YaxAB nanopore, nanopore system comprising same and application thereof

Abstract

The invention relates to a YaxAB nanopore, a system comprising the nanopore and application of the YaxAB nanopore. The YaxAB nanopore of the present invention has a funnel-shaped three-dimensional structure so that the change in current blocking according to the movement of an analyte captured inside the nanopore can be maximized, thereby enabling very sensitive measurement even of the structure and minute changes in physical and chemical properties (including dynamic changes and the like) of the analyte. And can cause very strong electroosmosis with high cation selectivity, such that various types of analytes can be captured and analyzed without being limited to the type of total charge. In addition, with respect to the nanopore of the present invention, the amino acid sequence of the monomer constituting the nanopore can be adjusted to improve protein purification yield and control an oligomerization mechanism, and thus a YaxAB nanopore having a pore size that cannot be formed by a wild-type monomer can be prepared. Furthermore, with respect to the nanopores of the present invention, YaxAB nanopores of various pore diameters can be secured using one-time purification, and nanopores of desired pore diameters can be selectively secured. Thus, limitations of conventional biological nanopores having fixed pore diameters can be overcome, so that for the YaxAB nanopore of the present invention, the size of the nanopore can be adjusted according to the size of an analyte. Further, the YaxAB nanopores of the present invention have a very wide analyte analysis range of dynamic detection range from small analytes to large analytes, which is caused by a funnel-shaped three-dimensional structure having a wide inlet and a narrow outlet. In addition, the YaxAB nanopore of the present invention has the largest nanopore structure among the hitherto biological nanopores, and a large-size analyte, which cannot be detected by conventional biological nanopores, can be detected and analyzed in a single molecule on the basis of a funnel structure having a large inlet. Furthermore, the present invention has established an optimized composition of a membrane in which YaxAB nanopores can be stably present, and by using a sensor implemented on the basis thereof, it is possible to qualitatively and quantitatively analyze various types of analytes having relatively various sizes and charges at a single molecule level with very high resolution and sensitivity.

Description

[0001] This application claims priority to Korean Patent Application No. 10-2022-0138724, filed on October 25, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to a YaxAB nanopore and a nanopore system comprising the YaxAB nanopore, and to a novel nanopore sensing platform capable of accurately analyzing analytes with various sizes and charges at the single-molecule level, screening drugs bound thereto, analyzing interactions with ligands, or detecting and quantitatively analyzing disease biomarkers. Background Art

[0003] Protein-protein interactions (PPIs) are targets for effective disease treatment in drug discovery; however, methods for screening PPI inhibitors by high-throughput screening have not yet been commercialized. Studies on structure-activity relationships (SARs) and modes of action (MOAs) are essential for monitoring protein-drug interactions (PDIs) in vitro. Although various methods exist for direct drug screening, such as nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET), low-cost and efficient drug screening for protein-protein interactions is urgently needed due to expensive equipment, inaccuracies due to labeling / immobilization, low-sensitivity detection of small molecule drugs, and limited solubility of target proteins and / or drugs.

[0004] Nanopores are a new type of high-precision biosensor that can detect subtle morphological changes in biomolecules at the single-molecule level. Nanopores, in which individual nanopores are embedded in a thin insulating film, usually serve only as channels for allowing ionic currents to flow between two liquid chambers. Nanopore experiments using the principle of a Coulter counter are related to changes in ionic currents, and when ionic currents are induced through the nanopores by electrophoresis in the presence of an external electric field, the translocation of analytes through the nanopores involves a temporary blockage of ionic currents. This is measured as current amplitude, etc., and the length, size, charge, structure, shape, etc. of the biomolecules are determined based on these electrical signals. Nanopore biosensors have been used for genome sequencing, detection of various individual biomolecules, and detection of biomolecular interactions. Cytolysin A (ClyA) is a known pore-forming protein that forms dodecamer nanopores to tetradecamer nanopores. ClyA nanopores have a constriction with a diameter of 3.3 nm or more, and have been studied to monitor various biomolecules, protein-ligand interactions, etc. Biomolecular analysis techniques using nanopores have the advantages of single-molecule level resolution, high sensitivity, label-free, and real-time measurement, and therefore, have been actively developed. However, these nanopores are based on the recognition of large structural changes induced by ligand binding, and have limitations for targets that cannot be used with drugs in a conventional manner, such as non-drugable targets. In addition, since proteins basically have various surface charges, the principle of electrophoretic force (EPF) that is basically used as a driving force to attract analytes in nanopores has had limitations in capturing protein analytes.

[0005] As a result of intensive efforts to develop a method for accurately and effectively screening protein-protein interaction inhibitors even with extremely small amounts of sample, the present inventors have discovered a novel biological nanopore that utilizes electroosmotic force (EOF) that occurs according to the surface charge inside the pore, and by measuring electrical signals using a novel nanopore sensing platform including the novel biological nanopore, single-molecule proteins or nucleic acids can be detected based on changes in electrical signals, protein-drug interactions, protein-protein interactions, and even small molecule drugs that inhibit such protein-protein interactions can be effectively screened, and furthermore, screening based on "drug fingerprints" at the single-molecule level that can sensitively distinguish subtle differences between different drugs binding to the same protein can be performed, thereby completing the present invention. Summary of the invention

[0006] Technical issues An aspect of the present invention is to provide a novel biological nanopore subunit.

[0007] Another aspect of the present invention is to provide a nanopore comprising nanopore subunits.

[0008] Another aspect of the present invention is to provide a nanopore-containing membrane, comprising: a membrane layer; and a nanopore, comprising at least one nanopore subunit and inserted into the membrane layer.

[0009] Another aspect of the present invention is to provide a nanopore system, comprising: a chamber; and a membrane containing nanopores, wherein the space in the chamber is divided into two compartments by the membrane containing nanopores.

[0010] Another aspect of the present invention is to provide a single molecule analysis method for a single analyte or multiple analytes using a nanopore system.

[0011] Another aspect of the present invention is to provide a method for analyzing the interaction between at least one analyte and at least one ligand or screening a ligand for the analyte using a nanopore system.

[0012] Another aspect of the present invention is to provide a method for analyzing or screening an inhibitor or promoter of interaction between interactable biomolecules using a nanopore system.

[0013] Yet another aspect of the present invention is to provide a method for identifying and quantifying an analyte in a sample using a nanopore system.

[0014] Yet another aspect of the present invention is to provide a method for providing information on diagnosing a biomarker-related disease using a nanopore system.

[0015] Technical Solution In order to achieve the above-mentioned purpose of the present invention, the present invention provides a YaxAB nanopore, which includes a first opening, a middle area and a second opening, and the YaxAB nanopore has a funnel shape, wherein the outer diameter of the lumen of the first opening is 5nm or greater, the second opening includes a narrowing portion, the diameter of the narrowing portion is 0.5nm or more, the outer diameter of the lumen of the second opening is 1nm or greater, and the depth of the lumen is 5nm or greater.

[0016] In addition, the present invention provides a YaxAB nanopore, comprising at least one subunit comprising a first monomer and a second monomer, the first monomer having an amino acid sequence having 70% or more homology to the amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34, and the second monomer having an amino acid sequence having 70% or more homology to the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25.

[0017] In addition, the present invention provides a membrane containing nanopores, the membrane containing nanopores comprising: a membrane layer; and a YaxAB nanopore inserted into the membrane layer, wherein the membrane layer comprises at least one selected from the group consisting of: 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPhPC ...choline (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPh -L-serine (DPPS), 1,2-adipoyl-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-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-dioleoyl-sn-glycero -3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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, and at least one selected from the group consisting of campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesteryl hemisuccinyl-sn- Glyceryl-3-phosphocholine (PChemsPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glyceryl-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesterylcarbonyl-sn-glyceryl-3-phosphocholine (PChcPC), 1,2-dicholesteryl hemisuccinyl-sn-glyceryl-3-phosphocholine (DChemsPC), 10,12-pentacosadiynoic acid (PCDA), 10,12-tricosadiynoic acid (TCDA), 5,7-hexadecanediynoic acid (HDDA), and 9,12-octadecadiynoic acid (ODDA).

[0018] In addition, the present invention provides a nanopore system, comprising: a chamber; and a membrane containing a YaxAB nanopore, wherein the space in the chamber is divided into two compartments by the membrane containing the nanopore.

[0019] In addition, the present invention provides a single molecule analysis method for a single analyte or multiple analytes, the single molecule analysis method comprising the following steps: placing at least one analyte in one compartment of a nanopore system; and measuring changes in electrical signals in two compartments before and after placing the analyte.

[0020] In addition, the present invention provides a method for analyzing the interaction between an analyte and a ligand or screening a ligand for an analyte, the method comprising the following steps: performing treatment with at least one analyte and at least one ligand candidate for the analyte in one or two compartments of a nanopore system; and measuring changes in electrical signals in the two compartments before and after treatment with the candidate.

[0021] In addition, the present invention provides a method for analyzing or screening inhibitors or promoters of interactions between biological molecules, the method comprising the following steps: reacting multiple biomolecules capable of interacting in one compartment of a nanopore system; performing treatment with candidates for interaction inhibitors or promoters in one compartment or two compartments; and measuring changes in electrical signals in the two compartments before and after treatment with the candidates.

[0022] In addition, the present invention provides a method for identifying and quantitatively analyzing analytes in a sample, the method comprising the following steps: placing the sample in a compartment of a nanopore system; and comparing changes in electrical signals caused by multiple analytes in the sample with a database of electrical signals for each substance.

[0023] In addition, the present invention provides a method for providing information about diagnosing biomarker-related diseases, the method comprising the following steps: placing a sample in a compartment of a nanopore system; and measuring changes in characteristic electrical signals caused by biomarkers for a specific disease among changes in electrical signals induced by the sample.

[0024] Beneficial Effects Because the amino acids constituting the inner wall of the pore are negatively charged under the condition of pH 7.5, the YaxAB nanopore of the present invention can induce electroosmosis by cations flowing mainly along the inner wall of the pore; therefore, the analyte can be captured inside the nanopore regardless of the total charge. In addition, the structure of the YaxAB nanopore, including the diameter, can be adjusted according to the number of heterodimers composed of two monomers, YaxA and YaxB, and YaxAB nanopores of various sizes can be ensured by only one purification process, so there is an advantage that the object of analysis is not limited by the charge or size of the analyte. In addition, since the nanopore has a funnel structure in which the entrance is wide but the exit is relatively much narrower, even large analytes can be captured one by one inside the nanopore, and in addition, due to the high ion density in the narrow area, the current blocking from the analyte changes significantly compared to the conventional cylindrical nanopore, so that even subtle changes in the physicochemical properties of the analyte can be detected more sensitively, and in addition, analytes of various sizes can also be analyzed.

[0025] Based on these unique physical and chemical properties, when the YaxAB nanopore of the present invention is used, a wider range of analytes can be detected with a higher resolution than conventional nanopores, and in addition, protein-small molecule compound complexes can be directly detected, and in addition, protein-small molecule compound complexes in which different small molecule compounds are bound can be sensitively distinguished at the single molecule level. Therefore, compared with conventional technologies, YaxAB nanopores can be effectively used in various ways in terms of time and cost for the interaction between analytes and ligands, analysis of the structure and dynamics of biomolecules, new drug screening, disease diagnosis, protein identification, protein post-translational modification (PTM) analysis, genomic and proteomic analysis, and protein sequencing and nucleic acid sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Bands of different oligomers obtained by electrophoresis of the modified YaxAB_dN nanopore using a gradient blue native polyacrylamide gel are shown. The modified YaxAB_dN nanopore is one of the modified YaxAB nanopores having an amino acid sequence modified from the wild-type YaxAB, and refers to a YaxAB oligomer formed by 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 modified YaxAB_dN-C was verified by three separate bands. 8 、YaxAB_dN-C 9 and YaxAB_dN-C 10 existence.

[0027] Figure 2Figures a to c show the observation of modified YaxAB_dN-C using negative staining EM (negative staining electron microscopy). 8 Nanopore, YaxAB_dN-C 9 Nanopores and YaxAB_dN-C 10 The results of nanopores, Figure 2 D shows a 3D reconstruction based on negative staining EM results. Figure 2 Figure e shows the modified YaxAB_dN-C based on the results of cryo-electron microscopy (Cryo-EM). 8 The structure of the nanopore.

[0028] Figure 3 is a cross-sectional view of a YaxAB_dN nanopore showing the modification of the structure of the nanopore.

[0029] Figure 4 a in FIG. 1 shows the result of molecular dynamics (MD) simulation, which shows that each of potassium ions, chloride ions, and water molecules flows in the modified YaxAB_dN nanopore when 100 mV is applied. Figure 4 Panels b and c show the results of molecular dynamics simulations when the Bcl-xL protein is located at a distance of 4 nm and 10 nm from the membrane, respectively.

[0030] Figure 5 A in FIG. 1 is a schematic diagram of an analyte capture event and shows a single molecule analyte signal when the Bcl-xL protein is captured in the modified YaxAB_dN nanopore. Figure 5 B in FIG. 1 is a schematic diagram of MD simulation results when the analyte exists at a distance of 4 nm and 10 nm from the membrane, and shows the free energy of the analyte according to the distance (z) from the membrane calculated by the MD simulation.

[0031] Figure 6 Results are presented showing that the modified YaxAB_dN nanopores achieved better stability of the DPhPC-sterol composite membranes compared to membranes formed with DPhPC alone. Figure 6 A in FIG. 5 shows the change in conductivity when the modified YaxAB_dN nanopore is inserted into a membrane formed of DPhPC alone and the conductivity after insertion, indicating that membrane rupture occurs. Figure 6 B shows the modified YaxAB_dN-C 8 Nanopore, YaxAB_dN-C 9 Nanopore, YaxAB_dN-C 10 Stable operation of the nanopore when inserted into the DPhPC-sterol composite membrane, wherein C represents a heterodimeric subunit composed of a first monomer and a second monomer, and C 8The YaxAB nanopore consists of eight pairs of heterodimeric subunits (hereinafter referred to as YaxAB-C 8 ).

[0032] Figure 7 It is shown that when voltage is applied under the conditions of modified YaxAB_dN nanopores with various sizes, a constant current flows compared to the applied voltage.

[0033] Figure 8 The results of purified and electrophoresed protein bands and the conductivity measurements of each nanopore show that the modified YaxAB_dN nanopores (C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 and C 15 ) formation trend.

[0034] Fig. 9 Figure a shows the modified YaxAB_dN-C inserted into the DPhPC-sterol composite membrane. 8 Schematic diagram of electrical measurements of a nanopore. Fig. 9 b shows the lumen structure connected by eight α-helices. Fig. 9 The c in the above is the modified YaxAB_dN-C 8 A cross section of a nanopore showing the surface charge becoming more negative as it approaches the trans opening, Fig. 9 d shows the modified YaxAB_dN nanopore (C 8 , C 9 and C 10 ) and the cation selectivity (P K + / P Cl - ).

[0035] Fig.10 The results of single molecule analysis of nine types of proteins (such as untagged proteins Hsp33, Bcl-xL, holotransferrin, FKBP12, MDM2, BSA, aldolase and ferritin, and thyroglobulin) using the modified YaxAB_dN nanopore are shown.

[0036] Fig.11 Shown are current blockade and current noise analysis of protein-ligand interactions using modified YaxAB_dN nanopores, Fig.11a shows a schematic diagram of Bcl-xL, Bcl-xL+Bak-BH3 complex and Bcl-xL+ABT-737 complex in the pore and the result of measuring the current blocking signal, Fig.11 (b) shows the dwell time ratio of each current blocking level (L1-L3), Fig.11 c shows the power spectral density of Bcl-xL, Bcl-xL+Bak-BH3 complex and Bcl-xL+ABT-737 complex, Fig.11 Figures d to f show the current noise (I N )’s analysis results.

[0037] Fig.12 The analysis of Bax-BH3 peptide with low binding affinity to Bcl-xL protein using modified YaxAB_dN nanopore (K D = 13 μM) binding results, Fig.12 a in the figure shows the change of the ion current signal depending on whether the Bax-BH3 peptide binds to the Bcl-xL protein. Fig.12 b shows the current noise (I N ), Fig.12 c is a scatter plot showing changes in the ion current trajectory pattern depending on whether the Bax-BH3 peptide binds to the Bcl-xL protein.

[0038] Fig.13 The modified YaxAB_dN nanopore was used to analyze small molecule drugs (quercetin, K D = 1.1 μM) binding results, Fig.13 a in the figure shows the change of ion current signal depending on whether quercetin binds to Bcl-xL protein. Fig.13 b shows the current noise (I N ), Fig.13 c is a scatter plot showing changes in the ion current trace pattern depending on whether quercetin binds to the Bcl-xL protein.

[0039] Fig.14 The results of electroosmosis-based GBP protein (glucose / galactose binding protein) detection using the modified YaxAB_dN nanopore are shown. Fig.14 A in FIG. 1 shows a schematic diagram of electroosmosis-based GBP protein detection in a modified YaxAB_dN nanopore. Fig.14B in the figure shows a current blockade signal for the GBP protein and a current blockade signal caused by a structural change of the GBP protein that occurs when the GBP protein binds to galactose as an allosteric activator. Fig.14 Panel C shows the current blockade signal for lysozyme protein. Fig.14 D shows the current blockade signal for whole myoglobin. Fig.14 Panel E shows a two-dimensional scatter plot showing the results of analyzing the current blockade signal, standard deviation, and dwell time factors for the lysozyme protein using a two-dimensional scatter plot.

[0040] Fig.15 a in FIG. 1 shows a Hill curve graph showing the binding rate of the Bcl-xL+Bak-BH3 complex according to the concentration of the Bak-BH3 peptide, Fig.15 b shows a Hill curve graph showing the binding rate of the Bcl-xL+ABT-737 complex according to the concentration of ABT-737, Fig.15 Panel c shows a Hill curve graph showing the binding rate of the Bcl-xL+A-1331852 complex according to the concentration of A-1331852.

[0041] Fig.16 a shows a schematic diagram of the analyte and electrical signal results in the pore when only free Bcl-xL is present, when Bcl-xL reacts 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 in a state where Bak-BH3 and Bcl-xL are pre-reacted, using the modified YaxAB_dN nanopore. Fig.16 b shows the results of measuring the signal frequencies of three types of analytes (Bcl-xL, Bcl-xL+Bak-BH3, and Bcl-xL+ABT-737) when treated with different concentrations of a small molecule compound (ABT-737) in a state where Bcl-xL and Bak-BH3 were pre-reacted. Fig.16 Panel c shows current traces showing three types of analyte signals.

