significantly lower compared to the degree of electrical interruption for Bcl-xL in the YaxAB nanopore of the present invention (30.1-41.0%). A comparison of the analytical resolution of YaxAB nanopores with conventional nanopores, ClyA and MspA, using the same sample revealed that the YaxAB nanopore of the present invention showed a variation in current levels within the GBP-derived signal of approximately 14.3%, which is more than 4.2 times higher than the variation of 3.4% obtained by analyzing GBP-derived signals with ClyA nanopores (see Figures 14a and 14b). Furthermore, the variation in current levels within the lysozyme and holo-myoglobin-derived signals of the YaxAB nanopore of the present invention was approximately 14.0% and 47.3%, respectively, which are 1.3 times and 2.6 times higher than the variation of 10.4% and 17.9% obtained by analyzing lysozyme and holo-myoglobin-derived signals with MspA nanopores. This confirmed the superior analytical performance of the YaxAB nanopore of the present invention (see Figures 14c and 14d).

[0166] Furthermore, the YaxAB nanopore of the present invention was used to compare the lysozyme signal characteristics derived from MspA nanopores with those of three types of proteins derived from lysozyme proteins. As a result, the YaxAB nanopore of the present invention showed a current drop magnitude (ΔI / I) relative to the fundamental current of approximately 65.5 ± 0.3% from the lysozyme protein. o The ΔI / I value, the standard deviation (SD) of the ion current of 15.9 ± 0.6 pA, and the trapping time in the range of 100–10000 ms are derived, which is the 33.9% ΔI / I value of conventional MspA nanopore-derived lysozyme protein signals. o ΔI / I compared to an SD of 9-10 pA level and an acquisition time in the range of 1-100 ms, respectively. o The YaxAB nanopore of the present invention demonstrated superior analytical performance compared to conventional MspA nanopores for the same sample, with a 1.9x improvement in analytical performance, a 1.7x improvement in standard deviation, and approximately 100 times faster sample capture time (see Figure 14e).

[0167] Furthermore, to confirm the range of proteins that can be analyzed using the YaxAB nanopore of the present invention, we analyzed the ion current patterns of the following proteins: Hsp33 protein (52.4 kDa) and BSA protein (66.5 kDa), which are not only larger in size than Bcl-xL protein (20.8 kDa) but also carry a larger negative charge; holo-Transferrin protein (77.1 kDa), which is large in size but carries a relatively small negative charge; Aldolase protein (158 kDa), which is large in size but carries a positive charge; Ferritin protein (440 kDa) and Thyrogobulin protein (669 kDa), which have surface charge properties similar to Bcl-xL but are larger in size; FKBP12 protein (13.0 kDa), which is smaller in size than Bcl-xL protein, can pass through the YaxAB nanopore of the present invention, and is electrically neutral; and MDM2 protein (12.3 kDa), which is electrically positively charged.

[0168] As a result, it was confirmed that each of the nine proteins exhibited a distinctly different ion current pattern (see Figure 10), demonstrating that single-molecule analysis is possible for proteins of various sizes and with various charges.

[0169] [Example 4] Presence or absence of sample-ligand interaction analysis via YaxAB nanopore sensor [4-1] Analysis of protein-protein or protein-small molecule interaction First, we confirmed whether it is possible to analyze protein-protein or protein-small molecule compound interactions using the YaxAB nanopore of the present invention.

[0170] In the cis compartment of the sensors prepared in Examples 1-4, i) Bcl-xL protein alone at a concentration of 100 nanomolars (nM), ii) Bcl-xL protein and Bak-BH3 protein, which is known to bind to Bcl-xL protein, were added together in a 1:2 molar ratio, and iii) Bcl-xL protein and ABT-737, a small molecule compound known to be a competitive inhibitor of Bak-BH3 bound to Bcl-xL protein, were added together in a 1:2:5 molar ratio. Then, (+) and (-) voltages were applied to the cis compartment and transform compartment, respectively.

[0171] As a result, the addition of Bak-BH3 protein or ABT-737 altered the pattern in which Bcl-xL protein alone caused a current-blocking effect. Since these two substances each exhibited different current-blocking patterns, it was confirmed that it is possible to distinguish between protein-protein complexes (Bcl-xL + Bak-BH3) and protein-small molecule compound complexes (Bcl-xL + ABT-737) (see Figures 11a and 11b).

[0172] Furthermore, as shown in Figures 11c to 11f, in addition to current interruption analysis, current noise (I NUsing base analysis, it was possible to distinguish free Bcl-xL from Bcl-xL / small molecule compound complexes using the YaxAB nanopore. In particular, it was confirmed that by signal filtering in the low-frequency range (~100 Hz), the ion current patterns of the specimens in the three cases as described above could be more clearly distinguished.

[0173] Next, it was confirmed whether the interaction between weak binders with low binding affinity to proteins could be analyzed using the YaxAB nanopore of the present invention.

[0174] To the cis compartment of the sensor fabricated in Examples 1-4, i) Bcl-xL protein alone at a concentration of 1 micromolar (μM), ii) Bcl-xL protein and the Bax-BH3 peptide and Quercetin small molecule compound known to bind to the Bcl-xL protein were added at molar ratios of 1:100 and 1:20, respectively. Then, (+) and (-) voltages were applied to the cis and trans compartments, respectively.<>

[0175] As a result, due to the addition of the Bax-BH3 peptide or Quercetin, intermittent changes occurred in the pattern in which the Bcl-xL protein alone caused a current interruption effect, and in both cases of these two substances, different current interruption patterns were shown, respectively. Thus, it was confirmed that the interaction between binders with low binding affinity to Bcl-xL could be analyzed (see FIGS. 12 to 13).

[0176] And as shown in FIGS. 12 to 13, in addition to current interruption analysis, current noise (I N ) Using base analysis, it was confirmed that the free Bcl-xL could be distinguished from the Bcl-xL / protein or small molecule compound complex using the YaxAB nanopore.

[0177] [4-2] Analysis of protein structural changes induced by ligand binding Based on the results of the protein-small molecule compound interaction analysis described above, the YaxAB nanopore of the present invention was used to determine when a glucose / galactose binding protein (GBP), which consists of two domains and undergoes an alloteric motion upon binding to glucose or galactose, binds to galactose, a small molecule compound that targets GBP. The current interruption signal generated at that time was measured (see Figure 14).

[0178] Specifically, 50 nM GBP was added to the cis compartment of a YaxAB nanopore, and a positive voltage of 80 mV was applied to the same compartment. The current cutoff signal generated by the capture of the GBP protein was observed. The current cutoff signal was observed to be at current levels 1 and 2, similar to gating. At this time, when galactose, a small molecule compound that targets GBP, was added to the trans compartment, it was confirmed that the frequency of gating generated from the GBP-derived current cutoff signal increased significantly. Furthermore, in conventional ClyA nanopores, the range of change between current levels was confirmed to be around 3.4% for signals derived from GBP and GBP-glucose (G. Maglia et al., "Direct electrical quantification of glucose and asparagine from bodily fluids using nanopores", Nat. Commun. (2018)) and signals derived from GBP-galactose (Chi et al., "Probing the Neuraminidase Activity of Influenza Virus Using a Cytolysin A Protein Nanopore", Anal. Chem. (2020)). However, in the YaxAB nanopore of the present invention, the range of change between current levels in signals derived from GBP and GBP-galactose was approximately 14.3%, which was significantly higher than that of ClyA nanopores (more than 4.2 times higher).

[0179] From the above results, by using the YaxAB nanopore of the present invention to measure and analyze the electrical signals specific to GBP and the GBP-galactose complex generated according to the presence or absence of galactose, the structural changes of the GBP protein due to galactose binding can be effectively analyzed, and it was confirmed that the analytical resolution of the YaxAB nanopore of the present invention is significantly superior to that of the conventional ClyA nanopore.