[0042] Fig.17 The modified YaxAB_dN nanopore is used to detect the interaction between Bcl-xL protein and small molecule compounds. Fig.17 a shows the ion current traces when Bcl-xL protein was treated with non-binding agents (LCL-161 and GDC-0152) and binding agents (ABT-737 and A-1331852) alone. Fig.17b shows the current blockade (ΔI / Io)-current noise (I N ) 2D density contour plot. Fig.17 Figure c shows the current blockade (ΔI / Io)-current noise (I N ) and the results of real-time monitoring.

[0043] Fig.18 The results of analyzing the competitive binding of drugs to Bcl-xL protein using the modified YaxAB_dN nanopore are shown. Fig.18 a in the figure is a schematic diagram of the competitive binding between the drug and the Bcl-xL protein. Fig.18 b shows the ion current trace when the Bcl-xL / Bak-BH3 complex is treated with ABT-737. Fig.18 c and d are real-time 2D density contour plots and violin plots showing competitive binding of Bak-BH3 and ABT-737 to Bcl-xL protein, respectively. Fig.18 e shows the ion current trace when the Bcl-xL+ABT-737 complex was treated with A-1331852, Fig.18 Panels f and g are real-time 2D density contour plots and violin plots showing competitive binding of ABT-737 and A-1331852 to Bcl-xL protein, respectively.

[0044] Fig.19 The results of detecting peptides bound to Bcl-xL protein using the modified YaxAB_dN nanopore and the results of peptide drug screening are shown. Fig.19 a in the figure shows the current blocking signal derived from the Bcl-xL protein and Bcl-xL+peptide drug complex that appeared after the Bcl-xL protein reacted with the wild-type Bak_BH3 peptide or three types of Bak_BH3 mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, and Bak_BH3_L78A). Fig.19 b shows a 2D scatter plot of current noise (SD)-current blockade (ΔI / Io) for the current blockade signal derived from Bcl-xL protein and Bcl-xL+peptide drug complex, Fig.19c shows a scatter plot analyzing current blockade signals that appeared after the Bcl-xL protein reacted with a solution obtained by mixing three types of mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, and Bak_BH3_L78A) and wild-type Bak_BH3.

[0045] Fig. 20 The results of electroosmosis-based detection of Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1) using modified YaxAB_dN nanopores are shown. Fig. 20 a to e in Figures 2 show the current blockade signal pattern and the 2D scatter plot of current noise (SD)-current blockade (ΔI / Io) for each protein. Fig. 20 f in FIG. 1 shows a current blockade signal that appeared after reacting a solution obtained by mixing equal amounts of Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1) and mTOR protein as a negative control, and a scatter plot result obtained by analyzing the current blockade signal. Fig. 20 g in FIG. 1 shows a current blockade signal that appears after A1155463, a small molecule drug targeting Bcl-xL at an equal ratio, is further added to the mixed solution, and a scatter plot result obtained by analyzing the current blockade signal.

[0046] Fig.21 The results of detecting ternary complexes (protein complexes containing three different molecules bound together) using the modified YaxAB_dN nanopore are shown. Fig.21 a in FIG. 1 is a schematic diagram showing that the mTOR-FRB domain and the FKBP12 protein can form a protein-protein interaction through rapamycin. Fig.21 b and c in the figure respectively show the current blocking signals for mTOR and FKBP12 proteins, and the scatter plots obtained by analyzing the current blocking signals. Fig.21 d and e in FIG. 3 show current blockade signals for the binary complex of mTOR and rapamycin and the binary complex of FKBP12 and rapamycin, respectively, and scatter plots obtained by analyzing the current blockade signals. Fig.21 f shows a current blockade signal that occurs when mTOR and FKBP12 are added together, and a scatter plot obtained by analyzing the current blockade signal. Fig.21 g shows a current blockade signal that occurs when mTOR, FKBP12, and rapamycin are added together, and a scatter plot obtained by analyzing the current blockade signal. Fig.21h in FIG. 1 shows the current blockade signal that appears when mTOR and FKBP12 are added together with eight types of negative small molecule compounds that do not bind to the mTOR-FRB domain (astemizole, mibefradil, terfenadine, pranlukast, capecitabine, dimethylmantanamide, sulfonamide, and monobenzone), and the scatter plot results obtained by analyzing the current blockade signal. Fig.21 i in FIG. 1 shows current blockade signals that appear when mTOR and FKBP12 are added together with eight types of negative small molecule compounds and rapamycin, and a scatter plot result obtained by analyzing the current blockade signals.

[0047] Fig. 22 The results of the interaction analysis between the BRD4-BD1 protein and post-translationally modified (PTM) histone peptides and the analysis of interaction inhibitors using the modified YaxAB_dN nanopore are shown. Fig. 22 A and B in the figure show the schematic diagram of the binding interaction between the H4 peptide (H4_1-12) composed of 12 amino acids from histone and the acetylated peptide (H4_1-12 K5acK8ac) acetylated at the fifth and eighth histone residues and BRD4-BD1. Fig. 22 C shows the current blockade signal measured after adding the H4 peptide (H4_1-12) or the peptide acetylated at residues 5 and 8 (H4_1-12 K5acK8ac) to the target protein BRD4-BD1. Fig. 22 D shows the change of the nanopore current blockade signal caused by the interaction of BRD4-BD1 protein and acetylated peptide (H4_1-12 K5acK8ac) as an analysis of the current noise (I) including the power spectral density (PSD) or standard deviation (SD) of the ion current. N ) value, and by the current noise (I N The binding affinity (K) of the acetylated peptide (H4_1-12 K5acK8ac) to the BRD4-BD1 protein was measured by quantitative analysis. D ), Fig. 22 E in FIG. 1 shows the result of measuring the nanopore current blockade signal after reacting each of four types of small molecule compounds (GSK778, Y06026, XMD8-92, and PLX51107) that can bind to the BRD4-BD1 protein with the BRD4-BD1 protein, Fig. 22 F in the figure shows the result of analyzing the dwell time ratio at each level (L1-L3) of current blockade when the BRD4-BD1 target protein, acetylated peptide (H4_1-12 K5acK8ac), and each small molecule compound were reacted, the analytes were captured in the nanopore, and then the change in the subtle ion current was measured.

[0048] Fig.23 Shown are the results of selective detection of cancer biomarkers in the presence of serum using modified YaxAB_dN nanopores. Fig.23 Figure A shows the nanopore current blockade signal and current noise (SD)-current blockade (ΔI / I o ), Fig.23 B shows the current blocking signal in the state of deproteinized fetal bovine serum (FBS) alone. Fig.23 C shows the result of adding 10 nM Bcl-xL protein in the presence of deproteinized FBS. Fig.23 D in FIG. 5 shows the result of measuring the nanopore current blockade signal by adding 20 nM Bcl-xL protein in the presence of deproteinized FBS.

[0049] Fig.24 Results of detecting and analyzing wild-type Bcl-xL protein as well as mutant proteins Bcl-xL (E31K, E36K) and Bcl-xL (R100E, R103E) using the modified YaxAB_dN nanopore are shown, indicating that the unique nanopore electrical signals of each of the three types of proteins are distinguishable from each other. Fig.24 A in FIG. 1 shows the structural features. Fig.24 B shows the current signal, Fig.24 C shows the scatter plot results obtained by analyzing the current blockade signal.

[0050] Fig.25 Shown are the results of electroosmosis-based mTOR protein detection using the modified YaxAB_dN nanopore, showing the results of capture signal or translocation signal occurring depending on the applied voltage.

[0051] Fig.26 Shown are the results of electrophoresis-based EGF protein detection using modified YaxAB_dN nanopores.

[0052] Fig. 27 The principle of electrophoresis using a modified YaxAB_dN nanopore to detect p53 as an intrinsically disordered protein (IDP) is shown. TAD1 Peptide (1.8 kDa, residues 15 to 29) and p53 TAD The results were for protein (8.3 kDa, residues 1 to 73).

[0053] Fig.28 The results of detecting double-stranded nucleic acids and single-stranded nucleic acids using the principle of electrophoresis using the modified YaxAB_dN nanopore are shown.

[0054] Fig.29Shown are the results of comparison of 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 the same conditions.

[0055] Fig.30 The bands of different oligomers obtained by electrophoresis of the modified YaxAB_dN nanopore on a gradient blue native polyacrylamide gel are shown, and it is shown that the oligomer formation trends of wild-type YaxAB (YaxAB_FL) and modified YaxAB_dN are different. Fig.30 In the figure, the wild-type YaxAB refers to an oligomer formed by YaxA_FL (a first monomer consisting of SEQ ID NO: 3) and YaxB_FL (a second monomer consisting of SEQ ID NO: 4). Fig.30 A in the figure shows the result of nanopore formation by oligomerization under low protein concentration conditions. Fig.30 Panel B shows the result of nanopore formation by oligomerization under high protein concentration conditions.

[0056] Fig.31 Shown are the trends of YaxAB nanopore formation from various protein bands obtained by electrophoresis of wild-type nanopores and modified YaxAB_dN nanopores, and the results comparing the conductivity measurements of the individual nanopores.

[0057] Fig.32 Bands of various YaxAB oligomers observed on a gradient blue native polyacrylamide gel for nanopores formed using YaxA and YaxB_(12-343) with adjusted sequence lengths are shown. The figure shows that the pattern of oligomer formation differs depending on the sequence length of YaxA by comparing the reaction between YaxB_(12-343) (SEQ ID NO: 2) and YaxA_(45-410) (SEQ ID NO: 1) or YaxA_(X-411) with adjusted sequence lengths.

[0058] Fig.33 The bands of YaxAB oligomers obtained by electrophoresis of YaxAB nanopores on a gradient blue native polyacrylamide gel are shown, and Fig.33 A in FIG. 1 shows the result of the tendency of the YaxAB oligomer formed after reacting the wild-type YaxA_FL or the first monomer YaxA_(45-410) consisting of SEQ ID NO: 1 with the second monomer YaxB_(12-343) consisting of SEQ ID NO: 2. Fig.33 B in the figure shows the bands of YaxAB oligomers obtained by electrophoresis of YaxAB nanopores on a gradient blue native polyacrylamide gel, and shows the results of the trend of YaxAB oligomers formed after reacting YaxA_(45-410), YaxA_(3-410), YaxA_(5-410), YaxA_(8-410) or YaxA_(9-410) alone with the second monomer YaxB_(12-343) consisting of SEQ ID NO: 2.

[0059] Fig.34 The results show that YaxAB oligomers were applied to the membrane and the Bcl-xL detection capability of the nanopore system was verified by electrical measurement experiments. Fig.34 A in the figure shows the results of verifying the detection ability of the YaxAB nanopore system for Bcl-xL protein by electrical measurement using YaxAB oligomers formed by complexing YaxB_(12-343) (SEQ ID NO: 2) with YaxA of various sequence lengths (YaxA_(11-411), YaxA_(21-411), YaxA_(31-411), YaxA_(41-411) and YaxA_(51-411)). Fig.34 B in the figure shows the result of using a YaxAB oligomer formed by combining YaxA_(3-410) and a second monomer YaxB_(12-343) consisting of the amino acid sequence of SEQ ID NO: 2, and verifying the detection ability of the YaxAB nanopore system for Bcl-xL protein through electrical measurement experiments.

[0060] Fig.35 The results of bands of different oligomers obtained by electrophoresis of four types of YaxAB oligomers (composed of nanopore subunits formed by binding YaxA_(57-398) or YaxA_(52-383) as the minimum sequence capable of forming the YaxAB oligomer with YaxB_(26-342) or YaxB_(12-316) respectively) and YaxAB_dN oligomer (composed of nanopore subunits formed by binding YaxA_(45-410) and YaxB_(12-343)) on gradient blue native polyacrylamide gel are shown.

[0061] Fig.36The YaxAB nanopore prepared with the minimal sequence capable of forming a YaxAB oligomer is shown, and the results of applying four types of YaxAB oligomers formed by combining YaxA_(57-398) or YaxA_(52-383) with each of YaxB_(26-342) or YaxB_(12-316) to a membrane and verifying the Bcl-xL detection ability of the nanopore system through electrical measurement experiments are shown.

[0062] Fig.37 Results showing that the YaxA minimal sequence and the YaxB minimal sequence are able to form YaxAB oligomers are shown.

[0063] Fig.38 The changes in current traces and current blockade ratio (ΔI / I) were observed when the electroneutral FKBP12 and FKBP12-FK506 complex were applied to the YaxAB nanopore system. o ) shows a histogram of relative event frequencies.

[0064] Fig.39 is a histogram showing the relative event frequency and the current blockade ratio (ΔI / I) based on the change in current trace when oxaliplatin alone and holo-transferrin-oxaliplatin were applied to the YaxAB nanopore system. o ).

[0065] Fig.40 Shown are the results of comparing the signals of the analyte-compound complex at low and high concentrations of ABT-737 compound in the YaxAB nanopore system.

[0066] Fig.41 The results of whether a non-specific signal is generated according to the presence or absence of an analyte in the YaxAB nanopore system when a non-targeting compound is added are shown.

[0067] Fig.42 Shown are comparison results of the signals of the analyte-compound complexes under various concentrations of DMSO in the YaxAB nanopore system. DETAILED DESCRIPTION

[0068] Hereinafter, the present invention will be described in detail.

[0069] 1. YaxAB nanopore subunit and YaxAB nanopore containing the YaxAB nanopore subunit Aspects of the present invention provide a YaxAB nanopore subunit and a YaxAB nanopore.

[0070] YaxAB is a heterodimer of YaxA and YaxB proteins and is a key component of Yersinia enterocolitica ( Yersinia enterocolitica) is one of the pore-forming toxins that binds to and invades host cells. Yersinia enterocolitica is an enteric pathogen that causes a variety of diseases ranging from mild diarrhea to severe systemic bacteremia. In Yersinia enterocolitica, RovA regulates the transcription of virulence genes, of which YE1984 and YE1985 were identified as genes upregulated by RovA and were identified as genes upregulated by RovA due to their interactions with Xenorhabditis nematophila ( Xenorhabdus nematophila ) xA and xB The YaxA and YaxB proteins are named yaxA and yaxB respectively due to their homology. Both the YaxA protein and the YaxB protein have membrane active parts, but only the YaxA protein can bind to the membrane by itself. The YaxB protein binds to the membrane-embedded YaxA protein to induce heterodimers to form transmembrane helices, and YaxAB can form YaxAB nanopores in the host cell membrane through additional oligomerization. The wild-type YaxAB nanopore mainly has a decamer structure composed of 10 YaxAB heterodimers, in which the YaxB protein is arranged in a manner facing the lumen of the hole, and the YaxA protein forms a structure surrounding the YaxB protein. That is, the toxicity mediated by YaxAB can induce osmotic lysis by forming holes in the membrane of the host cell.

[0071] In one aspect, the YaxAB nanopore of the present invention may be formed by comprising at least one subunit, the subunit comprising a membrane active portion or having a transmembrane α-helical structure, and being a part of a wild-type YaxAB (a pore-forming toxin), and specifically, the subunit constituting the YaxAB nanopore of the present invention may comprise a first monomer and a second monomer. In this case, the first monomer may have an amino acid sequence having 70% or more homology to the amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 34, and the second monomer may have an amino acid sequence having 70% or more homology to the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25.

[0072] In the present invention, "YaxAB nanopore" includes all types of proteins that can be called YaxAB proteins, and specifically, can include all subtypes of YaxAB proteins, as well as all homologous proteins, orthologous proteins and paralogous proteins. In addition, the YaxAB nanopore of the present invention can be a non-natural protein. Non-natural proteins refer to proteins composed of amino acid sequences that do not exist in nature, are usually not expressed by cells or organisms, and are artificially produced.

[0073] For example, in the present invention, the first monomer contained in the YaxAB nanopore can be, but is not limited to, a protein having an amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34, or an amino acid sequence of the following NCBI accession number, or a variant thereof: 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, HDL77486 05.1, HDL6737510.1, HEB4799346.1, WP_221871123.1, EKN3734298.1, EKN3569010.1, HDL7089514.1, WP_050160085.1, WP_2 21876226.1, EKN4747040.1, WP_050916836.1, HDM8435746.1, HDL8238855.1, WP_019079472.1, EKN3341278.1, WP_05016337 0.1, HDL7317648.1, HDM8081034.1, WP_050870897.1, EKN3737775.1, HDL7467110.1, HDL7471348.1, WP_046694975.1, WP_30 1211860.1, HDL7726911.1, WP_268215747.1, EKN5089609.1, WP_057620566.1, EKN6360258.1, WP_019083154.1, EKN5159302 .1, WP_050131624.1, ELI8015017.1, EKN4802793.1, EKN6165952.1, ELI8293390.1, ELI8097018.1, WP_050157801.1, EKN470 8697.1, EKN5102380.1, 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、WP_219643575.1、CQD54886.1、CNG18579.1、EKN4119565.1、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_145510697.1、WP_219646505.1、WP_145556850.1、WP_145537515.1、WP_219655358.1、CNG40075.1、EKN3837153.1、EKN3315583.1、WP_301178346.1、HDL8028676.1、HDL8052082.1、HDL7585724.1、HDL8104119.1、WP_005164820.1、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, and specifically, a protein consisting of the amino acid sequence or a variant thereof. The variant of the amino acid sequence may include a variant peptide having a different sequence by deletion, insertion or substitution of amino acids or a combination thereof within the range that does not affect the function of the amino acid sequence, or may be in the form of a protein fragment with the same function. Amino acid modifications at the protein and peptide levels that do not change the activity of the amino acid sequence are known in the art, and in some cases, the amino acid sequence may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc. Therefore, the variant of the amino acid sequence may have an amino acid sequence that is "substantially" the same as the above-mentioned amino acid sequence. The peptide having substantially the same amino acid sequence may be an amino acid sequence having 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 homology to each of the amino acid sequences, but is not limited thereto, and any protein having an amino acid sequence having 70% or more homology to each of the amino acid sequences and having an activity of binding to a lipid membrane is included in the scope of the present invention. .

[0074] In addition, in the present invention, the second monomer contained in the YaxAB nanopore can be, but is not limited to, a protein having an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25, or an amino acid sequence having the following NCBI accession number, or a variant thereof, etc.: WP_011816274.1 (SEQ ID NO: 4), WP_050336255.1 (SEQ ID NO: 36), WP_005169898.1, PNM17259.1, WP_050137918.1, HDL7748604.1, HDL6508072.1, WP_050137583.1, WP_050322893.1, HDL 7822655.1, HDL7646676.1, WP_050321988.1, WP_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, HDL75337 82.1, 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, EKN3734297.1, HDL6886350.1, HEB4799167.1, WP_263700684.1, EKN3341279. 1. HDL7478311.1, EKN3957448.1, WP_050327958.1, HDL7339638.1, HDL6946689.1, WP_019079471.1, HDV7159892.1, HDL74671 11.1, 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、HDL6959493.1、EKN5159301.1、HDL8096476.1、EKN3683754.1、EKN3973834.1、WP_219650266.1、EKN6365751.1、HDL8258876.1、EKN4119566.1、HDM8089399.1、WP_263696663.1、EKN6178815.1、ELI8097017.1、HDL7909664.1、WP_219643580.1、WP_050149278.1、CNH43347.1、HDL8000813.1、WP_020283191.1、HDL8788876.1、HDL7394360.1、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_265526189.1、WP_145537514.1、WP_050095454.1、WP_057634251.1、WP_050289682.1、WP_145531133.1、WP_145586506.1、WP_100286507.1、WP_219655343.1、WP_049557371.1、WP_087795913.1、WP_145510696.1、WP_271304184.1、WP_004390823.1、WP_145556851.1、WP_050120114.1、WP_219648204.1、WP_159678331.1, and specifically, may be a protein consisting of the amino acid sequence or a variant thereof. As in the first monomer, the variant of the amino acid sequence may include a variant peptide having a different sequence by deletion, insertion or substitution of amino acids or a combination thereof within the range that does not affect the function of the amino acid sequence, or may be in the form of a protein fragment with the same function. Amino acid modifications at the protein and peptide levels that do not change the activity of the amino acid sequence are known in the art, and in some cases, the amino acid sequence may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc. Therefore, the variant of the amino acid sequence may have an amino acid sequence that is "substantially" the same as the above-mentioned amino acid sequence. The peptide having substantially the same amino acid sequence may be an amino acid sequence having 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 homology to each of the amino acid sequences, but is not limited thereto, and any protein having an amino acid sequence having 70% or more homology to each of the amino acid sequences and having an activity of binding to a lipid membrane is included in the scope of the present invention. .

[0075] : Specifically, in the present invention, the subunit may include but is not limited to: a first monomer having any amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; a second monomer having any amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: Any amino acid sequence in the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0076] On the other hand, the YaxAB nanopore of the present invention includes a first opening, a middle area and a second opening, and has a funnel shape, wherein the outer diameter of the lumen of the first opening can be 5 nm or greater, the second opening can include a narrowing portion, the diameter of the narrowing portion can be 0.5 nm or greater, the outer diameter of the lumen of the second opening can be 1 nm or greater, and the depth of the lumen can be 5 nm or greater.

[0077] Specifically, the YaxAB nanopore includes a first opening, a middle region, and a second opening. The first opening is a portion that is not inserted into the membrane and is exposed and used as an entrance, and the outer diameter of the lumen located in the first opening may be 5 nm or more, for example, about 5 nm to 30 nm, 6 nm to 25 nm, 7 nm to 20 nm, 8 nm to 18 nm, or 10 nm to 17 nm, and specifically, in the YaxAB C 8 -C 10In the case of a nanopore, the outer diameter of the lumen of the first opening may be about 10 nm to 17 nm. The second opening is a portion inserted into the membrane and used as an outlet, and the outer diameter of the lumen of the second opening may be 1 nm or more, for example, about 1 nm to 20 nm, 1.1 nm to 15 nm, 1.2 nm to 10 nm, 1.3 nm to 5 nm, or 1.4 nm to 3 nm, and specifically, in YaxABC 8 -C 10 In the case of a nanopore, the diameter of the lumen at the second opening can be about 1.9nm to 3.1nm. The middle region is a portion connecting the first opening and the second opening, and the middle region can have a shape in which the diameter of the lumen gradually decreases from the first opening to the second opening. In the YaxAB nanopore, the depth of the lumen extending from the first opening through the middle region to the second opening can be 5nm or more, for example, about 5nm to 20nm, 6nm to 18nm, 7nm to 15nm or 10nm to 13nm. Therefore, the YaxAB nanopore can have a funnel shape with a wide entrance and a relatively much narrower exit. In addition, the lumen of the nanopore can have a funnel shape or a coffee dripper shape with a deep depth and a narrowed width.

[0078] The second opening includes a constriction. The constriction is a region in the lumen where the width is narrowed, and the diameter of the constriction may be 0.5 nm or more, for example, about 0.5 nm to 18 nm, 1 nm to 15 nm, 1.5 nm to 10 nm, 1.7 nm to 5 nm, or 1.9 nm to 3.1 nm, and specifically, in YaxAB C 8 -C 10 In the case of nanopores, the diameter may be about 1.9 nm to 3.1 nm.

[0079] The constriction may be electronegative or electronegative. When the constriction is electronegative, the surface charge of the lumen of the constriction has the characteristic of having a net negative charge. Specifically, in the YaxAB nanopore of the present invention, the subunits are arranged so that the second monomer faces the lumen, and when the surface charge of the second monomer has a negative charge, the constriction may have a negative surface charge at pH 7.5. Therefore, the electronegative constriction may impart selectivity to positively charged substances and / or cations in the direction from the first opening to the second opening or in the direction from the second opening to the first opening. In this case, the cation selectivity of the YaxAB nanopore may be 1.1 times or more, 1.4 times or more, 1.7 times or more, or 2.3 times or more of the anion selectivity. In a specific embodiment of the present invention, as a result of calculating the ion selectivity of the YaxAB nanopore of the present invention, it was verified that under the condition of pH 7.5, the cation selectivity of the YaxAB nanopore was 1.4 times or more of the anion selectivity (see Fig. 9 ).

[0080] When the constriction is electrically positive, the surface charge of the lumen of the constriction has the characteristic of having a net positive charge. Specifically, in the YaxAB nanopore of the present invention, the subunits are arranged so that the second monomer faces the lumen, and when the surface charge of the second monomer has a positive charge, the constriction can have a positive surface charge at pH 7.5. Therefore, the electrically positive constriction can impart selectivity to negatively charged substances and / or anions in the direction from the first opening to the second opening or in the direction from the second opening to the first opening. In this case, the anion selectivity of the YaxAB nanopore can be 1.1 times or more, 1.4 times or more, 1.7 times or more, or 2.3 times or more of the cation selectivity. Therefore, the YaxAB nanopore of the present invention can have an ion selectivity of 1.1 times or more.

[0081] At the same time, the first monomer can be expressed by a nucleic acid sequence encoding a protein or a variant thereof, wherein the protein has an amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34, or an amino acid sequence of the following NCBI accession number, etc.: 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, HDL774860 5.1, HDL6737510.1, HEB4799346.1, WP_221871123.1, EKN3734298.1, EKN3569010.1, HDL7089514.1, WP_050160085.1, WP_22 1876226.1, EKN4747040.1, WP_050916836.1, HDM8435746.1, HDL8238855.1, WP_019079472.1, EKN3341278.1, WP_050163370 .1, HDL7317648.1, HDM8081034.1, WP_050870897.1, EKN3737775.1, HDL7467110.1, HDL7471348.1, WP_046694975.1, WP_301 211860.1, HDL7726911.1, WP_268215747.1, EKN5089609.1, WP_057620566.1, EKN6360258.1, WP_019083154.1, EKN5159302. 1. WP_050131624.1, ELI8015017.1, EKN4802793.1, EKN6165952.1, ELI8293390.1, ELI8097018.1, WP_050157801.1, EKN4708 697.1, EKN5102380.1, EKN5913011.1, WP_050164534.1, WP_242365000.1, HDL6759266.1, WP_050327956.1, EKN3969341.1, H DL8127360.1, 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、WP_219643575.1、CQD54886.1、CNG18579.1、EKN4119565.1、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_145510697.1、WP_219646505.1、WP_145556850.1、WP_145537515.1、WP_219655358.1、CNG40075.1、EKN3837153.1、EKN3315583.1、WP_301178346.1、HDL8028676.1、HDL8052082.1、HDL7585724.1、HDL8104119.1、WP_005164820.1、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. For example, the first monomer can be expressed by a nucleic acid sequence encoding the following proteins: a protein having an amino acid sequence of SEQ ID NO: 7, a protein having an amino acid sequence of SEQ ID NO: 24, a protein having an amino acid sequence of SEQ ID NO: 1, a protein having an amino acid sequence of SEQ ID NO: 5, a protein having an amino acid sequence of SEQ ID NO: 6, a protein having an amino acid sequence of SEQ ID NO: 8, a protein having an amino acid sequence of SEQ ID NO: 9, a protein having an amino acid sequence of SEQ ID NO: 10, a protein having an amino acid sequence of SEQ ID NO: 11, a protein having an amino acid sequence of SEQ ID NO: 12, a protein having an amino acid sequence of SEQ ID NO: 13, a protein having an amino acid sequence of SEQ ID NO: 14, a protein having an amino acid sequence of SEQ ID NO: 15, a protein having an amino acid sequence of SEQ ID NO: 16, a protein having an amino acid sequence of SEQ ID NO: 17, a protein having an amino acid sequence of SEQ ID NO: 19, a protein having an amino acid sequence of SEQ ID NO: A protein having an amino acid sequence of SEQ ID NO: 20, a protein having an amino acid sequence of SEQ ID NO: 21, a protein having an amino acid sequence of SEQ ID NO: 22, a protein having an amino acid sequence of SEQ ID NO: 23, a protein having an amino acid sequence of SEQ ID NO: 30, a protein having an amino acid sequence of SEQ ID NO: 31, a protein having an amino acid sequence of SEQ ID NO: 32, a protein having an amino acid sequence of SEQ ID NO: 33, or a protein having an amino acid sequence of SEQ ID NO: 34. In addition, the nucleic acid sequence encoding the protein as described above may be codon-optimized for expression in a suitable host such as Escherichia coli, or may be chromosomal DNA encoding a wild-type first monomer extracted from a YaxAB nanopore-producing organism such as Yersinia enterocolitica, which may be extracted from a nanopore-producing organism such as Yersinia enterocolitica. The extracted nucleic acid sequence or the synthesized nucleic acid sequence may be amplified using PCR comprising specific primers, and the amplified sequence may be used to cause site-directed mutagenesis, thereby generating a mutant. Suitable methods for site-directed mutagenesis are known in the relevant art, and, for example, methods employing polymerase chain reaction can be used. A variant of the nucleic acid sequence can be a variant of a nucleic acid sequence encoding a gene having SEQ ID NO: 7,% 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 homology to a nucleic acid having the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 34, but is not limited thereto, and any nucleic acid sequence can be included without limitation as long as it encodes a nucleic acid having the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The protein having the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 34 can be used.

[0082] In addition, the first monomer can be expressed by a host cell into which a nucleic acid sequence encoding a protein is introduced, wherein the protein has an amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34, or an amino acid sequence of the following NCBI accession number, etc.: 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, HDL77486 05.1, HDL6737510.1, HEB4799346.1, WP_221871123.1, EKN3734298.1, EKN3569010.1, HDL7089514.1, WP_050160085.1, WP_2 21876226.1, EKN4747040.1, WP_050916836.1, HDM8435746.1, HDL8238855.1, WP_019079472.1, EKN3341278.1, WP_05016337 0.1, HDL7317648.1, HDM8081034.1, WP_050870897.1, EKN3737775.1, HDL7467110.1, HDL7471348.1, WP_046694975.1, WP_30 1211860.1, HDL7726911.1, WP_268215747.1, EKN5089609.1, WP_057620566.1, EKN6360258.1, WP_019083154.1, EKN5159302 .1, WP_050131624.1, ELI8015017.1, EKN4802793.1, EKN6165952.1, ELI8293390.1, ELI8097018.1, WP_050157801.1, EKN470 8697.1, EKN5102380.1, 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、WP_219643575.1、CQD54886.1、CNG18579.1、EKN4119565.1、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_145510697.1、WP_219646505.1、WP_145556850.1、WP_145537515.1、WP_219655358.1、CNG40075.1、EKN3837153.1、EKN3315583.1、WP_301178346.1、HDL8028676.1、HDL8052082.1、HDL7585724.1、HDL8104119.1、WP_005164820.1、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. For example, the first monomer can be expressed by a vector and a host cell containing the vector, the vector having a nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34. The vector can be obtained by introducing a nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34 into a recombinant vector such as a cloning vector or an expression vector. The nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34 can be operably linked to a promoter contained in the vector. The vector can be used to replicate and express the nucleic acid sequence in a host cell. Therefore, the first monomer can be obtained by introducing the nucleic acid sequence into a recombinant vector, introducing the vector into a compatible host cell, growing the host cell under conditions that induce replication of the vector, and recovering the vector from the host cell. As a host cell suitable for cloning or expressing a nucleic acid sequence, any host cell known in the relevant art can be used. .

[0083] At the same time, the second monomer can be expressed by a nucleic acid sequence encoding a protein or a variant thereof, wherein the protein has an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25, or an amino acid sequence of the following NCBI accession number, etc.: WP_011816274.1 (SEQ ID NO: 4), WP_050336255.1 (SEQ ID NO: 36), WP_005169898.1, PNM17259.1, WP_050137918.1, HDL7748604.1, HDL6508072.1, WP_050137583.1, WP_050322893.1, HDL78 22655.1, HDL7646676.1, WP_050321988.1, WP_221871120.1, HDL6961479.1, WP_050298175.1, CNG06681.1, EKN5919601.1, EKN5 074150.1, 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_0508751 82.1, HDL7335482.1, WP_050163371.1, EKN3734297.1, HDL6886350.1, HEB4799167.1, WP_263700684.1, EKN3341279.1, HDL747 8311.1, EKN3957448.1, WP_050327958.1, HDL7339638.1, HDL6946689.1, WP_019079471.1, HDV7159892.1, HDL7467111.1, WP_17 2654802.1, 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、HDL6959493.1、EKN5159301.1、HDL8096476.1、EKN3683754.1、EKN3973834.1、WP_219650266.1、EKN6365751.1、HDL8258876.1、EKN4119566.1、HDM8089399.1、WP_263696663.1、EKN6178815.1、ELI8097017.1、HDL7909664.1、WP_219643580.1、WP_050149278.1、CNH43347.1、HDL8000813.1、WP_020283191.1、HDL8788876.1、HDL7394360.1、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_265526189.1、WP_145537514.1、WP_050095454.1、WP_057634251.1、WP_050289682.1、WP_145531133.1、WP_145586506.1、WP_100286507.1、WP_219655343.1、WP_049557371.1、WP_087795913.1、WP_145510696.1、WP_271304184.1、WP_004390823.1、WP_145556851.1、WP_050120114.1、WP_219648204.1、WP_159678331.1. The second monomer may be expressed by a nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 18, a protein having an amino acid sequence of SEQ ID NO: 25, a protein having an amino acid sequence of SEQ ID NO: 2, a protein having an amino acid sequence of SEQ ID NO: 26, a protein having an amino acid sequence of SEQ ID NO: 27, a protein having an amino acid sequence of SEQ ID NO: 28, or a protein having an amino acid sequence of SEQ ID NO: 29. In addition, the nucleic acid sequence encoding the protein as described above may be codon-optimized for expression in a suitable host such as Escherichia coli, or may be chromosomal DNA encoding a wild-type second monomer extracted from a YaxAB nanopore-producing organism such as Yersinia enterocolitica, which may be extracted from a nanopore-producing organism such as Yersinia enterocolitica. The extracted nucleic acid sequence or the synthesized nucleic acid sequence may be amplified using PCR comprising specific primers, and the amplified sequence may be used to cause site-directed mutagenesis to generate a mutant. Suitable methods for site-directed mutagenesis are known in the relevant art, and, for example, methods utilizing polymerase chain reaction can be used. A variant of the nucleic acid sequence can be a variant of a nucleic acid sequence encoding a sequence having SEQ ID NO: 18 or SEQ ID NO: 25 amino acid sequence shows at least 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 homology to a nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25. In addition, the second monomer can be expressed using the vector described above and the host cell containing the vector in the description of the first monomer.

[0084] In addition, the second monomer can be expressed by a host cell into which a nucleic acid sequence encoding a protein is introduced, the protein having an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25, or an amino acid sequence of the following NCBI accession number, etc.: WP_011816274.1 (SEQ ID NO: 4), WP_050336255.1 (SEQ ID NO: 36), WP_005169898.1, PNM17259.1, WP_050137918.1, HDL7748604.1, HDL6508072.1, WP_050137583.1, WP_050322893.1, HDL78 22655.1, HDL7646676.1, WP_050321988.1, WP_221871120.1, HDL6961479.1, WP_050298175.1, CNG06681.1, EKN5919601.1, EKN5 074150.1, 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_0508751 82.1, HDL7335482.1, WP_050163371.1, EKN3734297.1, HDL6886350.1, HEB4799167.1, WP_263700684.1, EKN3341279.1, HDL747 8311.1, EKN3957448.1, WP_050327958.1, HDL7339638.1, HDL6946689.1, WP_019079471.1, HDV7159892.1, HDL7467111.1, WP_17 2654802.1, 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、HDL6959493.1、EKN5159301.1、HDL8096476.1、EKN3683754.1、EKN3973834.1、WP_219650266.1、EKN6365751.1、HDL8258876.1、EKN4119566.1、HDM8089399.1、WP_263696663.1、EKN6178815.1、ELI8097017.1、HDL7909664.1、WP_219643580.1、WP_050149278.1、CNH43347.1、HDL8000813.1、WP_020283191.1、HDL8788876.1、HDL7394360.1、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_265526189.1、WP_145537514.1、WP_050095454.1、WP_057634251.1、WP_050289682.1、WP_145531133.1、WP_145586506.1、WP_100286507.1、WP_219655343.1、WP_049557371.1、WP_087795913.1、WP_145510696.1、WP_271304184.1、WP_004390823.1、WP_145556851.1、WP_050120114.1、WP_219648204.1、WP_159678331.1. For example, the first monomer can be expressed by a vector or a host cell comprising the vector, the vector having a nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25. The vector can be obtained by introducing a nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25 into a recombinant vector such as a cloning vector or an expression vector. The nucleic acid sequence encoding a protein having an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 25 can be operably linked to a promoter contained in the vector. The vector can be used to replicate and express the nucleic acid sequence in a host cell. Therefore, the second monomer can be obtained by introducing a nucleic acid sequence into a recombinant vector, introducing the vector into a compatible host cell, culturing the host cell under conditions that induce replication of the vector, and recovering the vector from the host cell. As a host cell suitable for cloning or expressing a nucleic acid sequence, any host cell known in the relevant art can be used. .

[0085] At the same time, the first monomer and the second monomer can be combined with each other to form a heterodimer. The heterodimer is a YaxAB nanopore subunit, and the subunits can be assembled to form a YaxAB nanopore. Therefore, the subunit is the basic unit (protomer) constituting the YaxAB nanopore of the present invention.