[0180] [4-3] Analysis of the binding affinity of proteins or small molecule compounds to proteins Furthermore, based on the analysis of the current noise (I N ) measured in Example 3-2 above, a capacitance-dependent quantitative analysis was performed on the interaction between the Bcl-xL protein and the Bak-BH3 peptide or the ABT-737 compound, and the binding affinity (K D ) for the Bcl-xL protein was calculated.

[0181] Specifically, as a result of treating 100 nM of the Bcl-xL protein with the Bak-BH3 protein at concentrations of 10, 30, 50, 100, 200, and 500 nM respectively, it was confirmed that the ratios of forming complexes between the Bcl-xL protein and the Bak-BH3 protein at different concentrations were 21.6%, 35.8%, 42.3%, 63.6%, 85.3%, and 95.9% respectively. Therefore, the binding affinity (K D ) of the Bak-BH3 protein for the Bcl-xL protein was analyzed to be 65.78 ± 10.32 nM (see Fig. 15a).

[0182] Also, in the same manner as above, as a result of treating 100 nM of the Bcl-xL protein with the ABT-737 compound at concentrations of 10, 50, 70, 100, 200, and 1000 nM respectively, it was confirmed that the ratios of forming complexes between the Bcl-xL protein and the ABT-737 compound at different concentrations were 17.4%, 58.2%, 71.1%, 87.9%, 98.0%, and 99.6% respectively. Therefore, the binding affinity (K DThe concentration was analyzed to be 37.97 ± 7.91 nM (see Figure 15b).

[0183] Furthermore, using the same method as described above, 100 nM Bcl-xL protein was treated with compound A-1331852 at concentrations of 10, 30, 50, 70, 100, 200, and 1000 nM, respectively. The results showed that Bcl-xL protein and compound A-1331852 were treated at different concentrations. The proportions of complex formation between the two proteins were confirmed to be 34.5%, 65.7%, 78.1%, 98.2%, 99.5%, 100%, and 100%, respectively, thus determining the binding affinity (K) of the A-1331852 compound to the Bcl-xL protein. D The concentration was analyzed to be 19.16 ± 4.47 nM (see Figure 15c).

[0184] This demonstrates that, when using the YaxAB nanopore system of the present invention, the intensity of interactions between biomolecules such as proteins and drugs can also be analyzed, making it useful for drug screening.

[0185] [4-4] Activity analysis of protein-protein interaction inhibitors Furthermore, after reacting 100 nM Bcl-xL protein with 200 nM Bak-BH3 protein, the samples were treated with ABT-737 compound, a competitive inhibitor of Bak-BH3 protein, at concentrations of 100, 500, and 1000 nM. As a result, it was confirmed that as the concentration of added ABT-737 compound increased, the ion current pattern of the complex between Bcl-xL protein and Bak-BH3 protein (Bcl-xL + Bak-BH3) gradually decreased, while the ion current pattern of the complex between Bcl-xL protein and ABT-737 compound (Bcl-xL + ABT-737) gradually increased (see Figure 16).

[0186] The results described above show that when using the YaxAB nanopore of the present invention, the complex of protein and drug bound to the protein can be directly detected based on high resolution. This differs from conventional methods that indirectly analyze the complex of drug bound to protein using the degree of signal reduction of the protein-protein complex during drug treatment, as it allows for real-time, direct detection and analysis of the complex itself.

[0187] Next, we used the YaxAB nanopore sensor to analyze the interaction between the target protein and post-translational modification (PTM) peptides, and to confirm the presence or absence of protein-protein interaction inhibitors.

[0188] First, using the YaxAB-C8 nanopore sensor of the present invention, we confirmed the interaction between acetylated peptides and proteins or protein-small molecule compounds at the single-molecule level. The target proteins were bromodomain and extra-terminal We used BRD4, a member of the domain (BET) system of proteins. BRD4 is a chromatin-binding protein associated with cancer and autoimmune diseases, and it plays a role as a scaffold for transcription factors in both its promoter and super-enhancer. BRD4 is divided into bromodomain 1 (BD1) and bromodomain 2 (BD2). BD1 has an extended loop compared to BD2, and BD1 can specifically recognize and bind to the acetylated portion of the H4 moiety of histone proteins. Considering these binding characteristics, we used an H4 peptide consisting of 12 amino acids from histone proteins (H4_1-12) and an acetylated peptide at residues 5 and 8 (H4_1-12 K5acK8ac) to confirm the presence or absence of binding to the target protein BRD4-BD1 with or without PTM (see Figures 22a and 22b).

[0189] Specifically, after filling both compartments of the YaxAB-C8 nanopore sensor with a 10 mM Tris pH 7.5, 1 mM EDTA, and 1 M LiCl solution, 2 μM BRD4-BD1 protein was added to the cis compartment, and a (+) voltage was applied to observe the nanopore current cutoff signal. During this process, a unique nanopore current cutoff signal, expressed at the L1-L2 level and originating from the BRD4-BD1 protein due to its vibrational motion within the nanopore, was observed. Furthermore, the target protein and acetylated peptide (H4_1-12 K5acK8) were also analyzed. After adding ac) to the cis compartment in a molar ratio of 1:20, the nanopore current cutoff signal was measured. Furthermore, we confirmed that a new L3 level signal emerges from the L1-L2 level signal derived from the BRD4-BD1 protein (see Figure 22c).

[0190] Furthermore, BRD4-BD1 protein and acetylated peptide (H4_1-12 K5a The change in the nanopore current cutoff signal generated by the interaction of cK8ac is analyzed using PSD (Power Spectrum Density) to determine the current noise (I N This can be expressed as a value, and based on this, concentration-dependent quantitative analysis can be performed to determine the binding affinity (K) of the acetylated peptide (H4_1-12 K5acK8ac) to the BRD4-BD1 protein. D ) calculate We were able to do this. Specifically, against 2 μM BRD4-BD1 protein, H4_1- The results showed the binding affinity (K) of the acetylated peptide (H4_1-12 K5acK8ac) to the BRD4-BD1 protein at different concentrations, after treating with 12 K5acK8ac peptides at concentrations of 2, 4, 6, 10, 20, and 40 μM. D ) is 3.269±0.756 The result was in μM (see Figure 22d).

[0191] Based on the results described above, we confirmed that the YaxAB nanopore system of the present invention can qualitatively and quantitatively analyze the interaction between the target protein and its ligand, which is determined by the presence or absence of post-translational modifications of the target protein and its ligand.

[0192] Next, in the YaxAB nanopore system of the present invention, the interaction inhibitor between the BRD4-BD1 target protein and the acetylated peptide (H4_1-12 K5acK8ac) is activated. The presence or absence of sexuality was confirmed. Specifically, four small molecule compounds that bind to the BRD4-BD1 protein (GSK778, Y06026, XMD8-92, PLX51107) were reacted with the BRD4-BD1 protein, and the nanopore current cutoff signal was measured. As a result, a unique nanopore current cutoff signal for each protein-small molecule compound complex was observed (see Figure 22e).

[0193] Based on the above results, the BRD4-BD1 target protein was reacted with the acetylated peptide (H4_1-12 K5acK8ac) and their respective small molecule compounds, followed by nano- Pore ​​current cutoff signals were measured. As a result, we were able to confirm current cutoff signals specific to each target protein-small molecule compound complex. The signals from the four target protein-small molecule compound complexes differed significantly from those from the target protein-acetylated peptide complex, confirming that there are differences among the target protein-small molecule compound complexes (see Figure 22f).