[0086] In addition, the first monomer and the second monomer can improve the nanopore forming ability by adjusting the amino acid sequence. As used in the present invention, the term "nanopore forming ability" means that the subunits comprising the first monomer and the second monomer are repeatedly connected to each other to form a nanopore having a size of nanometers (nm, 10 -9 )-sized pores. Therefore, excellent nanopore-forming ability means that the subunits stably form pores, or that the subunits can form nanopores even when present at a low concentration.

[0087] In a specific embodiment of the present invention, in the wild-type YaxAB nanopore of the present invention, no oligomer bands appeared under low protein concentration conditions, while in the modified YaxAB_dN nanopore composed of the YaxAB subunit comprising the first monomer (YaxA_(45-410)) of SEQ ID NO: 1 and the second monomer (YaxB_(12-343)) of SEQ ID NO: 2, oligomer bands clearly appeared even under low protein concentration conditions; thus, it was verified that the nanopore was formed in a more stable state than the wild-type YaxAB (see Fig.30 ).

[0088] In addition, in a specific example of the present invention, as a result of preparing various shortened sequences and analyzing the oligomerization tendency in order to confirm the minimum amino acid sequence capable of forming the YaxAB nanopore of the present invention, in the case of a nanopore subunit comprising a first monomer having an amino acid sequence consisting of SEQ ID NO: 7 or SEQ ID NO: 24 and a second monomer having an amino acid sequence consisting of SEQ ID NO: 18 or SEQ ID NO: 25, YaxAB oligomers were stably formed, and the nanopore-containing membrane of the present invention was successfully implemented using the oligomers; therefore, it was verified that the analyte protein (Bcl-xL) was successfully captured using the nanopore system (see Fig.35 and Fig.36 ).

[0089] In another specific embodiment of the present invention, it was verified that in the case of a nanopore subunit comprising a first monomer comprising at least one of an amino acid sequence consisting 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 having an amino acid sequence consisting of SEQ ID NO: 2, a YaxAB oligomer was stably formed (see Fig.32 ).

[0090] The YaxAB nanopore of the present invention can be formed by assembling 2 or more, for example, 3 or more, 5 or more or 7 or more subunits, and specifically, can be formed by assembling 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more or 20 or more subunits, and specifically, can be formed by assembling 4 to 20 subunits. Depending on the number of subunits, the diameter of the lumen of the YaxAB nanopore can vary, and specifically, as the number of subunits increases, the diameter of the lumen of the YaxAB nanopore of the present invention can increase.

[0091] In the YaxAB nanopore of the present invention, the number of subunits constituting the nanopore can be easily adjusted by adjusting the length of the amino acid sequence of each of the first monomer and the second monomer. In other words, as the amino acid sequences of the first monomer and the second monomer are changed, the number of subunits forming the nanopore can also be changed. In a specific embodiment of the present invention, it was verified that there were differences in the types of gradient blue natural polyacrylamide gel electrophoresis bands that appeared when the wild-type YaxAB subunit and the modified YaxAB_dN subunit were used to form the nanopore, and it was verified that the number of subunits constituting the nanopore can be adjusted by changing the amino acid sequence of the subunit (see Fig.30 Thus, a new type of YaxAB-C is formed that cannot be formed in the wild-type YaxAB. 8 hole.

[0092] In addition, in a specific embodiment of the present invention, as a result of analyzing the gradient blue native polyacrylamide gel electrophoresis that appears when a nanopore is formed using a modified YaxAB-dN subunit, it was verified that although the YaxAB-C 8 、YaxAB-C 9 and YaxAB-C 10 The corresponding band (C 8 Strip, C 9 Strip and C 10 band), but located at C 10 The band above the band (C 11 Strip, C 12 Strip, C 13 Strip, C 14 Strip and C 15 The bands also appeared in a well-separated state, confirming that the number of subunits constituting the nanopore can be adjusted by changing the amino acid sequence of the subunits (see Figure 8 ). Thus, novel nanopores with various sizes that cannot be formed by the wild-type YaxAB sequence are formed. Therefore, the modified YaxAB_dN nanopore of the present invention not only ensures nanopores with various sizes through one purification, but also selectively ensures nanopores with desired sizes at the same time, and thus has significant advantages over conventional biological nanopores with fixed pore sizes.

[0093] In addition, the purification efficiency of the first monomer and the second monomer can be increased by adjusting the length of the amino acid sequence of the first monomer and the second monomer. Specifically, in the case where the first monomer is composed of the amino acid sequence of SEQ ID NO: 1, the protein purification efficiency can be 2 times or more, 3 times or more or 4 times or more of the protein purification efficiency of the wild-type YaxA protein. In addition, in the case where the second monomer is composed of the amino acid sequence of SEQ ID NO: 2, the protein purification efficiency can be 1.1 times or more. The protein purification process can be performed by known conventional protein purification methods. When the purification efficiency of each of the monomers is high, the nanopore can be formed in a more stable state than the wild-type YaxAB subunit.

[0094] In a specific embodiment of the present invention, it was verified that the purified protein yield of the first monomer having the amino acid sequence of SEQ ID NO: 1 of the present invention was 4.7 times that of the purified protein yield of the wild-type YaxA, and it was verified that the purified protein yield of the second monomer having the amino acid sequence of SEQ ID NO: 2 was 1.1 times that of the purified protein yield of the wild-type YaxB, thereby verifying the excellent purification efficiency under the same protein purification conditions (see Fig.29 ).

[0095] 2. Membranes with nanopores In addition, another aspect of the present invention provides a nanopore-containing membrane, which includes a membrane layer and a YaxAB nanopore inserted into the membrane layer.

[0096] The membrane may be included without limitation as long as it serves as a support to which the nanopore can be fixed and can divide the space or chamber in which the nanopore exists into two compartments. The membrane is used to block the passage of a fluid or a substance contained in the fluid without an additional device. The membrane layer may include phospholipids and amphiphiles.

[0097] Phospholipids are amphiphilic molecules containing at least one phosphorus group and are used as carriers constituting membranes. Phospholipid membranes refer to structures in which the hydrophobic head of a phospholipid points in one direction and the hydrophilic tail points in the opposite direction. The phospholipid may be 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-adipoyl-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-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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), mycolic acid, etc.

[0098] An amphiphile is a substance that has both water and lipid solubility and refers to a substance that stabilizes a membrane by maintaining the fluidity of a phospholipid-containing membrane at a constant level. When an amphiphile is included, the stability of the membrane can be increased, the number of nanopores formed can be affected, and the nanopore lifetime can be adjusted by changing the nanopore separation force (also by affecting the elasticity of the membrane).

[0099] The amphiphile may be, for example, campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (PChemsPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesteryl carbonyl-sn-glycero-3-phosphocholine (PChcPC), 1,2-dicholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (DChemsPC), 10,12-pentacosadiynoic acid (PCDA), 10,12-tricosadiynoic acid (TCDA), 5,7-hexadecanediynoic acid (HDDA), 9,12-octadecadiynoic acid (ODDA), and the like, but is not limited thereto, and may include any amphiphile that increases the stability of the membrane without limitation.

[0100] The membrane may include phospholipids and amphiphiles at a ratio of 50 mol% to 95 mol% and 5 mol% to 50 mol%, 55 mol% to 95 mol% and 5 mol% to 45 mol%, or 60 mol% to 95 mol% and 5 mol% to 40 mol%. Within the above ratio range, as the ratio of amphiphiles included in the membrane increases, the elasticity of the lipid membrane may decrease, and therefore, the separation force for forming nanopores may decrease, thereby increasing the life of the nanopores and thus increasing the formation frequency of the nanopores. When the phospholipids and amphiphiles in the membrane are outside the above ratio, the YaxAB nanopore of the present invention cannot be stably inserted into the membrane, and the membrane may rupture, or constant conductivity cannot be measured when a voltage is applied, and the ionic current according to Ohm's law may not flow.

[0101] In the present invention, the YaxAB nanopore is as described above. YaxAB nanopore subunit and a YaxAB Nanopore Therefore, the above description is cited and will not be repeated.

[0102] The YaxAB nanopore can be inserted into the membrane by a known method. For example, the nanopore can be inserted into the membrane by a change in ionic current. In the membrane containing nanopores, the stability of the YaxAB nanopore of the present invention is improved. The YaxAB nanopore of the present invention can be effectively inserted without rupturing the membrane, so that the frequency of nanopore formation can be high and the number of stably inserted nanopores can be increased. In addition, in the case where a membrane containing nanopores is used, a stable conductivity value and current according to Ohm's law can be exhibited through the YaxAB nanopore even when a voltage is applied.

[0103] In a specific example of the present invention, it was verified that the YaxAB nanopores of the present invention were stably inserted into the DPhPC-sterol composite membrane compared to the artificial membrane composed of DPhPC alone (see Figure 6 ).

[0104] 3. Nanopore system In addition, another aspect of the present invention provides a nanopore system using the YaxAB nanopore.

[0105] The nanopore system of the present invention comprises a chamber and a membrane containing YaxAB nanopores.

[0106] The chamber refers to a structure having an internal space formed to accommodate a certain volume of fluid. The space in the chamber is a fluid-filled compartment, and the fluid can be an electrolyte solution. In addition, the chamber may include an electrode capable of generating a transmembrane potential difference to facilitate the flow of electrolytes through the nanopores between the chambers. The internal space of the chamber is divided into two compartments by a nanopore-containing membrane, for example, a first compartment and a second compartment, or a cis compartment and a trans compartment. In the case where an analyte 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.

[0107] The YaxAB nanopores and nanopore-containing membranes are based on what has been described above and will not be described again.

[0108] The term "nanopore system" as used in the present invention refers to a new type of system that can detect or analyze changes in physicochemical properties (including changes in the fine structure and dynamics of biomolecules at the single-molecule level) and can be used as a sensor system, etc. In a nanopore system, a nanopore is usually inserted into an insulating membrane and serves only as a channel for allowing ion current to flow between two compartments divided by the insulating membrane. The operating principle of the nanopore system is that when a charged biomolecule translocates through a nanopore or is trapped in the lumen of a nanopore in the presence of an external electric field, the length, size, structure, dynamics, charge and shape of the biomolecule are determined by detecting tiny fluctuations in the ion current.

[0109] The nanopore system may include a liquid medium, and the liquid medium may be a saline solution. As the salt translocates through the nanopore between two compartments divided by the insulating membrane, the salt may cause an ionic current to flow. In the case where the salt is an ion, the salt may be a metal salt, a halide salt such as an alkali metal chloride salt, etc. Specifically, the salt may be a cation such as NH 4+ , K + 、Na + , Li + Mg 2+ , Ca 2+ or Gdm+ Cations such as F - 、SO 4 2- , HPO 4 2- , C 2 H 3 O 2- , Cl - Br - 、NO 3- , ClO 3- ,I - , ClO 4- or SCN - anions, and may be, for example, sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl), calcium chloride (CaCl 2 ), cesium chloride (CsCl), guanidine chloride (GdmCl), potassium ferrocyanide or potassium ferrocyanide. The salt may be an organic salt, for example, tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride. However, the salt is not limited thereto, and may include any salt having a charge without limitation.

[0110] Additionally, the concentration of the salt may be from 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. Additionally, the salt present in the two compartments may be present in asymmetric concentrations.

[0111] Additionally, the two compartments within the nanopore system may include electrodes. By applying one or more potentials to the electrodes, ion flow may be caused from one compartment to the other through the nanopore-containing membrane.

[0112] The nanopore system of the present invention can be operated by inducing electroosmotic force or electrophoretic force under the state of forming a gradient according to the difference in salt concentration in two compartments divided by the membrane containing the nanopore, applying voltage, or both, to allow the analyte to translocate through the hole or be captured through the hole. The nanopore system can be operated by inducing electroosmosis or electrophoresis, can be operated by inducing both electrophoresis and electroosmosis, or can be operated by a balance between electrophoresis and electroosmosis.

[0113] The term "electroosmosis" as used in the present invention refers to a phenomenon in which, when electrodes are attached to both sides of a porous membrane such as a nanopore-containing membrane and a direct current voltage is applied, a liquid moves toward one electrode through nanopores included in the membrane.

[0114] The term "electrophoresis" as used in the present invention refers to a phenomenon in which molecules move according to their charge and size in an electric field.

[0115] In the nanopore system, when the working electrode is located in the compartment with the cis opening of the YaxAB nanopore, the potential difference (voltage) between the two compartments can be -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 or +400mV to 500mV. Conversely, when the working electrode is located in the compartment with the trans opening of the YaxAB nanopore, the potential difference (voltage) between the two compartments can be +200mV, +180mV, +160mV, +140mV, +120mV, +100mV, +90mV, +85mV, +80mV, +75mV, +70mV, +65mV, +60mV, +55mV, +50mV, +45mV, +40mV, +35mV, +30mV. The applied potential refers to the potential of the working electrode, and when a positive applied potential is applied to the compartment where the cis opening of the YaxAB nanopore is located, a baseline current in the open state of steady flow can be observed. That is, when a voltage in the range of -100mV to +200mV or -200mV to +500mV is applied to the YaxAB nanopore when the working electrode is located at the cis opening of the pore in two compartments divided by the nanopore-containing membrane in the nanopore system, or when a voltage in the range of +100mV to -200mV or +200mV to -500mV is applied to the YaxAB nanopore when the working electrode is located at the trans opening of the pore, the YaxAB nanopore is stably maintained in an open state, which allows charged ions to move through the nanopore between the two compartments, thereby causing ionic current to flow.

[0116] Analyte is operated to be translocated through the nanopore of the nanopore system of the present invention, is captured in the lumen, or allows the nanopore to be gated. For example, when voltage is applied to the nanopore system to form an electric field, the analyte can be translocated, captured or gated. In this case, the flow of the ionic current flowing through the nanopore is blocked by the analyte, and the small fluctuations produced are expressed as a current signal, which can be used to detect the analyte or analyze the characteristics of the analyte. The characteristic of the analyte can be the electrical characteristic, chemical characteristic or physical characteristic of the analyte, and specifically, it can be selected from the surface charge of the analyte, the isoelectric point of the analyte, the stability of the analyte, isomers, length, size, molecular weight, structure, dynamics, the intensity and orientation of the dipole moment, orientation, flexibility, conformational heterogeneity, shape and the amount of the analyte and its variation composition at least one of the group.

[0117] Since the YaxAB nanopore has a wide funnel shape at the entrance (first opening), analytes of various sizes and molecular weights can be detected. The analyte may be a peptide, protein, lipid, carbohydrate, inorganic substance, small molecule compound, nucleic acid, or inorganic particle containing nanomaterial, and for example, the analyte may be a receptor, antibody, or aptamer. When the analyte is a protein, the molecular weight of the analyte may be 1 kDa to 1200 kDa, 1 kDa to 1000 kDa, or 1 kDa to 800 kDa. In a specific embodiment of the present invention, it was verified that proteins with a molecular weight of 1 kDa to 670 kDa were detected by the nanopore system of the present invention (see Fig.10 , Fig.18 and Fig.19 ). Because the lumen of the YaxAB nanopore has an electronegative surface charge, the nanopore system can capture analytes into the lumen of the nanopore regardless of the surface charge of the analyte, and such a property can induce an electroosmotic force that is greater than the electrophoretic force.

[0118] In addition, since the YaxAB nanopore has a funnel shape, the ion current density increases in the lumen of the nanopore as it approaches the constriction where the diameter becomes narrower, and therefore, the ion current blockade caused by the analyte can be maximized, and at the same time, due to the balance of electroosmotic and electrophoretic forces, the analyte can be retained in the nanopore lumen while exhibiting its own movement; therefore, a higher resolution signal can appear than in conventional nanopore systems.

[0119] 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 is verified that cations and water molecules mainly flow from the first opening of the YaxAB nanopore toward the second opening, verifying the induced electroosmosis (see Figure 4In addition, the YaxAB nanopore can detect proteins with various surface charges of isoelectric points of 2 to 12, 3 to 11, and 4 to 10 by utilizing electroosmotic forces as well as electrophoretic forces. In a specific embodiment of the present invention, it was verified that proteins with a wide range of surface charges (neutral, negative, positive) with isoelectric points of 4.1 to 9.6 were also captured in the lumen, verifying that detection can be performed regardless of the overall charge of the protein (see Fig.10 , Fig.18 and Fig.19 ).

[0120] In another specific embodiment of the present invention, it was observed that the nanopore system of the present invention exhibited a larger ion current blockade change than that caused by a conventional nanopore system, and exhibited significantly different electrical signal patterns depending on the type of analyte, verifying that the nanopore system of the present invention has excellent resolution (see Fig.11 and Fig.14 ).

[0121] In addition, in another specific embodiment of the present invention, Fig.10 As shown in FIG. , it is verified that the YaxAB-C 8 The detection range of the nanopore system is 1 kDa to 77 kDa; in YaxAB-C 11 The detection range of the system is 158 kDa or less; and in YaxAB-C 12 The detection range in the system is 669 kDa or less, which verifies that the nanopore system has a significantly wider analyte detection range than the detectable analyte range of conventional nanopore systems, which can detect analytes in the range of 25 kDa or less (FraC nanopore); 25 kDa to 42 kDa (ClyA nanopore) and 64.5 kDa or less (PlyAB nanopore). That is, the YaxAB nanopore system of the present invention not only produces pores of various sizes through a single purification process, but also allows the pore size to be adjusted. Therefore, it is verified that the YaxAB nanopore system of the present invention has an excellent analyte detection range compared to conventional nanopores.

[0122] 4. Applications of Nanopore Systems In addition, another aspect of the present invention provides a single molecule analysis method for a single analyte or multiple analytes, the single molecule analysis method comprising the following steps: placing at least one analyte in one compartment of a nanopore system; and measuring changes in electrical signals in two compartments before and after placing the analyte.

[0123] The analyte may be a peptide, protein, lipid, carbohydrate, inorganic substance, small molecule compound, nucleic acid or inorganic particle including nanomaterial, and for example, the analyte may be a receptor, antibody or aptamer. More specifically, the analyte may be a biomolecule, and specifically, may be a single compound such as nucleic acid, amino acid or monosaccharide, may be a polymer such as polynucleotide, peptide, protein, polysaccharide, carbohydrate or lipid, or may be a molecule such as 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, and the protein may be a linear protein whose structure is denatured using a surfactant such as SDS and a peptide, but is not limited thereto.

[0124] The single molecule analysis method for a single analyte or a plurality of analytes comprises the following steps: placing at least one analyte in one compartment divided by a membrane containing a nanopore, the step may be, for example, placing the analyte in one of the compartments, specifically, in one compartment facing the first opening of the nanopore. In addition, the processing of the analyte may be performed while inducing the analyte to move through the nanopore to another compartment (for example, by forming a gradient according to the difference in salt concentration in the two compartments divided by the membrane, applying a voltage, or providing both).

[0125] Additionally, the processing of the analyte includes the step of measuring changes in electrical signals in the two compartments before and after placement of the analyte.

[0126] The “electrical signal” refers to a signal generated when ions and / or charged small molecules pass from one compartment divided by a membrane to another compartment (e.g., from a first compartment to a second compartment), and specifically, may be, but is not limited to, a current pattern of an ion current, a baseline current in an open state (open pore current: I o ,I open ), the amplitude of the current drop (ΔI), the current blockade, the event duration, the frequency per unit time of the nanopore signal pattern, the current noise (I N ), etc. The frequency per unit time of the nanopore signal pattern may include, but is not limited to, the event frequency and the interval between events. The baseline current in the open state may refer to the current in the state where only the nanopore exists. The current drop amplitude (ΔI) may refer to the amplitude of the reduction in the amount of electrolyte (such as ions and / or charged small molecules) passing through the nanopore due to the analyte being trapped inside the nanopore or the analyte being translocated through the nanopore. The current blockade may be the ratio of ΔI to I o The ratio (ΔI / I o ), ΔI is the current drop caused by the analyte, I ois the baseline current (open pore current) when the nanopore is in an open state. The event duration may be the event duration when the analyte event is shown by the current drop amplitude (ΔI). The current noise may include a power spectral density (PSD) or an ion current standard deviation (SD), and the power spectral density is a representation of the noise power generated by the flow of the ion current on the spectrum, which represents the distribution of the power density according to the signal frequency. The standard deviation represents the standard deviation value of the current blockade signal itself generated when the ion current drops. In addition, the current noise (I N ) represents the square root of the integrated value of the power spectrum of the ion current when the current decreases or the standard deviation value of the ion current (which can mean the quantification of the electrical noise characteristics of the nanopore), and can include the current noise amplified by amplifying the difference of the current noise by signal filtering in the low oscillation section. In addition, the electrical signal can include a two-dimensional representation of the current blockade (ΔI / I o ) and current noise (I N ) density contour plot, two-dimensional scatter plot or violin plot. The analytical method for a single analyte or multiple analytes includes qualitative and quantitative analysis of the analytes. For example, the analytical method can be an analysis of the presence or absence of the analyte, the type of the analyte, the structure of the analyte, the kinetics and its transformation, and the amount of the analyte.

[0127] In the examples of the present invention, it was verified that analysis of single-molecule analytes is possible using a nanopore system, because distinctly different ion current change patterns appear between analytes having various sizes and various surface charges (see Fig.10 ).

[0128] In another embodiment of the present invention, the nanopore system is used to utilize electrophoretic force to allow EGF (which is a small-sized, negatively charged, single-molecule compound at pH 7.5), or p53 TAD1 peptide, which is an intrinsically disordered protein (IDP) with a small molecular weight of 1.8 kDa, and p53 TAD The translocation of the protein (which is an intrinsically disordered protein with a molecular weight of 8.3 kDa) was confirmed, thus verifying that the detection and analysis of intrinsically disordered protein analytes and peptide analytes is possible through the nanopore system (see Fig.26 and Fig. 27 ).

[0129] In yet another embodiment of the present invention, as a result of independently adding λDNA, a single-stranded nucleic acid (50mer), and a double-stranded nucleic acid (23 bp) to the nanopore system and observing whether the nucleic acid is translocated, ion current blockade was verified before and after the addition of each nucleic acid; thus, it was verified that by the nanopore system of the present invention, nucleic acid can be detected and analyzed by the nanopore system by utilizing electrophoretic force and electroosmotic force to translocate the nucleic acid (see Fig.28 ).

[0130] In another embodiment of the present invention, a nanopore system was used to perform protein profiling and drug identification based on the affinity of the target protein for Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1). As a result, it was observed that the nanopore current blockade signals for Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1) all appeared in a unique pattern that could be distinguished, and even when equal amounts of Bcl-2 family proteins were mixed, a unique current blockade signal originating from each protein was observed, and when A1155463, a small molecule drug targeting Bcl-xL, was added at an equal ratio, unique current blockade signals of the proteins appeared in the cases of Bcl-w, Bcl-2, Mcl-1, and mTOR, while the unique current blockade signal disappeared and a new current blockade signal appeared only in the case of the target protein Bcl-xL; therefore, it was verified that profiling of Bcl-2 family proteins by a nanopore system is possible, and identification of drugs that are selective for target proteins is also possible (see Fig. 20 ).

[0131] The analytical methods for single analytes or multiple analytes can be used to measure and analyze the presence or absence, type, behavior, charge, type, size, structure, dynamics, orientation, flexibility, structural heterogeneity or amount of single analytes or multiple analytes. The analytical methods can be used for biosensors for various analytes (such as proteins and nucleic acids), detecting analytes and analyzing structure and dynamics at the single molecule level, analyzing biomolecular interactions, and sequence analysis of nucleic acids and proteins. In addition, the analytical methods for single analytes or multiple analytes can be used for new drug screening, disease diagnosis, protein identification and proteomic analysis including post-translational modification of proteins. In addition, the analytical methods for single analytes or multiple analytes can be used to detect, identify, quantify and screen biomolecules by detecting characteristic electrical signals in the presence of multiple analytes and fingerprinting the target substance.

[0132] As an example, in the case where the analyte is a nucleic acid, since the ion current change pattern when the nucleic acid is translocated through the nanopore is different depending on the type of nucleotide, the nucleic acid can be identified by analyzing the pattern, which can be used for nucleic acid sequence analysis. In addition, analyzing the pattern makes it possible to identify the nucleotides that constitute the nucleic acid, thereby allowing accurate and rapid analysis of the nucleic acid sequence. The nucleotides that constitute the nucleic acid include a base, a sugar, and at least one phosphate group, and can be 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 (c GMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), or deoxycytidine triphosphate (dCTP).

[0133] Another aspect of the present invention provides a method for analyzing the interaction between an analyte and a ligand, or screening a ligand for an analyte, the method comprising the following steps: performing treatment with at least one analyte and at least one ligand candidate for the analyte in one or two compartments of a nanopore system; and measuring changes in electrical signals in the two compartments before and after treatment with the candidate.

[0134] The method for analyzing the interaction between an analyte and a ligand, or screening a ligand for an analyte comprises the following steps: treating at least one analyte with at least one ligand candidate for the analyte in one compartment or both compartments of a nanopore system.

[0135] Analyte means an object for detection, interaction analysis and screening of ligands by ligand interaction analysis or screening methods for ligands for analytes. Analytes can be peptides, polypeptides, proteins, inorganic substances, small molecule compounds or nucleic acids, but are not limited thereto. Specifically, small molecule compounds refer to organic compounds with a small molecular weight (e.g., 1000 Daltons or less) in the fields of molecular biology and pharmacology. Small molecule compounds are usually used as regulators or drugs in biological processes and are about 10 -9 m (which is a small size), and in the field of pharmacology, small molecules refer to molecules or drugs that bind to biopolymers (such as proteins or nucleic acids) and regulate the functions of biopolymers. Small molecule compounds have various biological functions as cell signaling substances. Small molecule compounds can be naturally occurring (secondary metabolites) or artificially synthesized. Nucleic acids can 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 nucleic acids.

[0136] Ligand candidate means a substance that is expected to interact with an analyte. Specifically, when a ligand interacts with an analyte in a free state, the oscillatory motion mode, the rotational motion mode or the nanopore translocation mode when the analyte is captured in the lumen of the nanopore may change due to physical or chemical reasons. For example, when a ligand interacts with an analyte in a free state to form a complex, the ratio of the analyte present in a free state is reduced. Therefore, not only the electrical signal exhibited by the analyte in a free state is significantly reduced, but also the new electrical signal exhibited by the complex of the analyte and the ligand is significantly increased, so that the complex of the analyte and the ligand can be directly detected.

[0137] The method for analyzing the interaction between an analyte and a ligand of the present invention, or screening a ligand for an analyte, can be used as a drug screening method. Specifically, when the analyte is a drug target and the ligand candidate is a drug candidate, it is possible to analyze or screen the interaction between the drug target and the drug candidate, and therefore, the drug candidate can be effectively screened.

[0138] In an embodiment of the present invention, the interaction between the target protein and the peptide bound to the target protein was analyzed using a nanopore system, thereby performing screening of peptide drugs. As a result of reacting the Bcl-xL protein with three types of Bak-BH3 mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, and Bak_BH3_L78A) and the wild-type Bak_BH3 peptide capable of binding to the Bcl-xL protein, and observing and analyzing the current blockade signal derived from the Bcl-xL protein and the Bcl-xL+peptide drug complex that appeared at this time, it was verified that the scatter plot results obtained by measuring and analyzing the Bcl-xL protein and the four types of mixed peptides were consistent with the scatter plot tendency observed in each experiment, and the signal frequency of the Bcl-xL-peptide complex was consistent with the order of the binding affinity between the Bcl-xL protein and each peptide. Therefore, it was verified that the YaxAB nanopore of the present invention can perform binding affinity-based screening for peptide drugs bound to proteins, and can effectively select peptide drugs with high binding affinity to target proteins even in a peptide mixed solution, and therefore can be effectively used as a drug screening platform (see Fig.19 ). The description of the electric signal is as described above in the analysis method for the analyte, and therefore, the above description is cited without repeating the description thereof.

[0139] The method for analyzing the interaction between an analyte and a ligand, or screening a ligand for an analyte may comprise the steps of simultaneously performing a treatment with at least one analyte and at least one ligand candidate for the analyte in one or both compartments of a nanopore system.

[0140] In the step of simultaneously performing treatment with at least one analyte and at least one ligand candidate for the analyte in one or two compartments of the nanopore system, at least one analyte can be first placed in one compartment or two compartments, and then, multiple ligands of the analyte, for example, 2 or more, 4 or more, 8 or more, 10 or more, 20 or more, 50 or more or 100 or more ligands, can be simultaneously treated in the same compartment.

[0141] The method for analyzing the interaction between an analyte and a ligand, or screening a ligand for an analyte of the present invention comprises the following steps: measuring the change in the electrical signal of the analyte before and after treatment with a candidate. Measuring the electrical signal exhibited by the nanopore oscillation mode or rotational motion mode of the analyte captured in the nanopore lumen in a free state, or the nanopore translocation mode of the analyte, makes it possible to determine whether the analyte interacts with the candidate, for example, whether the analyte and the candidate bind to form a complex.

[0142] In addition, the method for analyzing the interaction between the analyte and the ligand, or screening the ligand for the analyte, may also include the following steps: in the case of significant changes, the candidate is determined as a ligand that interacts with the analyte. The case of significant changes may include the case in which a new signal that has not appeared before or after treatment with the ligand candidate appears.

[0143] In the examples of the present invention, using the YaxAB nanopore of the present invention, when treated with Bak-BH3 (which is a peptide that binds to the Bcl-xL protein) or when treated with a small molecule compound (ABT-737) that binds to the protein, it was verified that a significant change in the ion current pattern was observed compared to the negative control (see Fig.11 ), and even in the case of treatment with Bax-BH3 peptide, or with quercetin small molecule compound (which is a weak binder with low binding affinity to Bcl-xL protein), it was verified that changes in the ion current pattern were observed (see Fig.12 and Fig.13 ).

[0144] In another embodiment of the present invention, as a result of measuring a current blockade signal generated when glucose / galactose binding protein (GBP, which undergoes allosteric movement when bound to glucose or galactose) binds to galactose (which is a small molecule compound targeting GBP) using the YaxAB nanopore of the present invention, it was verified that when galactose was added, the gating frequency occurring in the current blockade signal derived from GBP increased significantly, verifying that the GBP-galactose interaction according to the presence or absence of galactose was effectively analyzed (see Fig.14 ).

[0145] In another embodiment of the present invention, when the target protein Bcl-xL was treated with non-binders (LCL-161 and GDC-0152) and binders (ABT-737 and A-1331852) separately using the YaxAB nanopore system, the current noise (I N ) signal did not change, and only the current noise (I N ) signal specifically increased. This verified that the current noise (I N ) signal analysis to specifically detect the interaction between Bcl-xL protein and small molecule compounds (see Fig.17 a).

[0146] In yet another embodiment of the present invention, it was verified that single molecule fingerprint analysis of protein-drug interactions can be performed at high resolution by a 2D density contour map that represents the ratio of complexes with small molecule compounds (LCL-161, GDC-0152, ABT-737, and A-1331852) bound to the target protein Bcl-xL as a current blockade (ΔI / I o ) and current noise (I N ). Thus, it was verified that the signal of the Bcl-xL+small molecule compound complex varied depending on the type of small molecule compound bound to Bcl-xL, and therefore, that various small molecule compounds bound to the Bcl-xL protein were clearly distinguished (see Fig.17 b), which verifies that the interaction between the analyte Bcl-xL protein and various ligands can be analyzed.

[0147] In another specific embodiment of the present invention, it was verified that when the Bcl-xL+Bak-BH3 complex was treated simultaneously with four types of small molecule compounds (LCL-161, GDC-0152, ABT-737 and A-1331852) that can bind to Bcl-xL, the ratio of the Bcl-xL+ABT-737 complex gradually decreased over time, while the ratio of the Bcl-xL+A-1331852 complex continued to increase, verifying that the YaxAB nanopore system of the present invention can be used to simultaneously analyze the binding activity between the analyte and multiple small molecule compounds in real time (see Fig.17 c) in.

[0148] In another specific embodiment of the present invention, when treated with a ligand (Bak-BH3) capable of binding to the target protein Bcl-xL and a small molecule compound (ABT-737 and A-1331852), the 2D density contour map and current blockade (ΔI / I o )-Current noise (I N) analyzed the ratio of individual complexes of ligands and small molecule compounds that competitively bind to Bcl-xL in real time at a single molecule fingerprint level with high resolution. Thus, it was verified that after ABT-737 was added 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, and that after A-1331852 was added 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, and thus, it was verified that ABT-737 and A-1331852 competitively bind to the same binding site in the Bcl-xL protein as the site bound by Bak-BH3 (see Fig.18 ).

[0149] The ratio of the molecular weight of the analyte to the molecular weight of the ligand may be 1:1 or greater, 1:2 or greater, 1:5 or greater, 1:10 or greater, 1:20 or greater, 1:20 or greater, 1:50 or greater, 1:150 or greater, 1:170 or greater, 1:190 or greater, 1:250 or greater, 1:500 or greater, or 1:1000 or greater. In a specific embodiment of the present invention, it was verified that, since the nanopore system of the present invention can clearly distinguish the ion current patterns of the analyte and the analyte-ligand complex even if the molecular weight ratio of the analyte to the ligand differs by 16 times or 194 times, it can be sensitively analyzed whether there is an interaction between the analyte and the ligand (see Fig.38 and Fig.39 ).

[0150] The concentration ratio of the analyte 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, even if the ratio of ligand binding to analyte is 1:2 or 1:100 (which is a large concentration difference), the difference in ion current pattern is clearly distinguished from the case where the analyte is captured alone; therefore, it is verified that the presence or absence of interaction between the analyte and the ligand can be sensitively analyzed (see Fig.40 and Fig.41 ).

[0151] In addition, the method for analyzing the interaction between the analyte and the ligand, or screening the ligand for the analyte can be a qualitative analysis and / or quantitative analysis of the interaction between the analyte and the ligand. For example, the method can be an analysis of whether the ligand candidate binds to the analyte, an analysis of the type and amount of the ligand, and their real-time analysis. It can be seen that the degree or frequency of the interaction between the analyte and the ligand becomes stronger or higher according to the change of the electrical signal. In addition, the degree or frequency of the interaction between the proteins as described above can be, for example, measured by binding affinity (binding dissociation constant (K D )) to quantify.

[0152] In a specific embodiment of the present invention, the YaxAB nanopore is used to perform current noise dose-dependent quantitative analysis on the target protein Bcl-xL and the ligand Bak-BH3 peptide, or the small molecule drugs ABT-737 and A-1331852, and the binding affinity (K) for Bcl-xL binding is calculated. D )(See Fig.15 ).

[0153] The method for analyzing the interaction between an analyte and a ligand, or screening a ligand for an analyte can be used for protein-protein interaction, protein-ligand interaction research and drug screening, and can be used for new drug discovery and design through drug screening of protein-protein interaction inhibitors or promoters, small molecule compounds, etc., and protein binding site mapping, because when multiple candidates are processed simultaneously, the protein binding site mapping can analyze the competitive binding or structure-activity relationship (SAR) between the analyte and the ligand candidate.

[0154] Another aspect of the present invention provides a method for analyzing or screening inhibitors or promoters of interactions between biological molecules, the method comprising the following steps: reacting multiple biological molecules capable of interacting in one compartment of a nanopore system; performing treatment with candidates for interaction inhibitors or promoters in one compartment or both compartments; and measuring changes in electrical signals in the two compartments before and after treatment with the candidates.

[0155] The method for analyzing or screening for inhibitors or promoters of interactions of the present invention comprises the following steps: treating a biomolecule in a free state in one of the two compartments of a nanopore system. In an embodiment of the present invention, the biomolecule may be first placed in one of the compartments to allow the reaction to proceed, and then the candidate may be treated in the reactant or the relative compartment. In other words, the candidate may be treated after the biomolecule in a free state is placed in one of the compartments. In addition, the biomolecule in a free state and the candidate may be treated simultaneously in one of the compartments. In addition, the treatment with the candidate may be performed by inducing the biomolecule to move to another compartment through the YaxAB nanopore contained in the nanopore system (e.g., forming a gradient, applying a voltage, or providing both according to the difference in salt concentration in the two compartments divided by the separation membrane).

[0156] In addition, biomolecules can be used without limitation, as long as they can interact with each other, for example, can bind to each other to form a complex, and can be, for example, peptides, proteins, lipids, carbohydrates, small molecules, nucleic acids or inorganic particles formed by nanomaterials. For example, the biomolecule can be a receptor, an antigen, an antibody or an aptamer.

[0157] A ligand candidate can be any type of interaction that is expected to inhibit or promote biomolecules, for example, a single compound (such as a nucleic acid, an amino acid, or a monosaccharide) or a polymer (such as a polynucleotide, a peptide, a protein, or a polysaccharide), and means a molecule such as a natural organic or synthetic organic, inorganic, or a complex thereof. For example, a ligand candidate can be a receptor, an antigen, an antibody, or an aptamer.