[0194] Based on the results described above, the YaxAB nanopore system of the present invention allows for the analysis of target protein-acetylated peptide interactions, the analysis of the presence or absence of activity of interaction inhibitors that inhibit target protein-acetylated peptide interactions, and the identification of small molecule compounds that bind to target proteins. Therefore, the YaxAB nanopore system of the present invention enables effective screening of interactions between target proteins and acetylated peptides and their interaction inhibitors.

[0195] [4-5] Screening of protein-drug interactions and single molecule fingerprinting analysis Using the YaxAB nanopore of the present invention, it was confirmed how precisely the interaction between a protein and a low molecular weight compound can be analyzed.

[0196] Two low molecular weight compounds (ABT-737 and A-1331852) that strongly bind to the Bcl-xL protein and two low molecular weight compounds (LCL-161 and GDC-0152) that do not bind to the Bcl-xL protein were selected and treated with the Bcl-xL protein respectively. As a result, as shown in Fig. 17a, when treated with the strong binding agents ABT-737 and A-1331852 compounds, the ion current pattern changed significantly. On the other hand, when treated with the non-binding agents LCL-161 and GDC-0152 compounds, it was confirmed that there was no significant difference from the negative control group. In particular, as shown in Fig. 17b, in the two-dimensional density contour map of current blockade (ΔI / I o ) - current noise (I N ), it was confirmed that the event distribution of the non-binding agent and Bcl-xL protein mixture was the same as that of free Bcl-xL. In contrast, the strong binding agent and Bcl-xL protein complex were clearly distinguished from the negative control group by two individual peaks like single molecule fingerprinting.

[0197] Also, when using the YaxAB nanopore of the present invention, in order to investigate whether drugs can be screened from a mixture of various compounds, the protein-drug interaction (Protein-Drug Interaction, PDI) was monitored in real time using the current recording of the YaxAB nanopore.

[0198] Specifically, as shown in Figure 17c, Bcl-xL was simultaneously treated with a mixture of compounds containing non-binding agents (LCL-161 and GDC-0152) and strong binding agents (ABT-737 and A-1331852). After 35 and 70 minutes, the distribution of free Bcl-xL protein, representing the negative control group, was significantly reduced (2.5% and 0.7%, respectively). In contrast, the signal distribution of the Bcl-xL + strong binding agent complex gradually appeared, and finally, after 70 minutes, two distinct signal distributions for the Bcl-xL + A-1331852 and Bcl-xL + ABT-737 complexes emerged. At the same time point, the proportion of the Bcl-xL + ABT-737 complex gradually decreased (10.7% and 6.3%), while the proportion of the Bcl-xL + A-1331852 complex steadily increased (86.8% and 93.1%). These experimental results confirm that the YaxAB nanopore of the present invention can screen the binding between proteins and drugs that bind to proteins at a single-molecule level with high resolution.

[0199] This suggests that YaxAB nanopores can sensitively distinguish between different small-molecule drugs bound to the same protein with excellent resolution, and that this allows for single-molecule fingerprinting of protein-drug interactions using biological nanopore sensors. This provides a concept and examples of molecular fingerprinting. These results demonstrate that the system using the YaxAB nanopore of the present invention can detect and analyze small molecular weight compounds more inexpensively, quickly, and with higher sensitivity compared to conventional biophysical analysis methods such as ITC, NMR, and SPR.

[0200] [4-6] Structure-activity relationship (SAR) analysis and binding site mapping. On the other hand, we confirmed whether the YaxAB nanopore of the present invention could be used to analyze the binding sites where protein-small molecule interactions occur. To this end, we first reacted 200 nM Bak-BH3 protein with 100 nM Bcl-xL protein, and then treated the mixture with ABT737 compound, a competitive inhibitor of Bak-BH3 protein, at a concentration of 200 nM.

[0201] As a result, it was confirmed that as the reaction time increased, the signal of the complex between Bcl-xL protein and Bak-BH3 protein (Bcl-xL+Bak-BH3) gradually decreased (87.6%, 34.0%, 2.7%), while the signal of the complex between Bcl-xL protein and ABT737 compound (Bcl-xL+ABT737) increased (0%, 60.3%, 94.9%) (Figure 18b). Current cutoff (ΔI / I o )-current noise (I N In the 2D density contour plot of ), the distribution of the Bcl-xL+Bak-BH3 complex gradually shifts over time to the distribution location of the newly formed Bcl-xL+ABT-737 complex, and 40 minutes after the addition of ABT-737, only the Bcl-xL+ABT-737 complex remains. This means that ABT-737 completely substituted the Bak-BH3 peptide bound to Bcl-xL (see Figures 18b-18d). From these results, we were able to confirm that ABT-737 cleaves the already bound Bak-BH3 and binds to its position.

[0202] Furthermore, after reacting 100 nM Bcl-xL protein with 200 nM ABT737 compound, the mixture was treated with 200 nM A-1331852 compound. As a result, it was confirmed that as the reaction time increased, the signal of the complex between Bcl-xL protein and ABT737 compound (Bcl-xL + ABT737) gradually decreased (93.0%, 52.5%, 26.6%), while the signal of the complex between Bcl-xL protein and A-1331852 compound (Bcl-xL + A-1331852) increased (6.0%, 46.6%, 72.5%) (see Figures 18e to 18g). These results confirmed that A-1331852 competes with ABT-737 for binding to the same site where ABT-737 is already bound.

[0203] This indicates that when using the YaxAB nanopore of the present invention, it is possible to screen for drug binding activity to proteins at the single-molecule level, and furthermore, structure-activity relationship (SAR) analysis is also possible.

[0204] [4-7] Peptide drug screening that binds to target proteins We used the YaxAB nanopore sensor of the present invention to analyze the interaction between target proteins and peptides, thereby performing peptide drug screening.

[0205] First, a solution containing 5 mM HEPES (pH 7.5), 1 mM EDTA, and 1 M KCl was placed in each compartment of the YaxAB nanopore sensor, along with Bcl-xL protein, three Bak-BH3 mutant peptides that bind to Bcl-xL protein (Bak_BH3_D84A, Bak_BH3_I85A, Bak_BH3_L78A), and the wild-type Bak_BH3 peptide. Each compound was reacted in a molar ratio of 1:15 and added to the cis compartment, after which a (+) voltage was applied. The current cutoff signals from the Bcl-xL protein and Bcl-xL+ peptide drug conjugates that appeared at this time were observed and analyzed, and the current noise (SD) - current cutoff (ΔI / I) was calculated. oWe checked the 2D scatter plot of ) (see Figures 19a and 19b).

[0206] Based on that, Bcl-xL protein and three mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, Bak_BH3_L78A) and wild-type Bak_BH The solution containing the three peptides was reacted in a molar ratio of 1:15, and the current cutoff signal was measured. As a result, a unique current cutoff signal was observed when each peptide was bound, and it was confirmed that the scatter plot results, measured and analyzed with Bcl-xL protein and the four mixed peptides, matched the scatter plot trends observed in the individual experiments (see Figure 19c).

[0207] Furthermore, the signal frequency of the Bcl-xL-peptide complex was 51% for wild-type Bak_BH3. Bak_BH3_D84A showed 39%, Bak_BH3_I85A 7%, and Bak_BH3_L78A 3%, which corresponds to the order of binding affinity between the Bcl-xL protein and each peptide (wild-type Bak_BH3 0.34 μM; Bak_BH3_D84A 0.14 μM; It matched Bak_BH3_I85A (93μM) and Bak_BH3_L78A (270μM).

[0208] Based on the results described above, the YaxAB nanopore of the present invention enables binding affinity-based screening for peptide drugs that bind to proteins, and effectively selects peptide drugs that have a high binding affinity to target proteins even in peptide mixed solutions. Because of this capability, we confirmed that it can be usefully utilized as a drug screening platform.

[0209] [4-8] Drug binding selectivity profiling for multiple proteins Experiments were conducted to confirm whether the YaxAB nanopore sensor of the present invention is capable of profiling the protein binding selectivity of drugs against multiple Bcl-2 system proteins (Bcl-xL, Bcl-w, Bcl-2, Mcl-1).

[0210] First, a solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 M KCl was placed in each compartment of the YaxAB nanopore sensor. In the cis compartment, 50 nM of Bcl-xL, Bcl-w, Bcl-2, and Mcl-1 (all Bcl-2 system proteins) and the mTOR protein used as a negative control group were added, and a (+) voltage was applied.

[0211] In this process, we observed that the nanopore current cutoff signal for each protein appeared in a unique pattern that allowed us to distinguish them all, and from this, we determined the current noise (SD) - current cutoff (ΔI / I o When the 2D scatter plot of ) was shown, we were able to confirm the signal distribution results from each protein (see Figures 20a to 20e).

[0212] Furthermore, when equal amounts of Bcl-2 proteins (Bcl-xL, Bcl-w, Bcl-2, Mcl-1) and the negative control group mTOR protein were mixed and added to the cis compartment at a concentration of 50 nM, and then a (+) voltage was applied, we were able to observe unique current interruption signals originating from Bcl-xL, Bcl-w, Bcl-2, Mcl-1, and mTOR proteins. In addition, as a result of nanopore current interruption signal analysis from the mixed solution, the signal distributions of the four Bcl-2 proteins and the one negative control protein could be confirmed on a 2D scatter plot (see Figure 20f).

[0213] After adding A1155463, a small molecule drug targeting Bcl-xL, at a concentration of 50 nM in the same proportion, the nanopore current cutoff signal was analyzed. The results showed that while protein-specific current cutoff signals appeared for Bcl-w, Bcl-2, Mcl-1, and mTOR, the specific current cutoff signal for the target protein Bcl-xL disappeared, and a new current cutoff signal appeared. The signal analysis results, as measured above, confirmed the signal distributions for Bcl-w, Bcl-2, Mcl-1, and mTOR proteins on a 2D scatter plot, while the signal distribution for the target protein Bcl-xL disappeared, and a new signal distribution, presumably for the complex of Bcl-xL and A1155463, appeared (see Figure 20g).

[0214] Based on the results described above, we verified that the YaxAB nanopore sensor enables profiling of Bcl-2 proteins and allows for the identification of drugs with selectivity for target proteins.

[0215] [Example 5] Presence or absence of ternary complex analysis including protein-protein interaction enhancement analysis using molecular adhesives with YaxAB nanopore sensor We investigated whether the YaxAB nanopore system of the present invention can be used to analyze the promotion of protein-protein interactions by molecular adhesives.

[0216] First, the mTOR-FRB domain and the FKBP12 protein cannot interact with each other on their own; protein-protein interactions are mediated by rapamycin, a molecular adhesive. Specifically, after the FKBP12 protein and rapamycin bind, the mTOR-FRB domain becomes FKBP12-rapamycin. It binds to the in complex to form a ternary complex (see Figure 21a).

[0217] Using the characteristics described above, a solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 M KCl was placed in each compartment of the YaxAB nanopore system. After adding 160 nM of mTOR-FRB domain protein (hereinafter referred to as mTOR protein) or FKBP12 protein to the cis compartment, a (+) voltage was applied to the cis compartment and the nanopore current cutoff signal was observed (see Figures 21b and 21c).

[0218] As a result, unique current cutoff signals appeared from both the mTOR protein and the FKBP12 protein, and scatter plot analysis confirmed that the signal distributions originating from each protein were clearly distinguishable from one another. Furthermore, when mTOR protein and FKBP12 protein were added to the cis compartment in a 1:1 molar ratio as another negative control group, and a (+) voltage was applied to observe the nanopore current cutoff signal, the current cutoff signals from the mTOR protein alone and the FKBP12 protein alone appeared mixed, and scatter plot analysis confirmed that the unique signal distributions originating from each protein were reproduced (see Figure 21f).

[0219] Based on the experimental results described above, when mTOR protein, FKBP12 protein, and rapamycin (which acts as a molecular adhesive) were added to the cis compartment in a molar ratio of 1:1:10, and a (+) voltage was applied to observe the current cutoff signal, a novel current cutoff signal was observed along with the current cutoff signals corresponding to mTOR protein and FKBP12 protein (see Figure 21g).

[0220] Furthermore, to confirm that the novel signal was not a binary complex formed by the binding of mTOR or FKBP12 protein to the molecular adhesive, but rather a current-cutting signal from a ternary complex in which mTOR, FKBP12, and the molecular adhesive were all bound, mTOR protein or FKBP12 protein and rapamycin were added in a 1:1 ratio to the cis compartment of the YaxAB nanopore system, and then a (+) voltage was applied to observe the current-cutting signal (Figures 21d and 21e).

[0221] As a result, the presence or absence of rapamycin did not significantly alter the current-blocking signals derived from the mTOR protein and FKBP12 protein. Therefore, it was confirmed that the novel current-blocking signal originated from a ternary complex in which the mTOR protein, FKBP12 protein, and rapamycin were all bound.

[0222] From the results described above, it was found that when using the YaxAB nanopore system of the present invention, it is possible to distinguish between proteins and ternary complexes, thereby enabling the analysis of the promotion of protein-protein interactions by molecular adhesives.

[0223] Next, to determine whether molecular adhesives present in a mixed solution of various small molecules could be screened, negative small molecules that do not bind to the mTOR-FRB domain (Astemizole, Mibefradil, Terfenadine, Pranlukast, Capecitabine, Dimantine, Sulfanilamide, and Monobenzone) were selected using nuclear magnetic resonance spectroscopy (NMR spectroscopy). The presence or absence of rapamycin, a molecular adhesive, in the mixed solution of negative small molecules was observed to confirm whether molecular adhesive screening was possible.

[0224] Specifically, when a mixed solution of eight negative small molecule compounds was reacted with equal amounts of mTOR protein and FKBP12 protein in a molar ratio of 1:1:10, and then the nanopore current cutoff signal was observed, only current cutoff signals specific to mTOR protein and FKBP12 protein were observed. (See Figure 21h). On the other hand, when rapamycin, a molecular adhesive, was reacted with the aforementioned experimental conditions in a molar ratio of 1:1:10:10, a current interruption signal corresponding to a ternary complex was observed along with the mTOR protein and FKBP12 protein (see Figure 21i).

[0225] In summary, the results of the present invention demonstrate that the YaxAB nanopore system allows for the distinction between single proteins, binary complexes of proteins and small molecules, and ternary complexes of proteins, small molecules, and proteins. Furthermore, the YaxAB nanopore system can screen for the presence or absence of molecular adhesives in various small molecule compound mixtures without being inhibited by negative small molecules. Therefore, it was confirmed that the YaxAB nanopore system of the present invention can be used to screen for molecular adhesives that can promote protein-protein interactions.

[0226] [Example 6] Availability of selective detection and quantitative analysis of biomarker proteins using YaxAB nanopores We investigated whether selective detection and quantitative analysis of specific biomarker proteins are possible using the YaxAB nanopore system of the present invention in the presence of serum.

[0227] The Bcl-xL protein selected as the sample is a protein that is overexpressed in various types of malignant tumors, is well known as a target for cancer treatment agents, and can also be used as a biomarker for cancer diagnosis (Morales-Martinez et al., "Roles and Regulation of BCL-xL in Hematological Malignancies", Int.J.Mol.Sci. (2022)).