[0158] Among the ligand candidates, a ligand candidate that inhibits the interaction between biomolecules (particularly a ligand candidate that competitively binds to some of the biomolecules and inhibits the interaction between the biomolecules) is called an "inhibitor", and the inhibitor can reduce the interaction between the biomolecules through such competitive binding. In other words, the inhibitor can bind to some of the biomolecules and increase the ratio of biomolecules that exist in a free state in which they do not interact with each other, and can also increase the ratio of complexes in which the inhibitor interacts with some of the biomolecules. As described above, when the ratio of some of the biomolecules that exist in a state in which they do not interact with each other increases, the electrical signal generated by the interaction between the biomolecules decreases, and a new electrical signal pattern exhibited by the complex in which the inhibitor interacts with some of the biomolecules can appear, making it possible to directly detect or analyze the inhibitor that inhibits the interaction between the biomolecules.

[0159] In addition, among the ligand candidates, the ligand candidates that promote the interaction between biomolecules are called "promoters", and the promoters include molecular glues and protein degraders (PROTACs), and can induce or increase the interaction between biomolecules. For example, a promoter is a molecular glue and can be bound to a biomolecule to induce or increase the interaction between biomolecules. As described above, when the ratio of biomolecules present in a state where the biomolecules interact with each other increases, the electrical signal generated by the interaction between the biomolecules can increase, and a new electrical signal pattern exhibited by a complex in which the biomolecule and the promoter interact may appear, making it possible to directly detect or analyze the promoter that promotes the interaction between biomolecules.

[0160] Therefore, the method for analyzing or screening inhibitors or promoters of interactions between biomolecules may further include the following step: when the change in the electrical signal is significant, determining the candidate as an inhibitor that inhibits interactions between biomolecules, or a promoter that promotes interactions.

[0161] The case where the change of the electrical signal is significant may include the case where a new signal that has not appeared before or after treatment with a candidate for interaction inhibitor or promoter appears, or the case where the electrical signal generated by the interaction between biomolecules increases or decreases. Specifically, the case where the change of the electrical signal may include the current blockade (ΔI / I o ) and current noise (I N ) in the two-dimensional density contour map.

[0162] The method for analyzing or screening inhibitors or promoters of interactions between biomolecules can be used for new drug discovery and design through drug screening of protein-protein interaction inhibitors or promoters and protein binding site mapping.

[0163] In a specific embodiment of the present invention, the activity of a protein-protein inhibitor targeting the Bcl-xL+Bak-BH3 complex was analyzed using the YaxAB nanopore system. When a small molecule compound (ABT-737) capable of binding to the Bcl-xL protein was treated, it was verified that the signal of the Bcl-xL+Bak-BH3 complex decreased, and the signal of the Bcl-xL+ABT-737 complex increased, and it was verified that the binding activity of the inhibitor on the interaction between the analyte and the ligand can be analyzed in real time (see Fig.16 ).

[0164] In another embodiment of the present invention, the activity of inhibitors on the interaction between the BRD4-BD1 target protein and the acetylated peptide (H4_1-12 K5acK8ac) was analyzed using the YaxAB nanopore system. As a result, in the case of each reaction of the BRD4-BD1 target protein with the acetylated peptide (H4_1-12 K5acK8ac) and the four types of small molecule compounds (GSK778, Y06026, XMD8-92, and PLX51107) that can bind to the BRD4-BD1 protein, a unique current blocking signal of the target protein-small molecule compound complex was verified, and the signals derived from the four types of target protein-small molecule compound complexes were significantly different from the signals of the target protein-acetylated peptide complex, and differences also appeared between the target protein-small molecule compound complexes; therefore, it was verified that the activity of the interaction inhibitor that inhibits the target protein-acetylated peptide interaction can be analyzed, and further, the small molecule compounds that bind to the target protein can be distinguished (see Fig. 22 ).

[0165] In another embodiment of the present invention, the YaxAB nanopore system is used to analyze the promotion of protein-protein interactions by molecular glue. When mTOR protein, FKBP12 protein and rapamycin acting as a molecular glue are added together, a new current blocking signal appears together with the current blocking signal corresponding to the mTOR protein and the FKBP12 protein, verifying that this signal is a current blocking signal caused by a ternary complex in which mTOR, FKBP12 and the molecular glue are all combined, rather than a binary complex formed by the combination of the mTOR protein or the FKBP12 protein and the molecular glue; therefore, it is verified that proteins and ternary complexes can be distinguished, and thus, the promotion of protein-protein interactions by molecular glue can be analyzed (see Fig.21 ).

[0166] The analysis can be a qualitative or quantitative analysis of inhibitors for the interaction between the analyte and the ligand. For example, the analysis can be, but is not limited to, the presence or absence of an inhibitor of the interaction, the amount of the inhibitor, the presence or absence of competitive binding with the ligand for the analyte, the identification of the binding site of the inhibitor to the target substance (binding site mapping), or the binding affinity of the analyte and the inhibitor (binding dissociation constant, K D ).

[0167] In addition, the analysis can be a qualitative analysis or a quantitative analysis of the interaction promoter of at least one analyte and at least one ligand. For example, the analysis can be, but is not limited to, the presence or absence of the interaction promoter, the amount of the promoter, the presence or absence of a binary complex, or the presence or absence of a ternary complex.

[0168] Another aspect of the present invention provides a method for identifying and quantitatively analyzing analytes in a sample, the method comprising the following steps: placing the sample in one of the compartments of a nanopore system; and comparing the changes in characteristic electrical signals caused by multiple analytes in the sample with a database of electrical signals for each substance.

[0169] In addition, another aspect of the present invention provides a method for providing information for diagnosing biomarker-related diseases, the method comprising the following steps: placing a sample in one of the compartments of a nanopore system; and measuring changes in characteristic electrical signals caused by biomarkers for a specific disease among changes in electrical signals induced by the sample.

[0170] Sample means a sample that can contain an analyte or a biomarker without limitation, and can be, for example, one or more selected from the group consisting of: a cell sample, a tissue sample, a blood sample, a urine sample, a saliva sample, a lymph fluid sample, a cerebrospinal fluid sample, an amniotic fluid sample, a pleural fluid sample, a pericardial fluid sample, an ascites sample, an aqueous humor sample, a bone marrow sample, a semen sample, a biopsy sample, a cancer sample, a tumor sample, a forensic sample, an archaeological sample, a paleontological sample, an infection sample, a production sample, a plant sample, a microbial sample, a viral sample, a soil sample, a marine sample, and a freshwater sample.

[0171] The tissue sample may be derived from one or more tissues selected from the group consisting of epididymis, eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchus, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, bladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, urinary bladder, urethra, gonad, testis, ovary, uterus, fallopian tube, thymus, pituitary, thyroid, adrenal gland and parathyroid gland. In addition, the tissue sample may be derived from any of the various organs of a human or another organism.

[0172] The cell sample may be derived from animal cells, plant cells, fungal cells, bacterial cells or protoplast cells, specifically, may be derived from animal cells, and more specifically, may be derived from human cells.

[0173] For example, the cell sample can be derived from one or more cells selected from the group consisting of germ cells (egg cells, sperm, etc.), ovarian epithelial cells, ovarian fibroblasts, immune cells, B cells, T cells, natural killer cells, dendritic cells, cancer cells, eukaryotic cells, stem cells, blood cells, muscle cells, fat cells, skin cells, nerve cells, bone cells, 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 erythrocytes, bone marrow dendritic cells, bone marrow adipocytes, bone marrow osteocytes, bone marrow chondrocytes, preosteoblasts, bone marrow promegakaryocytes, brain B lymphocytes, brain glial cells, neurons, brain astrocytes, neuroectodermal cells, brain macrophages, brain microglia, brain epithelial cells, cortical neurons, brain fibroblasts, mammary epithelial cells, colon epithelial cells, colon B lymphocytes, mammary myoepithelial cells cells, breast fibroblasts, colon intestinal epithelial cells, cervical epithelial cells, ductal epithelial cells, tongue epithelial cells, tonsillar dendritic cells, tonsillar B lymphocytes, peripheral blood lymphoblasts, peripheral blood T lymphoblasts, peripheral blood skin T lymphocytes, peripheral blood natural killer cells, peripheral blood B lymphoblasts, peripheral blood monocytes, peripheral blood myeloblasts, peripheral blood mononuclear cells, peripheral blood preosteoblasts, peripheral blood macrophages, peripheral blood basophils, liver endothelial cells, Hepatic mast cells, hepatic epithelial cells, hepatic B lymphocytes, splenic endothelial cells, splenic epithelial cells, splenic B lymphocytes, hepatocytes, hepatic fibroblasts, lung epithelial cells, bronchial epithelial cells, lung fibroblasts, lung B lymphocytes, lung Schwann cells, lung squamous cells, lung macrophages, lung osteoblasts, neuroendocrine cells, alveolar cells, gastric epithelial cells, gastric fibroblasts, stem cells, fetal cells, tumor cells, suspected cancer cells, cancer cells and cells that have undergone gene editing procedures.

[0174] In the method for identifying and quantitatively analyzing an analyte in a sample of the present invention, the analyte means an object used to analyze the presence or absence and the amount of the analyte in the sample by the method, and the analyte can be but is not limited to a peptide, a polypeptide, a protein, an inorganic substance, a small molecule compound or a nucleic acid.

[0175] In the method for identifying and quantitatively analyzing an analyte in a sample of the present invention, the sample is placed in a compartment of a nanopore system, and changes in characteristic electrical signals caused by a plurality of analytes in the sample are measured, and the measured changes are compared with a database of electrical signals for each substance, so that identification and quantitative analysis of the analyte in the sample can be performed. The database of electrical signals for each substance can be constructed by measuring the characteristic electrical signals of each substance using the nanopore system of the present invention.

[0176] Even when multiple types of analytes are included in multiple samples, the method for identifying and quantitatively analyzing analytes in samples of the present invention can accurately measure the presence or absence and the amount of the analyte by detecting changes in the characteristic electrical signals of the specific analyte to be identified among the changes in the electrical signals of the entire sample, and can therefore be effectively used for analyte identification and quantitative analysis, and further for drug screening, etc.

[0177] In addition, in the present invention, "biomarker" means an anatomical parameter, a physiological parameter, a biochemical parameter or a molecular parameter associated with the presence or progression of a specific physiological state. A biomarker may include an organic biological molecule that shows an increase or decrease in an individual or in a specific sample derived from an individual, such as a polypeptide, a protein or a nucleic acid (e.g., mRNA, etc.), a lipid, a glycolipid, a glycoprotein or a sugar (e.g., a monosaccharide, a disaccharide, an oligosaccharide, etc.).

[0178] The method of the present invention for providing information for diagnosing biomarker-related diseases can accurately measure the presence or absence and the amount of biomarkers in a sample by placing the sample in a compartment of a nanopore system and measuring changes in characteristic electrical signals caused by biomarkers known to be associated with a specific disease among changes in electrical signals induced by the sample, and can therefore be effectively used for the diagnosis of biomarker-related diseases and the prediction of disease prognosis.

[0179] The disease is not limited as long as it is a disease identified to be associated with a specific biomarker, and may be an infectious disease or a non-infectious disease. Infectious diseases are diseases caused by microorganisms such as bacteria and viruses, and may be, for example, sepsis, septic shock, severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome (MERS), salmonellosis, food poisoning, typhoid fever, paratyphoid fever, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), pneumonia, tuberculosis, tuberculosis, cold, flu, respiratory tract infection, rhinitis, nasopharyngitis, otitis media, bronchitis, lymphadenitis, mumps, lymphadenitis , 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, urethritis, cystitis, vaginitis, cervicitis, salpingitis, infectious erythema, bacillary dysentery, abscesses and ulcers, bacteremia, diarrhea, dysentery, gastroenteritis, gastrointestinal inflammation , genitourinary abscess, open wound or wound infection, suppurative inflammation, abscess, furuncle, pyoderma, impetigo, folliculitis, cellulitis, postoperative wound infection, scalded skin syndrome, skin burn syndrome, thrombotic thrombocytopenia, hemolytic uremic syndrome, renal failure, pyelonephritis, glomerulonephritis, nervous system abscess, otitis media, sinusitis, pharyngitis, tonsillitis, mastoiditis, cellulitis (phlegmon), odontogenic infection, dacryocystitis, pleurisy, abdominal abscess, liver abscess, cholecystitis, spleen abscess, pericarditis, heart Myositis, placentaitis, amniotic sac, mastitis, mastitis, puerperal fever, toxic shock syndrome, Lyme disease, gas gangrene, atherosclerosis, Mycobacterium avium complex (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.

[0180] The non-infectious disease can be cancer, autoimmune disease, non-infectious inflammatory disease, neurodegenerative disease, heart disease, stroke, diabetes, metabolic disease, chronic kidney disease or chronic respiratory disease. The cancer can be 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, stomach cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell / histiocyte-rich B cell lymphoma, Epstein-Barr virus (EBV, or simply "EB virus") positive and negative post-transplantation lymphoproliferative disease (PTLD), EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, human herpes virus 8 (HHV8)-related primary effusion lymphoma, other hematological malignancies including Hodgkin's lymphoma, primary central nervous system (CNS) lymphoma, spinal tumors, brainstem glioma, etc. Autoimmune diseases can be 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, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Gram's Fuzzy's disease (also known as "diffuse toxic goiter"), IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, granulomatous polyangiitis, other 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, leukoplakia, myasthenia gravis, systemic sclerosis, etc. Neurodegenerative diseases can be cognitive impairment, brain tumors, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain, memory loss, etc.

[0181] In a specific embodiment of the present invention, it was verified whether it is feasible to selectively detect and quantitatively analyze specific biomarker proteins present in serum samples. It was verified whether the unique characteristic electrical signals of Bcl-xL at concentrations of 10nM and 20nM appeared in the presence of fetal bovine serum (FBS) similar to human serum, and when a voltage of 80mV was applied in the presence of FBS from which some proteins were removed (deproteinized FBS), in addition to the various nanopore electrical signals measured in the deproteinized FBS, the unique characteristic electrical signal of the 10nM Bcl-xL protein was observed; therefore, the signal distribution consistent with the unique electrical signal of the Bcl-xL protein among the various scattered signals in the deproteinized FBS was verified. In addition, as a result of performing electrical measurement under the condition of 20 nM Bcl-xL protein in the presence of deproteinized FBS, more unique electrical signals of Bcl-xL were measured compared to the condition of 10 nM Bcl-xL protein, and it was verified in the two-dimensional scatter plot results that the Bcl-xL protein signal distribution was obviously densely distributed in a concentration-dependent manner; therefore, it was verified that the nanopore system of the present invention can accurately measure the presence or absence of an analyte protein and the amount of the analyte protein in a mixed sample containing a plurality of types of substances (see Fig.23 ).

[0182] Hereinafter, the present invention will be described in detail with reference to examples.

[0183] However, the following examples are intended to specifically illustrate the present invention, and the present invention is not limited to the following examples.

[0184] [Example 1] Fabrication of YaxAB nanopore sensor [1-1] Sample preparation The compounds used in the nanopore measurement buffer were purchased from Sigma-Aldrich (St. Louis, 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 (Daejeon, South Korea). 6 (Sigma-Aldrich) to prepare small molecule compounds ABT-737 (Cayman chemical), LCL-161 (Selleckchem), GDC-0152 (Selleckchem), A-1331852 (Selleckchem) and quercetin (Sigma-Aldrich).

[0185] [1-2] Purification of the first monomer (YaxA) and the second monomer (YaxB) The first monomer (YaxA) and the second monomer (YaxB) used in the experiment were synthesized by Cosmogenetech. For the first monomer (YaxA), the amino acid sequence of SEQ ID NO: 1 (YaxA_(45-410)) and the wild-type amino acid sequence of SEQ ID NO: 3 (YaxA_FL) were synthesized and purified, and for the second monomer (YaxB), the amino acid sequence of SEQ ID NO: 2 (YaxB_(12-343)) and the wild-type amino acid sequence of SEQ ID NO: 4 (YaxB_FL) were synthesized and purified.

[0186] Specifically, YaxA and YaxB were inserted into a pET15b vector containing a thrombin cleavage site with an N-terminal 6xHis-SUMO tag and a 6xHis tag, respectively. YaxA was expressed in E. coli BL21 (DE3) pLysS cells, which were grown overnight in 2xYT medium at 18°C ​​after induction with 0.5mM IPTG. YaxB was also expressed under the same conditions as YaxA. To purify the expressed YaxA protein, the cell pellet was resuspended in lysis buffer (50mM Tris pH 8.0, 300mM NaCl, 1mM PMSF, 1g / ml DNase I and 0.2mg / ml lysozyme) and lysed using a microfluidizer. The YaxA protein was dissolved in 3% DDM at 4°C for 2 hours. The purified lysate was added to a 5 mL Ni-NTA affinity column equilibrated with buffer A (50 mM Tris pH 8.0, 300 mM NaCl, 0.05% Cymal-6, and 10 mM imidazole), and the protein bound to the Ni-NTA resin was eluted with a linear gradient from 10 mM to 1 M imidazole. Dialysis was performed at 4 ° C against buffer B (50 mM Tris pH 8.0 and 300 mM NaCl) in the presence of thrombin protease. YaxA was further purified with a secondary Ni-NTA step to remove the enzyme tag and protease. In the case of the 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, and 0.2 mg / mL lysozyme) and lysed using an ultrasonic generator. The clarified lysate was added to a 5 mL Ni-NTA affinity column equilibrated with buffer C (50 mM Tris pH 8.0, 300 mM NaCl, and 10 mM imidazole), and the bound protein was eluted with a linear gradient from 10 mM to 1 M imidazole. Dialysis was performed at 4 ° C against buffer E (20 mM HEPES pH 7.0 and 25 mM NaCl) in the presence of thrombin protease. Next, the sample was loaded onto a 5 mL HiTrap Q column equilibrated with buffer E and eluted with a linear salt gradient from 0 to 1 M NaCl. The peak fraction was further purified using a Superdex 75 16 / 600 gel filtration column (GE Healthcare) using buffer F (25 mM HEPES pH 7.0 and 150 m M NaCl).

[0187] [1-3] Fabrication of YaxAB nanopores The modified YaxAB_dN nanopore was prepared using the first monomer (YaxA_(45-410)) of SEQ ID NO: 1 and the second monomer (YaxB_(12-343)) of SEQ ID NO: 2 purified in Example 1-2. In the same manner, a wild-type YaxAB nanopore containing a wild-type first monomer (SEQ ID NO: 3, YaxA_FL) and a wild-type second monomer (SEQ ID NO: 4, YaxB_FL) was prepared.