[0228] First, 800 μl each of 10 mM Tris pH 7.5, 1 mM EDTA, and 1 M KCl buffer were added to both compartments of the YaxAB nanopore system. Then, to confirm the electrical signal specific to the Bcl-xL protein, 50 nM of Bcl-xL protein was added to the cis compartment, and the resulting change in electrical signal was measured after applying a voltage of 80 mV. This was then statistically analyzed to determine the current noise (SD) - current cutoff (ΔI / I). o When a two-dimensional scatter plot composed of the above was shown, we were able to confirm the results of the two-dimensional scatter plot, which showed a uniform current pattern and a dense distribution, as shown in Figure 23a.

[0229] Next, in order to utilize the YaxAB nanopore system of the present invention for cancer diagnosis, an experiment was conducted to determine whether it is possible to detect trace amounts of Bcl-xL protein present in a mixed solution. First, in the presence of FBS (Fetal Bovine Serum), which is similar to human serum, the presence or absence of characteristic electrical signals specific to Bcl-xL at concentrations of 10 nM and 20 nM was confirmed. The FBS solution was used after removing some proteins such as albumin and immunoglobulin present in the serum using a depletion minispin column. Subsequently, 5 μl of the FBS solution was added to the cis compartment of the YaxAB nanopore system, which contained 800 μl each of 10 mM Tris pH 7.5, 1 mM EDTA, and 1 M KCl buffer in both compartments, and the resulting change in electrical signal was measured after applying a voltage of 80 mV.

[0230] As a result, various electrical signals were observed, and when these were statistically analyzed and shown in a two-dimensional scatter plot, it was confirmed that they were dispersed as shown in Figure 23b. Specifically, when a voltage of 80mV was applied to the cis compartment of the YaxAB nanopore system in the presence of depleted FBS (Flat Boomer Sulfide), in addition to the various nanopore electrical signals measured by the depleted FBS, a characteristic electrical signal specific to the 10nM Bcl-xL protein was observed. When this signal was analyzed and shown in a two-dimensional scatter plot, deple We were able to identify a signal distribution that matched the Bcl-xL protein-specific electrical signal within the various dispersed signals of ted FBS (see Figure 23c).

[0231] Furthermore, electrical measurements performed under 20 nM Bcl-xL protein conditions in the presence of depleted FBS showed that even more Bcl-xL-specific electrical signals were detected compared to under 10 nM Bcl-xL protein conditions. This was confirmed by the two-dimensional scatter plot results, which clearly showed a concentration-dependent clustering of Bcl-xL protein signals (see Figure 23d).

[0232] This confirmed whether it is possible to detect trace amounts of Bcl-xL protein present in the mixed solution. This verified that, when using the YaxAB nanopore system of the present invention, it is possible to detect and quantitatively analyze minute amounts of biomarker proteins in the mixed solution in a short time with ultra-high sensitivity and specificity. This confirmed the potential of the system to be used in the diagnosis of various diseases, including cancer and genetic disorders, and in early diagnosis where the detection of minute amounts of biomarkers is required.

[0233] On the other hand, as shown in Figure 24, experiments were conducted to confirm whether mutant Bcl-xL proteins, which are frequently observed in cancer patients, can be detected using the YaxAB nanopore system of the present invention. Specifically, the arginine residues at positions 100 and 103 of the Bcl-xL protein, which can be used as a biomarker for cancer diagnosis, were replaced with (-) charged glutamic acid, resulting in Bcl-xL_R100E and R100E, whose overall net charge changed from -11.5e to -14.8e. Bcl-xL_E31K and E36K mutant proteins were created by substituting the 3E mutant protein with positively charged lysine residues at positions 31 and 36, resulting in an overall net charge of -7.4e. Using mutant proteins, we confirmed whether it is possible to measure changes in electrical signals when amino acid residues in a protein are altered due to mutation (see Figure 24a).

[0234] Using the YaxAB nanopore system, Bcl-xL wild-type protein, Bcl-xL E31K, E36K mutant proteins, and Bcl-xL R100E, R103E mutant proteins were added under 10 mM Tris pH 7.5, 1 mM EDTA, and 1 M KCl buffer, and then the nanopore electrical signals were measured. As a result, it was observed that three distinct nanopore electrical signals, unique to each of the three proteins, appeared (see Figure 24b). This is because the net charge of the Bcl-xL protein affects the oscillation phenomenon of the sample protein within the pore, which is generated by the harmony of the electro-osmotic force (EOF) and the electrophoretic force (EPF) due to the applied voltage in the YaxAB nanopore.

[0235] For Bcl-xL_R100E and R103E mutant proteins, the smallest current shielding is observed. The signal frequency of L1, which is at the break level, increased. Bcl-xL_R100E, R103E sudden The mutation, due to an increased (-)charge compared to the wild-type Bcl-xL protein, was expected to generate a strong repulsive force by EPF, causing the protein to not remain in the YaxAB nanopore for long and to tend to escape. This tendency was confirmed by electrical signal analysis. Furthermore, the signal frequency of L3, which is the largest current cutoff level in Bcl-xL_E31K and E36K, was analyzed. When the degree increases, the (+)charge increases compared to the Bcl-xL wild-type protein, resulting in less influence from EPF and a tendency to occupy a deeper position inside the YaxAB nanopore, which was also revealed by the increased electrical signal at L3.

[0236] Thus, when the net charge of a protein changes, the protein within the nanopore Because it affects the vibrational motion of the protein, it can also affect the generated electrical signals. As a result, we were able to confirm a change in the electrical signal that was remarkably distinguishable even with a change of just two residues. Therefore, when using the YaxAB nanopore system of the present invention, it is possible to accurately detect and distinguish not only Bcl-xL wild-type protein but also minute amounts of mutant proteins, demonstrating the diagnostic potential for various diseases such as cancer or genetic disorders in which the detection of mutant proteins observed in cancer patients is required.

[0237] [Example 7] Whether electroosmotic or electrophoretic-based detection and analysis of samples passing through YaxAB nanopores is available. In the YaxAB nanopore of the present invention, we confirmed whether it is possible to detect and analyze single-molecule proteins and nucleic acids in samples passing through the nanopore using electroosmosis or electrophoresis.

[0238] First, in the case of mTOR, a small monomolecule compound that carries a weak negative charge at pH 7.5, the strong cation selectivity of the YaxAB nanopore allowed it to pass through the nanopore by electroosmosis, and the change in ion current generated at this time was measured. As a result, as shown in Figure 25, it was confirmed that the time it took for the monomolecule protein to pass through the nanopore increased as the voltage applied to the cis and transform compartments increased, and that the time it took to pass through the nanopore became faster when a certain threshold voltage was reached. This confirmed that, in the YaxAB nanopore, when the sample is similar in diameter to the pore, it is possible to detect and analyze the sample by using electroosmosis to pass the sample through, rather than being limited to a method of vibration after the sample flows in using electroosmosis.

[0239] Next, in the case of EGF, a small monomolecule compound that is negatively charged at pH 7.5, after being added to the trans compartment, it was passed through the YaxAB nanopore of the present invention by electrophoresis, and the change in the ion current generated at this time was measured. As a result, as shown in Figure 26, it was confirmed that the time it takes for the monomolecule protein to pass through the nanopore increases as the voltage applied to the cis and trans compartments increases. Furthermore, p53, an intrinsically disordered protein (IDP) with a small molecular weight of 1.8 kDa, was also tested. TAD1 In the case of peptides, after being added to the trans compartment, they were passed through the YaxAB nanopore of the present invention by electrophoresis, and the change in the ion current generated at this time was measured. As shown in Figure 27, p53 TAD1 We were able to confirm the ion current blockage phenomenon before and after the addition of the peptide. Furthermore, p53, which has a molecular weight of 8.3 kDa, was also observed. TAD In the case of proteins, as shown in Figure 27, p53 TAD We were able to confirm the ion current blockage phenomenon before and after protein addition. This indicates that it is possible to detect and analyze intrinsically disordered protein and peptide samples using electrophoresis in the YaxAB nanopore.

[0240] On the other hand, as shown in Figure 28, when Lambda DNA, single-stranded nucleic acid (50mer), and double-stranded nucleic acid (23bp) were independently added to the trans compartment of the YaxAB nanopore and the pass-through was observed, an ion current blockage phenomenon was confirmed before and after the addition of each nucleic acid. From these results, it was found that with the YaxAB nanopore of the present invention, intrinsically disordered proteins and nucleic acids can be detected and analyzed not only by electroosmosis but also by electrophoresis.

[0241] [Example 8] Analysis of the purification efficiency of modified YaxAB nanopores In the YaxAB nanopore of the present invention, the purification efficiency of each monomer protein was compared based on amino acid sequence length to confirm whether the purification efficiency of modified monomers is improved compared to wild-type monomers.

[0242] Specifically, the first modified monomer (YaxA_(45-410)) having the amino acid sequence of SEQ ID NO: 1, purified in Examples 1-2, and the second modified monomer having the amino acid sequence of SEQ ID NO: 2 The monomer (YaxB_(12-343)) has the amino acid sequence of SEQ ID NO: 3 and is wild-type. 1 monomer (YaxA_FL), and wild-type 2 monomer having the amino acid sequence of SEQ ID NO: 4 The nomer (YaxB_FL) was purified in the same manner as in Examples 1-2. During preparation, YaxA_FL was prepared using 6 liters of E. coli culture solution, and YaxB_FL was prepared using 1 liter of E. coli culture solution. YaxA_(45-410) was prepared using 2 liters of E. coli culture solution. For YaxB_(12-343), 1 liter of E. coli culture solution was used.

[0243] As a result, as shown in Figure 29, after purification, the yield of each monomer per liter was 12 mg for YaxA_FL, 66.2 mg for YaxB_FL, and 66.2 mg for YaxA_(45-410). 56.1 mg and YaxB_(12-343) were 72.3 mg, and under similar purification conditions, The yield of the first modified monomer (YaxA_(45-410)) is the same as that of the first modified monomer of wild-type YaxAB. The yield was 4.7 times higher compared to Nomar (YaxA_FL). Also, under similar purification conditions... The yield of the modified second monomer (YaxB_(12-343)) is greater than that of the wild-type second monomer (Y The yield was 1.1 times higher compared to axB_FL). Based on the above results, modified Yax For each modified monomer constituting the AB_dN nanopore, under similar purification conditions, the wild-type monomer It was found to show a higher yield compared to the other method.

[0244] [Example 9] Analysis of the oligomerization mechanism of modified YaxAB nanopores The monomers purified in Example 6 were oligomerized into YaxAB nanopores (wild-type YaxAB, modified YaxAB_dN) using the same method as in Examples 1-3, and each nanopore was processed into a g After electrophoresis on radiant blue polyacrylamide gel, we identified the distinct oligomeric bands of wild-type nanopores and modified nanopores.

[0245] As a result, as shown in Figure 30, when low concentrations of the first and second monomers were used, no bands were observed in wild-type YaxAB nanopores, while modified YaxAB_dN nanopores were observed. It was found that the pores were well distinguishable according to the oligomer type. Furthermore, when high concentrations of the first and second monomers were used, the difference between modified YaxAB_dN and wild-type YaxAB nanopores was observed. It was found that the types of bands appearing are different. From the results above, the first monomer (YaxA_(45-410)) has the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 2 The second monomer (YaxB_(12-343)) having an acid sequence was purified under similar conditions. Compared to the wild-type YaxAB nanopore, it was found that not only was the yield higher, but more stable oligomerization was possible. Furthermore, it was confirmed that YaxAB-C8, composed of eight subunits that cannot be formed with the wild-type monomer, could be formed.

[0246] Furthermore, as shown in Figure 31, the electrical conductivity distribution of purified YaxAB nanopores for each band was shown, and in wild-type YaxAB nanopores, C9 and C were found to be different for each band. 10 , C 11 While C8, C9, and C2 were mainly observed in the modified YaxAB_dN nanopore, C8, C9, and C2 were observed in each band. 10 , C 11 It was found that this was the most prominent feature. Thus, it was discovered that the oligomerization mechanism of YaxAB nanopores can be regulated by altering the amino acid sequence of the first or second monomer.

[0247] Furthermore, the sequence length of Ya has been adjusted based on the sequence of YaxA_FL, the wild-type first monomer. The oligomerization tendency between xA_(X-411) and the second monomer YaxB_(12-343) was confirmed.

[0248] Specifically, YaxA_(X-411) is N-terminal in YaxA_FL(1-411). YaxA_(X) is the first monomer from which the sequence from the first amino acid to the Xth amino acid from the end has been removed, where X is 11, 21, 31, 41, 51, 61, 71, or 81. After preparing -411), YaxB_(12-343) is prepared in the same manner as in Examples 1-3 above. And it was oligomerized.

[0249] As a result, as shown in Figures 32 and 33, wild-type YaxA_FL(1-411), YaxA_(11-411), YaxA_(21-411), YaxA_(31-411) YaxA_(41-411), YaxA_(51-411), and YaxA_(61- When 411) is combined with YaxB_(12-343), YaxAB-C8 and Y axAB-C9, and YaxAB-C 10 YaxA_(71-411), Furthermore, it was confirmed that when YaxA_(81-411) and YaxB_(12-343) bind, the YaxAB-C7 oligomer is formed.

[0250] Furthermore, using YaxA_(X-410) with an adjusted YaxA sequence length, the second monomer Y The tendency for oligomerization with axB_(12-343) was confirmed. Specifically, YaxA_(X-410) is the Xth amino acid from the first amino acid at the N-terminus. After creating YaxA_(X-410), which is the first monomer obtained by removing the sequence up to the amino acid and the first amino acid from the C-terminus, and where X is 3, 5, 8, or 9, YaxB_(12-343) was oligomerized using the same method as in Examples 1-3.

[0251] As a result, as shown in Figure 33, YaxA_(3-410), YaxA_(5-410), YaxA_(8-410), and YaxA_(9-410) become YaxB_(12- It was confirmed that YaxAB oligomers are formed when each of the following compounds (343) is bound to YaxAB.

[0252] Furthermore, as shown in Figure 34, YaxA_(11-411), YaxA_(21-411), YaxA_(31-411), YaxA_(41-411), YaxA_(51- We confirmed that the YaxAB nanopore system, utilizing YaxAB oligomers formed when YaxA_(3-410) and YaxB_(12-343) are bound, respectively, can detect Bcl-xL proteins.

[0253] Subsequently, in order to confirm the smallest amino acid sequence capable of forming YaxAB nanopores, the C-terminus of the YaxA amino acid sequence was fixed, and sequences from YaxA_(52-410) to YaxA_(60-410) were created and the oligomerization tendency was analyzed. The result showed that YaxA_(57- When 410) and YaxB_(12-343) bind, a YaxAB oligomer is formed. However, it was confirmed that when YaxA_(58-410) and YaxB_(12-343) combine, no oligomer is formed. Furthermore, from YaxA_(57-409) The sequence up to YaxA_(57-391) was generated and the oligomerization tendency was analyzed, and the result was Y It was confirmed that YaxAB oligomers are formed in the case of axA_(57-398), but not in the case of YaxA_(57-397).

[0254] Furthermore, YaxB_(12-343) can form YaxAB nanopores using the method described above. Analysis of the minimum amino acid sequence of ) confirmed that it is YaxB_(26-342). Furthermore, when YaxA_(57-398) and YaxB_(26-342) bind, a YaxAB oligomer is formed, and this oligomer can successfully realize the nanopore-containing membrane in the present invention. This confirmed that the Bcl-xL protein can be successfully captured using the nanopore system.