[0188] Specifically, 6 mg of each monomer was incubated together at 25°C for 30 minutes to allow complex formation. 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 before being injected onto a Superose 6 column running in gel filtration buffer (25 mM HEPES pH 7.0, 150 mM NaCl and 0.05% w / v Cymal-6). Peak fractions were separated into different sizes of YaxAB oligomeric proteins with different numbers of YaxAB nanopore subunits by 4% to 16% blue native polyacrylamide gel electrophoresis (BN-PAGE, Invitrogen, Thermo Fisher Scientific, Korea) (see Figure 1 ). The bands corresponding to the YaxAB oligomeric proteins were cut out from the gel and soaked in gel filtration buffer, and the YaxAB oligomeric proteins were extracted from each band at 4° C. The wild-type YaxAB nanopores were extracted in the same manner, and the supernatants containing the modified YaxAB_dN nanopores or the wild-type YaxAB nanopores were used in the experiments.

[0189] [1-4] Fabrication of nanopore system and measurement method of nanopore experimental data The YaxAB-C prepared in Example 1-3 and comprising the first monomer (YaxA_(45-410)) of SEQ ID NO: 1 and the second monomer (YaxB_(12-343)) of SEQ ID NO: 2 was used. 8 Nanopores are used to make nanopore sensors.

[0190] Specifically, two compartments were assembled by positioning a Teflon membrane with 100 μm pores between two Teflon chambers, and each compartment was filled with 0.8 mL of a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 M KCl. Regarding the composition of the solution, various types of buffers (such as HEPES, phosphate buffer, PBS, sodium acetate, etc.) as well as Tris-HCl can be used. Prior to assembly, the nanopores were pre-coated with a lipid bilayer.

[0191] For electrical measurements, among the Ag / AgCl electrodes, the ground electrode was connected to the trans side, and the working electrode was connected to the cis side. 8 To YaxAB-C 13 Addition to the cis compartment allowed a single YaxAB nanopore to be inserted into the lipid membrane, and the nanopore current signal was measured by applying a voltage of +100 mV to the cis compartment. After insertion of the YaxAB nanopore, 10 nM to 1000 nM of various proteins (Bcl-xL, Hsp33, FKBP12, holotransferrin, MDM2, BSA, aldolase, ferritin, and thyroglobulin) or Bcl-2 family proteins were added, and then, a single peptide ligand or multiple different peptide ligands (Bak-BH3, Bak-BH3-I83A, Bak-BH3-D84A, and Bak-BH3-L78A), or a single small molecule compound or multiple different small molecule compounds (ABT-737, LCL-161, GDC-0152, A-1331852, quercetin, A1155463, FK506, and oxaliplatin) were added to the cis compartment. Electrical signals were recorded using a patch clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA, USA) at bias voltages ranging from 40 mV to 120 mV, and the collected data were analyzed using the single channel search function of pClamp 11 software (Molecular Devices). Current signals recorded at a sampling rate of 100 kHz were filtered at 10 kHz using a built-in 8-pole low-pass Bessel filter. The sampling rate and filter were optimized for the signal under various conditions. All nanopore experiments were performed at 25 °C.

[0192] [Example 2] YaxAB nanopore formation and electrical characterization [2-1] Formation of YaxAB nanopores In order to verify whether the YaxAB nanopore protein prepared in Example 1-3 can be used as a nanopore sensor, the structure of the modified YaxAB_dN nanopore was analyzed by negative staining EM and cryo-electron microscopy (cryo-EM). Figure 2 As shown in a to c, it was verified that the modified YaxAB_dN nanopore is an α-helical pore complex assembled by 8 to 10 subunits.

[0193] Based on EM data, export Figure 2 The modeling structure of d in Figure 2 YaxAB-C shown in e 8The results verified that the YaxAB nanopore has a distinct symmetrical structure, and has a wide first opening, a narrow second opening, and an intermediate region connecting the first opening and the second opening. In addition, it was verified that the lumen of the nanopore has a narrowing portion with a narrowed diameter. Specifically, it was verified that the YaxAB-C 8 The nanopore has a funnel structure with a wide entrance and a narrow exit, wherein 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 narrowed portion of the lumen at the second opening is 1.9 nm, and the lumen of the nanopore has a wavy shape due to the alpha helix in the shape of a coffee dripper with a narrowed portion. Based on this, Figure 3 The structure of the YaxAB nanopore is shown in FIG.

[0194] [2-2] Molecular dynamics simulation analysis Prior to the experiment, molecular dynamics (MD) simulations were performed to analyze the properties of the YaxAB nanopore. 8 The simulation was performed by inserting a setting into a composite membrane having a size of 25×25 nm and including 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and sterols. Molecular dynamics simulations were performed by GROMACS 2020.2 software package at a temperature of 303 K, with 1 M KCl solution and without surface tension.

[0195] In order to pass YaxAB-C at 100mV 8 The free energy of Bcl-xL protein was measured by adding Bcl-xL at a distance of z = 10 nm from the membrane containing the nanopore, and then MD simulations were performed by adjusting the distance (z) from the membrane in the range of 2 nm to 13 nm by 1 nm intervals. The Bcl-xL protein was free to move in the x-direction and the y-direction, and the free energy was calculated by integrating the average force at each set z value.

[0196] Results, such as Figure 4 As shown in a, the local distribution of water and ion molecules inside the nanopore is verified, and it is verified that when a positive voltage is applied, the flow rate of potassium ions and water becomes faster as they approach the constriction of the nanopore. In the constriction of the lumen, the flow rate of potassium ions (0.16 nm -2 ns -1 ) is faster than the flow rate of chloride ions (0.04nm -2 ns -1 ) is fast, which means that when a positive voltage is applied, the electroosmotic force (EOF) acts in both the cis and anti directions.

[0197] In addition, as a result of measuring the free energy through MD simulation, such as Figure 5As shown in , the free energy shows a dual minimum when the Bcl-xL protein is located at a distance of z = 4 nm and 10 nm. It is predicted to oscillate between several residence sites (R1 to R3), while various current blockade levels (L1 to L3) are induced due to the transient residence of the Bcl-xL protein at the free energy minimum. The minimum free energy value of Bcl-xL observed at a distance of z = 4 nm means that the protein preferentially resides at this location once trapped within the YaxAB nanopore. MD simulations predict that the flow of potassium ions and water will be significantly reduced in the constriction when the Bcl-xL protein is trapped at z = 4 nm compared to the empty YaxAB nanopore ( Figure 4 b). Therefore, due to the weakened EOF, the Bcl-xL protein will move toward the cis side (z = 10 nm). MD simulations show that the flow of potassium ions and water is greater when the Bcl-xL protein is trapped at a distance of z = 10 nm than when the Bcl-xL protein is trapped at a distance of z = 4 nm ( Figure 4 This indicates that the restored EOF caused the Bcl-xL protein to move back to the trans side (z = 4 nm).

[0198] [2-3] Improvement of stability of nanopore-containing membranes It was verified that there were problems such as that the YaxAB nanopores were not well inserted into the DPhPC membrane, which is an artificial biomembrane material that has been widely used in the related art, or that even if inserted therein, the YaxAB nanopores had low stability and, therefore, the membrane was easily broken (see Figure 6 A in the figure).

[0199] To supplement this, DPhPC-sterol composite membranes were constructed by combining 5 mol% to 50 mol% of sterol molecules with DPhPC, and in the case of such composite membranes, not only the C 6 Nanopore, C 7 Nanopore, C 8 Nanopore, C 9 Nanopore, C 10 Nanopore, C 11 Nanopore, C 12 Nanopores and C 13 All of the nanopores were successfully inserted, and as shown in Table 1, it was verified that even after insertion into the membrane, a certain amount of current flowed remarkably stably following Ohm's law (see Figure 6 B and Figure 7 ).

[0200] [Table 1]

[0201] In addition, the conductivity results measured when a voltage of 100 mV was applied to the cis compartment were matched to each of the modified YaxAB_dN nanopores. The following constant conductivities were observed: 4 , is 1.0±0.1nS; for YaxAB-C 5 , is 1.8±0.1nS; for YaxAB-C 6 , is 3.0±0.1nS; for YaxAB-C 7 , is 4.1±0.1nS; for YaxAB-C 8 , is 5.0±0.1nS; for YaxAB-C 9 , is 7.8±0.2nS; for YaxAB-C 10 , is 10.9±0.2nS; for YaxAB-C 11 , is 14.6±0.2nS; for YaxAB-C 12 , is 18.5nS; and for YaxAB-C 13 , is 24.4nS (see Figure 7 and Figure 8 ). The experimental values ​​are verified to be consistent with the theoretical values ​​of the currents derived from the YaxAB nanopore structure (for YaxAB-C 8 , the current value is 5.4nS; for YaxAB-C 9 , the current value is 8.2nS; and for YaxAB-C 10 , the current value is 11.7nS), and thus, it is verified that the YaxAB nanopores of the present invention follow Ohm's law and exist significantly and stably on the DPhPC-sterol composite membrane. 8 The DPhPC-sterol composite membrane (hereinafter, referred to as YaxAB nanopore) was inserted therein to fabricate a nanopore sensor, and then, experiments were performed.

[0202] [2-4] Measurement of cation selectivity of YaxAB nanopore To measure the movement of ions through the YaxAB nanopore of the present invention, the ion current was measured by applying various voltages.

[0203] The ion selectivity (P) representing the permeability ratio of potassium ions to chloride ions was calculated using the Goldman-Hodgkin-Katz equation of the following equation 1: K + / P Cl - ).

[0204] <Equation 1>

[0205] Here, [α K+ / Cl- ] 顺 / 反 represents the activity of potassium ions in the cis chamber or the activity of chloride ions in the trans chamber, R represents the ideal gas constant, F represents the Faraday constant, and Vr represents the reversal potential measured under asymmetric salt conditions.

[0206] The reversal potential (Vr) of the YaxAB nanopore was measured in the compartments on both sides of the nanopore using different concentrations of KCl solution in 10 mM Tris HCl buffer at pH 7.5 (trans / cis: 2 M KCl / 0.5 M KCl). Specifically, 800 μL of 1 M KCl solution was added to both the cis compartment and the trans compartment, and then the YaxAB nanopore was inserted into the membrane. After adjusting the offset and balancing the electrodes, 400 μL of 1 M KCl solution was removed from both compartments, 400 μL of 3 M KCl was added to the trans compartment and an equal volume of salt-free buffer was added to the cis compartment to induce a salt gradient. For each ion activity, the cation selectivity was calculated by multiplying the molar concentration of a given ion by the average ion activity coefficient (2 M KCl: 0.573, 0.5 M KCl: 0.649).

[0207] Results, such as Fig. 9 As shown in Figure , three modified YaxAB_dN nanopores (C 8 Nanopore, C 9 Nanopore, C 10 Nanopores) all have asymmetric IV curves. 8 The cation selectivity (P K + / P Cl - ) is 2.34; YaxAB-C 9 The cation selectivity (P K + / P Cl - ) is 1.72; and YaxAB-C 10 The cation selectivity (P K + / P Cl - ) is 1.47, and this high cation selectivity of the YaxAB nanopore of the present invention can induce an electroosmotic force that acts mainly in the opposite direction to the electrophoretic force. In the case of applying a positive voltage under the condition of pH 7.5, the amino acids forming the inner wall of the pore are negatively charged, which leads to strong electroosmosis caused by cations flowing mainly along the inner wall of the pore from the cis direction to the anti-direction (due to excellent cation selectivity).

[0208] Thus, since the YaxAB nanopore of the present invention can induce electroosmosis, the analyte can be captured inside the nanopore regardless of the total charge, and since the diameter of the YaxAB nanopore can be controlled by adjusting the number of heterodimers composed of a portion of the monomers YaxA and YaxB in addition to having a wide entrance, even large analytes can be captured inside the nanopore; therefore, there is an advantage in that the object to be analyzed is not limited by the charge or size of the analyte. In addition, since the YaxAB nanopore has a funnel structure in which the entrance is wide but the exit is relatively much narrower, when the analyte is captured inside the nanopore, the current blockade caused by the analyte can be changed more drastically compared to a conventional cylindrical nanopore, and as a result, the YaxAB nanopore has the advantage of being able to more sensitively detect even small changes in the analyte by maximizing the difference in the current blockade caused by the analyte.

[0209] [Example 3] Detection of analytes at the single-molecule level using the YaxAB nanopore sensor In order to verify whether the YaxAB nanopore sensor of the present invention can detect proteins at the single molecule level, the YaxAB-C prepared in Example 1-4 was used. 8 Nanopore sensor experiments were performed.

[0210] First, in order to verify the principle of the change of ion current caused by the analyte in the YaxAB nanopore of the present invention, the Bcl-xL protein (3.4 nm × 5.1 nm) with a total negative charge, larger than 2 nm and unable to pass through the YaxAB nanopore of the present invention was selected as a representative analyte. The Bcl-xL protein thus selected was placed in the cis compartment, and (+) and (-) 100 mV voltages were applied to the cis compartment and the trans compartment, respectively.

[0211] In the case of Bcl-xL protein, since the interior of the pore is negatively charged at pH 7.5, Bcl-xL protein cannot flow into the interior of the nanopore by the electrophoretic force (EPF) induced by the voltage applied to the two compartments. However, Bcl-xL protein flows into the pore by the electroosmotic force (EOF) induced by the YaxAB nanopore of the present invention (see Figure 5 The capture of A in (I) and at this time, the open pore current (I open )(See Figure 5 After being trapped in the pore, the Bcl-xL protein undergoes an oscillatory motion due to the balance between electroosmotic and electrophoretic forces (see Figure 5 The oscillatory motion induces an open pore current (I open) in the range of 68.4±3.6% to 79.0±1.8% (see Figure 5 In addition, when the Bcl-xL protein undergoing oscillatory motion as described above escapes from the nanopore (see Figure 5 The escape from the pore (III) no longer induces current blockade, and the ionic current returns to the open pore current (I open ) level.

[0212] Finally, it was verified that the Bcl-xL protein captured in the YaxAB nanopore exhibited three types of ion current patterns (L1, L2, and L3), and specifically, the current blockade between L1 and L2 / L3 was 30.1% to 41.0%, which was greater than that of conventional nanopores (e.g., ClyA, PlyAB, MspA, NeoTrap, etc.).

[0213] The current blockade levels of conventional ClyA nanopores were verified to be: 1.3% for SBP1; 1.7% for SBP2; 8.3% for BtuF; 3.4% for GBP; 4.8% for SiaP; 2.5% for TbpA; 6.2% for LBP; 3.6% for SpuD; and 4.1% for MBP (Zernia et al., “ Current Blockades of Proteins inside Nanopores for Real-Time Metabolome Analysis ”, ACS Nano, 14(2): 2296-2307(2020)), and verified the current blockade levels of conventional PlyAB nanopores: 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)).

[0214] In addition, although the specific current blockade levels of conventional MspA nanopores are not separately indicated, their current blockade levels were verified to be within 15% for lysozyme and within 18% for whole myoglobin (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)).

[0215] In addition, although the specific current blocking level of the NeoTrap nanopore is not separately indicated, it was verified that the current blocking level for Hsp90 and avidin was within 20%, thus verifying that the YaxAB nanopore of the present invention has 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)).

[0216] Meanwhile, the current blockade of whole myoglobin (within 18%) or HbA (within 23.2%) in the MspA nanopore having a relatively high current blockade level among conventional nanopores was significantly lower than that of Bcl-xL in the YaxAB nanopore of the present invention (30.1% to 41.0%). As a result of comparing the analytical resolution of the YaxAB nanopore and the conventional nanopores ClyA and MspA for the same analyte, in the case of the YaxAB nanopore of the present invention, the change between the current levels of the signal derived from GBP was approximately 14.3%, which was significantly higher than the change of 3.4% in the signal derived from GBP analyzed in the ClyA nanopore, and was 4.2 times or more of the change of 3.4% (see Fig.14 A and B in the figure). In addition, in the case of the YaxAB nanopore of the present invention, the changes between the current levels of the signals derived from lysozyme and whole myoglobin were about 14.0% and 47.3%, respectively, which were about 1.3 times and 2.6 times the changes in the signals derived from lysozyme and whole myoglobin analyzed by the MspA nanopore (which were 10.4% and 17.9%, respectively); therefore, the excellent analytical performance of the YaxAB nanopore of the present invention was verified (see Fig.14 C and D in the figure).

[0217] In addition, based on the three analysis parameters, the signal characteristics of lysozyme using the YaxAB nanopore of the present invention were compared with the signal characteristics of lysozyme obtained using the MspA nanopore. As a result, in the YaxAB nanopore of the present invention, the current blockade (ΔI / I o ) of about 65.5 ± 0.3%, the standard deviation (SD) of the ionic currents was 15.9 ± 0.6 pA, and the capture time ranged from 100 ms to 10000 ms, which were 33.9% of the lysozyme protein signal from the conventional MspA nanopore and 14.8% of the ΔI / I of the ion current. o , SD levels from 9pA to 10pA, and capture times from 1ms to 100ms. o In terms of analyte capture time, it is 100 times that of the conventional MspA nanopore for the same analyte, thus verifying that the analytical performance of the YaxAB nanopore of the present invention is excellent (see Fig.14 E in.

[0218] In addition, in order to verify the range of proteins that can be analyzed using the YaxAB nanopore of the present invention, the patterns of ion currents were analyzed using Hsp33 protein (52.4 kDa) and BSA protein (66.5 kDa), holotransferrin (77.1 kDa), aldolase protein (158 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa), FKBP12 protein (13.0 kDa) and positively charged MDM2 protein (12.3 kDa), where Hsp33 protein (52.4 kDa) and BSA protein (66.5 kDa) were significantly higher than those of Bcl-xL protein. The protein (20.8 kDa) has a large size and has a large negative charge; the whole transferrin (77.1 kDa) has a large size but has a relatively small negative charge; the aldolase protein (158 kDa) has a large size but has a positive charge; ferritin (440 kDa) and thyroglobulin (669 kDa) have surface charge properties similar to those of Bcl-xL, but have a large size; the FKBP12 protein (13.0 kDa) is electrically neutral, and because it is smaller than the Bcl-xL protein, the FKBP12 protein (13.0 kDa) can be translocated through the YaxAB nanopore of the present invention.

[0219] As a result, it was verified that each of the nine proteins exhibited distinct ion current patterns (see Fig.10 ), which demonstrates that single-molecule analysis of proteins of various sizes and charges is feasible.

[0220] [Example 4] Analysis of analyte-ligand interactions using the YaxAB nanopore sensor [4-1] Analysis of protein-protein interactions or protein-small molecule interactions First, it was verified whether the YaxAB nanopore of the present invention can be used to analyze protein-protein interactions or protein-small molecule compound interactions.