[0255] Alternatively, in the opposite direction to the above method, the N-terminus of the YaxA amino acid sequence can be fixed, and YaxA_ The sequence from (45-390) to YaxA_(45-369) was constructed to determine the oligomerization tendency. Analysis of the direction revealed that in the case of YaxA_(45-383), YaxAB oligomers are formed. Furthermore, in the case of YaxA_(45-382), YaxAB oligomers are not formed. This was confirmed. Furthermore, by creating sequences from YaxA_(46-383) to YaxA_(58-383) and analyzing the oligomerization tendency, it was found that in the case of YaxA_(52-383) YaxAB oligomers are formed in the case of YaxA_(53-383), and in the case of YaxA It was confirmed that B oligomers were not formed.

[0256] Furthermore, by creating sequences from YaxB_(12-325) to YaxB_(12-314) using the method described above and analyzing the oligomerization tendency, we found that YaxB_(12-316) In this case, YaxAB oligomer is formed, and in the case of YaxB_(12-315), Ya It was confirmed that xAB oligomers were not formed. Furthermore, YaxB_(13-316 We created sequences from ) to YaxB_(18-316) and analyzed the oligomerization tendency. As a result, it was confirmed that YaxAB oligomers were formed in the case of YaxB_(12-316), but not in the case of YaxB_(13-316). Furthermore, when YaxA_(52-383) and YaxB_(12-316) bound, YaxAB oligomers were formed, and these oligomers were able to successfully realize the nanopore-containing membrane in the present invention. This confirmed that Bcl-xL protein can be successfully captured using the nanopore system.

[0257] As a result, as shown in Figures 32, 35, 36, and 37, the minimum amino acid sequence of YaxA that can form a YaxAB nanopore capable of constructing the nanopore-containing membrane of the present invention is YaxA_(57-398) having the amino acid sequence of SEQ ID NO: 7 and SEQ ID NO: 24 YaxA_(52-383) has the amino acid sequence, and YaxB has the minimum amino acid sequence The column contains YaxB_(26-342) with the amino acid sequence of SEQ ID NO: 18 and SEQ ID NO: 25 YaxB_(12-316) has the amino acid sequence, and Ya is a combination of these. We confirmed that oligomers can be produced using both xAB. Furthermore, even in the case of the oligomers formed by combining them, the nanopore-containing membrane of the present invention can be successfully realized, and this confirmed that the Bcl-xL protein can be successfully captured using the nanopore system (see Figure 36).

[0258] Furthermore, we confirmed that a nanopore subunit comprising a first monomer consisting of at least one of the amino acid sequences composed of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, and a second monomer containing the amino acid sequence of SEQ ID NO: 2, stably forms a YaxAB oligomer (see Figure 32).

[0259] Based on the above results, the minimum amino acid sequences of YaxA that can form YaxAB nanopores capable of constructing the nanopore-containing membrane of the present invention are YaxA_(57-398) having the amino acid sequence of SEQ ID NO: 7, YaxA_(52-383) having the amino acid sequence of SEQ ID NO: 24, and YaxA_(81-411) having the amino acid sequence of SEQ ID NO: 34. It was found that the minimum amino acid sequences of YaxB are YaxB_(26-342) having the amino acid sequence of SEQ ID NO: 18 and YaxB_(12-316) having the amino acid sequence of SEQ ID NO: 25, and based on the above examples, the minimum amino acid sequence that can form the YaxAB nanopore of the present invention was investigated (see Figures 32 to 36).

[0260] [Example 10] Confirmation of the analytical precision of sample-ligand interactions in YaxAB nanopores. [10-1] Is it possible to perform precise interaction analysis even when there is a large molecular weight difference between the sample and the ligand? The range of protein-small molecule compound complexes that can be analyzed using the YaxAB nanopore of the present invention I checked the enclosure.

[0261] Specifically, ii) 100 nM of FKBP12 protein (13.0 kDa) was added alone to the cis compartment of the sensor prepared in Examples 1-4, or ii) 100 nM of FKBP12 protein and 1 uM of FK506 were added in a molar ratio of 1:10, and (+) and (-) voltages were applied to the cis compartment and transform compartment, respectively.

[0262] As a result, as shown in Figure 38, it was confirmed that, unlike when FKBP12 protein was added alone, a change occurred in the current interruption pattern when FK506 was added together, confirming that it is possible to distinguish between the protein and the protein-small molecule compound complex (FKBP12 + FK506).

[0263] Furthermore, using the method described above, the cis compartment was subjected to either i) the addition of holo-transferrin protein (77.1 kDa) alone, or ii) the addition of oxaliplatin to holo-transferrin, followed by the application of voltage. As a result, as shown in Figure 39, a clear change in the flow blockage pattern appeared with the addition of oxaliplatin, confirming that it is possible to distinguish between the addition of the protein alone and the addition of the protein-small molecule compound complex (holo-transferrin + oxaliplatin).

[0264] In the protein-small molecule complex described above, the molecular weight ratio of FKBP12 to FK506 is 16:1, and the molecular weight ratio of holo-transferrin to oxaliplatin is 194:1. Therefore, we confirmed that the nanopore system of the present invention can sensitively distinguish between the signals of proteins and protein-small molecule complexes, despite the difference in molecular weight ratios of proteins to small molecule compounds ranging from 16 to 194 times.

[0265] [10-2] Confirmation of whether precise interaction analysis is possible even when there is a large concentration difference between the sample and the ligand or under high drug concentration conditions. To confirm whether the YaxAB nanopore system of the present invention can be usefully used for drug screening even under conditions where drugs are present at various concentrations, including high concentrations, the signals of protein-compound complexes were compared under conditions of target-specific compounds or target-nonspecific compounds at various concentrations.

[0266] Specifically, the interaction between the Bcl-xL protein and the small molecule compound ABT-737 was analyzed using the same method as in Example 4-1, but it was also confirmed whether drug binding activity could be screened under conditions where ABT-737 concentrations (binding ratios) of 200 nM (1:2) and 10 μM (1:100) were added.

[0267] As a result, as shown in Figure 40, the same results were obtained regardless of the concentration of the ABT-737 compound. This indicates that when using the YaxAB nanopore of the present invention, it is possible to screen for drugs by analyzing drug binding activity to proteins even under conditions where drugs are present at high (10 μM or higher) or low concentrations at the single-molecule level.

[0268] Furthermore, to confirm the presence or absence of nonspecific signal generation by non-target compounds, as shown in Figure 41, four types of low molecular weight compounds (ABT-737, A-1331852, LCL-161, and GDC-0152) were added to the YaxAB nanopore system of the present invention at concentrations of 200 nM or 1 μM, and the ion-blocking current signal was measured for 30 minutes. In addition, with the Bcl-xL protein trapped in the YaxAB-C8 nanopore, four types of non-target compounds (LCL-161, GDC-0152, Birinapant, and Phentolamine) were added to the cis compartment at a concentration of 10 μM (1:100) (concentration ratio), and the ion-blocking current signal was observed for 30 minutes.

[0269] As a result, as shown in Figure 41a, it was confirmed that adding only the compound did not affect the ion current flowing through the YaxAB-C8 nanopore. Furthermore, as shown in Figures 41b and 41c, the ion current signal when Bcl-xL was captured alone and when Bcl-xL and a non-target compound were added together were measured to be almost identical. This confirmed that the YaxAB nanopore system in the present invention can specifically detect the signal of the captured sample (Bcl-xL) even under conditions where four types of high concentrations of non-target compounds were added.

[0270] [Example 11] Confirmation of whether the nanopore system of the present invention operates even in solutions containing DMSO. Furthermore, in order to confirm whether the YaxAB nanopore system of the present invention operates stably even under conditions in which DMSO, which is necessary for detecting poorly soluble drugs, was added to Bcl-xL protein and three ligands (Bak-BH3 peptide, ABT-737, and A-1331852) in the same manner as in Example 4-1, under various DMSO concentrations (0, 0.01, 0.1, 0.5, 1, 2, 5, and 10%), and the signals of the complexes were observed.

[0271] As a result, as shown in Figure 42, the YaxAB nanopore system of the present invention clearly distinguishes the Bcl-xL capture signal alone and the ion current signals of each complex with four types of Bcl-xL ligands under DMSO concentration conditions of 5% or less, confirming that stable and precise analysis is possible.

Claims

1. A YaxAB nanopore having a funnel shape and including a first opening, an intermediate region, and a second opening, The outer diameter of the inner lumen of the first opening is 5 nm or more. The second opening includes a constricted portion, The diameter of the constricted portion is 0.5 nm or more. The outer diameter of the lumen of the second opening is 1 nm or more. A Yax AB nanopore having a lumen depth of 5 nm or more.

2. YaxAB nanopore comprising at least one subunit comprising a first monomer having an amino acid sequence identical by 70% or more to the amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34, and a second monomer having an amino acid sequence identical by 70% or more to the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:

25.

3. The YaxAB nanopore according to claim 1 or 2, wherein the YaxAB nanopore comprises 4 to 20 of the subunits.

4. The YaxAB nanopore according to claim 1 or 2, wherein the cation selectivity for anions is 1.1 times or more, or the anion selectivity for cations is 1.1 times or more.

5. The subunit comprises a first monomer containing any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 30, 31, 32, 33, and 34, A YaxAB nanopore according to claim 1 or 2, comprising a second monomer having one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO:

29.

6. The membrane layer, A nanopore-containing membrane comprising YaxAB nanopores inserted into the membrane layer, The membrane layer contains 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (1,2-dipalmitoyl-sn-glycero-3-phospho 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (1,2-dihexanoyl-sn-g lycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine (1,2-Dimyristoyl-sn-gl 1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPE), 1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPG), 1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (1,2-dioleoyl-sn-glycero-3-phosphoglycerol) Lycerol (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and mycolic acid (mycolic At least one selected from the group consisting of (acid), and campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine yl-sn-glycero-3-phosphocholine, PChemsPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine, ne, PChcPC), 1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), 10,12-pentacosadiic acid (PCDA), 10,12-tricosadiic acid (TCDA), 5,7-hexadecadiic acid (5,7-Hexadecadiic A nanopore-containing membrane comprising at least one selected from the group consisting of acid (HDDA) and 9,12-octadecadiynic acid (ODDA).

7. The YaxAB nanopore comprises a first monomer containing an amino acid sequence that is 70% or more identical to any one of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 24, SEQ ID NO: 34, or SEQ ID NO: 3, The nanopore-containing membrane according to claim 6, comprising at least one subunit comprising a second monomer having an amino acid sequence that is 70% or more identical to the amino acid sequence of any one of SEQ ID NO: 18, SEQ ID NO: 25, or SEQ ID NO:

4.

8. Chamber and It contains a Yax AB nanopore-containing membrane, A nanopore system in which the space within the chamber is divided into two compartments by the nanopore-containing membrane.

9. The nanopore system operates so that a sample (analyte) is transmitted, trapped, or gated through the pore. The passage, capture, or opening / closing of the aforementioned sample is performed by electrophoresis or electroosmosis. The characteristics of the sample are analyzed by detecting the oscillation movement or rotational motion of the sample that occurs when the sample is trapped inside the pore, clogging originating from the sample, the passage of the sample through the pore, or the opening and closing of the pore by the sample. The nanopore system according to claim 8, wherein the characteristics of the sample are at least one selected from the group consisting of surface charge, isoelectric point, stability, isomers, length, size, molecular weight, structure, dynamics, dipole moment intensity and orientation, orientation, flexibility, conformational heterogeneity, shape, quantity, and variations thereof.

10. The nanopore system according to claim 9, wherein the sample has a molecular weight of 1 to 1200 kDa.

11. The nanopore system according to claim 8, wherein the nanopore system is used for at least one selected from the group consisting of interaction analysis between a sample and a ligand, drug screening, nucleic acid sequencing, protein sequencing, peptide sequencing, protein identification, disease diagnosis, post-translational modification (PTM) analysis, proteome analysis, and structural analysis of biomolecules.

12. The steps of placing at least one specimen (analyte) in one compartment of the nanopore system according to claim 8, The steps include measuring the change in electrical signals in the two compartments before and after the placement of the sample, A monomolecule analysis method for a single or multiple samples, including [specific component].

13. The aforementioned sample is an inorganic particle composed of peptides, proteins, lipids, carbohydrates, inorganic substances, low molecular weight compounds, nucleic acids, or nanomaterials. The aforementioned electrical signal is an ion current pattern, and the basic current in the open state (I ｏ ), magnitude of current drop (ΔI), degree of current interruption (ΔI / I ｏ ), event duration, frequency per unit time of nanopore signal patterns, or current noise (I) Ｎ ) and The monomolecule analysis method for a single or multiple samples according to claim 12, wherein the analysis is the detection or identification of the sample.

14. The steps of processing one or two compartments of the nanopore system according to claim 8 with at least one sample (analyte) and at least one ligand candidate substance for the sample, The steps include measuring the change in electrical signals in the two compartments before and after processing the candidate substance, A method for analyzing the interaction between a sample and a ligand, or for screening ligands for a sample, including the analysis of the interaction between the sample and the ligand.

15. The aforementioned analysis, as a quantitative analysis, determines the binding affinity (dissociation constant, K) between the sample and the ligand. Ｄ A quantitative analysis of the interaction between a sample and a ligand or a method for screening a ligand for a sample, according to claim 14, which calculates the following:

16. The method for analyzing the interaction between a sample and a ligand or screening a ligand for a sample according to claim 14, wherein the interaction is competitive binding between a sample and ligands of multiple samples or competitive binding between multiple samples and ligands of samples, ligand binding site mapping of a sample, or structure-activity relationship (SAR).

17. The aforementioned sample is an inorganic particle composed of peptides, proteins, lipids, carbohydrates, inorganic substances, low molecular weight compounds, nucleic acids, or nanomaterials. The method for analyzing the interaction between a sample and a ligand or screening a ligand for a sample according to claim 14, wherein the ligand is a peptide, protein, lipid, carbohydrate, small molecule compound, or nucleic acid.

18. The steps of reacting multiple interacting biomolecules in one compartment of the nanopore system according to claim 8, The steps include treating one or two of the aforementioned compartments with a candidate substance for an interaction inhibitor or promoter, The steps include measuring the change in electrical signals in the two compartments before and after processing the candidate substance, A method for analyzing or screening for inhibitors or promoters of interactions between biomolecules, including those mentioned above.

19. The steps of placing a sample in one compartment of the nanopore system according to claim 8, The steps include comparing the characteristic changes in electrical signals caused by multiple samples in the aforementioned sample with a database of electrical signals for each substance, A method for identifying and quantitatively analyzing a sample in a specimen, including [specific details omitted].

20. The steps of placing a sample in one compartment of the nanopore system according to claim 8, The steps include measuring characteristic changes in electrical signals caused by a biomarker for a specific disease, among the changes in electrical signals induced by the aforementioned sample, A method for providing information for the diagnosis of biomarker-related diseases, including [specific examples of biomarker-related information].

21. The method for analyzing or screening biomolecular interaction inhibitors or promoters according to claim 18, wherein the analysis is a quantitative analysis that calculates the degree of binding affinity (dissociation constant, KD) between the plurality of biomolecules.

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