[0221] To the cis compartment of the sensor fabricated in Example 1-4, i) Bcl-xL protein was added alone at a concentration of 100 nanomolar (nM), ii) Bcl-xL protein and Bak-BH3 protein known to bind to Bcl-xL protein were added together at a molar ratio of 1:2, and iii) Bcl-xL protein and ABT-737, a small molecule compound known to bind to Bcl-xL protein and a competitive inhibitor for Bak-BH3, were added together at a molar ratio of 1:2:5; and (+) and (-) voltages were applied to the cis compartment and the trans compartment, respectively.

[0222] As a result, it was verified that the pattern of current blocking effect induced by Bcl-xL protein alone changed due to the addition of Bak-BH3 protein or ABT-737, and different current blocking patterns appeared for these two substances; therefore, it was verified that protein-protein complex (Bcl-xL+Bak-BH3) and protein-small molecule compound complex (Bcl-xL+ABT-737) could be distinguished (see Fig.11 a and b in ).

[0223] In addition, if Fig.11 As shown in Figures c to f, in addition to the current blocking analysis, a method based on current noise (I N ) analysis using the YaxAB nanopore to distinguish between free Bcl-xL, Bcl-xL / small molecule compound complexes, and Bcl-xL / peptide complexes. Specifically, it was verified that the ion current patterns of the analytes in the three cases described above could be more clearly distinguished by filtering the signal in the low oscillation range (100 Hz or less).

[0224] Next, it was verified whether the YaxAB nanopore of the present invention can be used to analyze the interaction between proteins and molecules with low binding affinity.

[0225] To the cis compartment of the sensor fabricated in Example 1-4, i) Bcl-xL protein alone was added at a concentration of 1 micromolar (μM); and ii) Bcl-xL protein and Bax-BH3 peptide, and Bcl-xL protein and a quercetin small molecule compound known to bind to Bcl-xL protein were added at molar ratios of 1:100 and 1:20, respectively. Thereafter, (+) and (-) voltages were applied to the cis compartment and the trans compartment, respectively.

[0226] As a result, the addition of Bax-BH3 peptide or quercetin caused intermittent changes in the current blockade pattern induced by Bcl-xL protein alone, and the two substances also showed current blockade patterns different from each other; thus, it was verified that the interaction between Bcl-xL and binders with low binding affinity can be analyzed (see Fig.12 and Fig.13 ).

[0227] In addition, if Fig.12 and Fig.13 As shown in Figure 2, it was demonstrated that in addition to current blockade analysis, the YaxAB nanopore can utilize the current noise (I N ) analysis was used to distinguish between free Bcl-xL and Bcl-xL complexes with peptides or small molecules.

[0228] [4-2] Analysis of protein structural changes induced by ligand binding Based on the results of protein-small molecule compound interaction analysis, the YaxAB nanopore of the present invention is used to measure the current blocking signal generated when glucose / galactose binding protein (GBP) binds to galactose. Glucose / galactose binding protein (GBP) is composed of two domains and undergoes allosteric movement when bound to glucose or galactose. Galactose is a small molecule compound that targets GBP (see Fig.14 ).

[0229] Specifically, 50 nM GBP was added to the cis compartment of the YaxAB nanopore, and a (+) voltage of 80 mV was applied to the same compartment to verify the current blockade signal generated by the capture of the GBP protein. Current blockades corresponding to current level 1 and current level 2, as well as specific gating behaviors, were observed. At this time, when galactose, a small molecule compound targeting GBP, was added to the trans compartment, it was verified that the gating frequency occurring in the current blockade signal derived from GBP was significantly increased. In addition, the signals derived from GBP and GBP-glucose in the conventional ClyA nanopore (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)), it was verified that the change between the current levels was approximately 3.4%, but in the case of the YaxAB nanopore of the present invention, the change between the current levels in the signals originating from GBP and GBP-galactose was approximately 14.3%, which is significantly 4.2 times or more of the change between the current levels of the ClyA nanopore.

[0230] Through the above results, the unique electrical signal of GBP and the unique electrical signal of GBP-galactose complex generated according to the presence or absence of galactose were analyzed using the YaxAB nanopore of the present invention. As a result, the structural changes of the GBP protein caused by galactose binding can be effectively analyzed, and it is verified that the analysis resolution of the YaxAB nanopore of the present invention is significantly better than that of the conventional ClyA nanopore.

[0231] [4-3] Analysis of the binding affinity of proteins or small molecules to proteins In addition, based on the current noise (I N ) analysis, perform dose-dependent quantitative analysis of the interaction between Bcl-xL protein and Bak-BH3 peptide or ABT-737 compound, and calculate the binding affinity (K) for Bcl-xL protein D ).

[0232] Specifically, as a result of treating 100 nM Bcl-xL protein with each of 10 nM, 30 nM, 50 nM, 100 nM, 200 nM and 500 nM of Bak-BH3 protein, it was confirmed that the ratios of complex formation between 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, and thus the binding affinity (K) of Bak-BH3 protein to Bcl-xL protein was analyzed. D ) was 65.78±10.32nM (see Fig.15 a) in the figure.

[0233] In addition, as a result of treating 100 nM Bcl-xL protein with each of 10 nM, 50 nM, 70 nM, 100 nM, 200 nM and 1000 nM of the ABT-737 compound in a similar manner to the above, it was confirmed that the ratios of complex formation 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, and thus the binding affinity (K) of the ABT-737 compound to the Bcl-xL protein was analyzed. D ) was 37.97±7.91nM (see Fig.15 b) in the above.

[0234] In addition, as a result of treating 100 nM Bcl-xL protein with each of the A-1331852 compound at concentrations of 10 nM, 30 nM, 50 nM, 70 nM, 100 nM, 200 nM and 1000 nM in a similar manner to the above, it was confirmed that the ratios of complex formation between the Bcl-xL protein and the A-1331852 compound at different concentrations were 34.5%, 65.7%, 78.1%, 98.2%, 99.5%, 100% and 100%, respectively, and the binding affinity (K) of the A-1331852 compound to the Bcl-xL protein was calculated therefrom. D ) was 19.16±4.47nM (see Fig.15 c) in.

[0235] It was thus found that the YaxAB nanopore system of the present invention can analyze the strength of the interaction between a drug and a biomolecule such as a protein, and can therefore be effectively used for drug screening.

[0236] [4-4] Analysis of the activity of protein-protein interaction inhibitors In addition, Bcl-xL protein at a concentration of 100 nM was reacted with 200 nM Bak-BH3 protein, and then treated with ABT-737, a competitive inhibitor of Bak-BH3 protein, at concentrations of 100 nM, 500 nM, and 1000 nM. As a result, it was verified that as the concentration of the added ABT-737 compound increased, the occurrence ratio of the ion current mode of the complex of Bcl-xL protein and Bak-BH3 protein (Bcl-xL+Bak-BH3) gradually decreased, and the occurrence ratio of the ion current mode of the complex of Bcl-xL protein and ABT-737 compound (Bcl-xL+ABT-737) gradually increased (see Fig.16 ).

[0237] According to the above results, it is verified that the protein-drug complex can be directly detected with high resolution using the YaxAB nanopore of the present invention. This shows that the method of directly detecting drug binding by measuring the signal reduction of the protein-protein complex has a significant improvement over the conventional method, making it possible to directly and in real time detect and analyze the protein-drug complex.

[0238] Next, we validated whether the YaxAB nanopore sensor could be used to analyze the interactions between target proteins and post-translational modification (PTM) peptides as well as interactions between inhibitors of protein-protein interactions.

[0239] First, using the YaxAB-C of the present invention 8 Nanopore sensors verify the interaction between acetylated peptides and proteins, or protein-small molecule compounds at the single-molecule level. BRD4, a bromodomain and extra-terminal domain (BET) family protein, was used as a target protein. BRD4 is a chromatin-binding protein associated with cancer and autoimmune diseases, and acts as a scaffold for transcription factors at promoters and super enhancers. BRD4 is divided into bromodomain 1 (BD1) and bromodomain 2 (BD2), and BD1 has a more extended loop than BD2. BD1 can specifically recognize and bind to the acetylated part of the H4 part of the histone. Considering the binding characteristics, the H4 peptide consisting of 12 amino acids in the histone (H4_1-12) and the peptide acetylated at residues 5 and 8 (H4_1-12 K5acK8ac) were used to verify the effect of the presence or absence of PTM on binding to the target protein BRD4-BD1 (see Fig. 22 A and B in the figure).

[0240] Specifically, a solution containing 10 mM Tris (pH 7.5), 1 mM EDTA, and 1 M LiCl was filled into the YaxAB-C 8Two compartments of the nanopore sensor. Then, 2 μM BRD4-BD1 protein was added to the cis compartment, (+) voltage was applied, and the nanopore current blockade signal was observed. At this time, a unique nanopore current blockade signal derived from the L1-L2 level represented as BRD4-BD1 protein, which was generated by the oscillatory motion of the BRD4-BD1 protein inside the nanopore, was observed. In addition, when the target protein and the acetylated peptide (H4_1-12 K5acK8ac) were added to the cis compartment at a molar ratio of 1:20 and the nanopore current blockade signal was measured, the emergence of the L3 level in the L1-L2 level derived from the BRD4-BD1 protein was verified (see Fig. 22 C in.

[0241] In addition, the change in the nanopore current blockade signal caused by the interaction between the BRD4-BD1 protein and the acetylated peptide (H4_1-12 K5acK8ac) can be expressed as current noise (I N ) values, and based on this, concentration-dependent quantitative analysis was performed to calculate the binding affinity (K) of the acetylated peptide (H4_1-12 K5acK8ac) to the BRD4-BD1 protein. D Specifically, as a result of treating 2 μM BRD4-BD1 protein with H4_1-12K5acK8ac peptide at concentrations of 2 μM, 4 μM, 6 μM, 10 μM, 20 μM, and 40 μM, the binding affinity (K) of the acetylated peptide (H4_1-12 K5acK8ac) to the BRD4-BD1 protein was analyzed. D ) was calculated to be 3.269 ± 0.756 μM (see Fig. 22 D in.

[0242] The above results demonstrate that the YaxAB nanopore system of the present invention is capable of qualitatively and quantitatively analyzing the interaction between a target protein and a ligand, which is determined according to the state of its post-translational modification (PTM).

[0243] Next, the activity of the inhibitor of the interaction between the BRD4-BD1 target protein and the acetylated peptide (H4_1-12 K5acK8ac) was verified in the YaxAB nanopore system of the present invention. Specifically, each of four types of small molecule compounds (GSK778, Y06026, XMD8-92, and PLX51107) capable of binding to the BRD4-BD1 protein was allowed to react with the BRD4-BD1 protein, and then the nanopore current blockade signal was measured. As a result, a specific nanopore current blockade pattern was observed for each protein-small molecule compound complex (see Fig. 22 E in.

[0244] Based on the above results, the BRD4-BD1 target protein was reacted with each of the acetylated peptide (H4_1-12 K5acK8ac) and the small molecule compound, and then the nanopore current blockade signal was measured. As a result, the unique current blockade signal of the target protein-small molecule compound complex was verified, and it was verified that the signals derived from the four types of target protein-small molecule compound complexes were significantly different from the signals of the target protein-acetylated peptide complex, and there were differences among the target protein-small molecule compound complexes (see Fig. 22 F in.

[0245] Through the above results, the YaxAB nanopore system of the present invention can be used not only to analyze the target protein-acetylated peptide interaction, but also to analyze the activity of the interaction inhibitors that inhibit the target protein-acetylated peptide interaction. In addition, small molecule compounds bound to the target protein can be distinguished; therefore, the YaxAB nanopore system of the present invention can be used to effectively perform the interaction between the target protein and the acetylated peptide and the screening of their interaction inhibitors.

[0246] [4-5] Screening of protein-drug interactions and single-molecule fingerprint analysis Verify how the YaxAB nanopore of the present invention can be used to accurately analyze protein-small molecule compound interactions.

[0247] Two types of small molecule compounds (ABT-737 and A-1331852) that strongly bind to the Bcl-xL protein and two types of small molecule compounds (LCL-161 and GDC-0152) that do not bind to the Bcl-xL protein were selected, and the Bcl-xL protein was treated with each of the selected small molecule compounds. Fig.17 As shown in a, when treated with ABT-737 and A-1331852 compounds as strong binders, the pattern of ion current changed significantly, while when treated with LCL-161 and GDC-0152 compounds as non-binding agents, there was no significant difference from the negative control. Specifically, as Fig.17 As shown in b, it is verified that the current blocking (ΔI / I o )-Current noise (I N ), the event distribution of the mixture of Bcl-xL protein and non-binder was identical to that of free Bcl-xL. In contrast, the strong binder-Bcl-xL protein complex was clearly distinguished from the negative control by two separated peaks (similar to a single molecule fingerprint).

[0248] In addition, to investigate whether the YaxAB nanopore of the present invention can be used to screen drugs from a mixture of various compounds, protein-drug interaction (PDI) was monitored in real time using current recordings of the YaxAB nanopore.

[0249] Specifically, Fig.17 As shown in c, after Bcl-xL was treated with a mixture of compounds containing non-binders (LCL-161 and GDC-0152) and strong binders (ABT-737 and A-1331852) for 35 and 70 minutes simultaneously, the distribution of free Bcl-xL protein as a negative control was significantly reduced (2.5% and 0.7%). In contrast, the event distribution of the Bcl-xL / strong binder complex gradually appeared, and after 70 minutes, two different event distributions corresponding to the Bcl-xL+A-1331852 complex and the Bcl-xL+ABT-737 complex were clearly observed. At the same time point, the ratio of the Bcl-xL+ABT-737 complex gradually decreased (10.7% and 6.3%), while the ratio of the Bcl-xL+A-1331852 complex continued to increase (86.8% and 93.1%). Based on these experimental results, it was verified that the YaxAB nanopore of the present invention is capable of screening drugs bound to target proteins at a single molecule level with high resolution.

[0250] Thus, the YaxAB nanopore can sensitively distinguish different small molecule compounds bound to the same protein with excellent resolution, which can provide the concept and example of single molecule fingerprints of protein-drug interactions using biological nanopore sensors. From these results, it can be seen that the system using the YaxAB nanopore of the present invention can detect and analyze small molecule compounds at a lower cost, faster speed and higher sensitivity than conventional biophysical analysis methods (such as ITC, NMR or SPR).

[0251] [4-6] Structure-activity relationship (SAR) analysis and binding site mapping At the same time, it was verified whether the YaxAB nanopore of the present invention can be used to analyze the binding sites where protein-small molecule compound interactions occur. To this end, 100 nM concentration of Bcl-xL protein was pre-reacted with 200 nM Bak-BH3 protein, and then treated with 200 nM concentration of ABT-737 as a competitive inhibitor of Bak-BH3 protein.

[0252] As a result, it was observed that as the reaction time increased, the signal of the complex of Bcl-xL protein and Bak-BH3 protein (Bcl-xL+Bak-BH3) gradually decreased (87.6%, 34.0% and 2.7%), and the signal of the complex of Bcl-xL protein and ABT-737 compound (Bcl-xL+ABT-737) increased (0%, 60.3% and 94.9%) ( Fig.18 b). In the current blocking (ΔI / I o )-Current noise (IN ), the distribution of Bcl-xL+Bak-BH3 complexes gradually shifted to the newly formed distribution position of Bcl-xL+ABT-737 complexes over time, and only Bcl-xL+ABT-737 complexes remained 40 minutes after the addition of ABT-737, which means that ABT-737 completely replaced the Bak-BH3 peptide bound to Bcl-xL (see Fig.18 From these results, it was confirmed that ABT-737 disengaged the previously bound Bak-BH3 and bound to this site.

[0253] In addition, the Bcl-xL protein at a concentration of 100 nM was reacted with the ABT-737 compound at a concentration of 200 nM, and then treated with the A-1331852 compound at a concentration of 200 nM. As a result, it was observed that as the reaction time increased, the signal of the complex of the Bcl-xL protein and the ABT-737 compound (Bcl-xL+ABT-737) gradually decreased (93.0%, 52.5%, and 26.6%), and the signal of the complex of the Bcl-xL protein and the A-1331852 compound (Bcl-xL+A-1331852) increased (6.0%, 46.6%, and 72.5%) (see Fig.18 From these results, it was confirmed that A-1331852 competes with previously bound ABT-737 for binding to the same site.

[0254] It can be seen from this that the YaxAB nanopore of the present invention can be used to screen the binding activity of drugs to proteins at the single molecule level and further analyze the structure-activity relationship (SAR).

[0255] [4-7] Screening of peptide drugs that bind to target proteins The YaxAB nanopore sensor of the present invention is used to analyze the interaction between the target protein and the peptide, and thereby perform peptide drug screening.

[0256] First, a solution containing 5mM HEPES (pH 7.5), 1mM EDTA, and 1M KCl was placed in each compartment of the YaxAB nanopore sensor. Bcl-xL protein was then reacted with each of three Bak-BH3 mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, and Bak_BH3_L78A) known to bind to Bc-xL at a molar ratio of 1:15. The product mixture was added to the cis compartment, followed by application of a (+) voltage. At this point, the current blockade signal derived from the Bcl-xL protein and the current blockade signal of the Bcl-xL+peptide drug complex were observed and analyzed to be expressed as current noise (SD)-current blockade (ΔI / I o) of a 2D scatter plot (see Fig.19 a and b in ).

[0257] Based on this, a solution obtained by mixing Bcl-xL protein with three types of mutant peptides (Bak_BH3_D84A, Bak_BH3_I85A, and Bak_BH3_L78A) and wild-type Bak_BH3 at a molar ratio of 1:15 was reacted, and then, the current blockade signal was measured. As a result, specific current blockade signals that appeared when various peptides were bound were observed, and it was verified that the scatter plot results measured and analyzed by adding Bcl-xL protein and the four mixed peptides were consistent with the scatter plot tendency that appeared during individual experiments (see Fig.19 c) in.

[0258] In addition, the signal frequencies of the Bcl-xL-peptide complexes were 51% for wild-type Bak_BH3, 39% for Bak_BH3_D84A, 7% for Bak_BH3_I85A, and 3% for Bak_BH3_L78A, which were consistent with the order of binding affinities between Bcl-xL protein and various peptides (0.34 μM for wild-type Bak_BH3; 0.14 μM for Bak_BH3_D84A; 93 μM for Bak_BH3_I85A; and 270 μM for Bak_BH3_L78A).

[0259] The above results verify that the YaxAB nanopore of the present invention can perform binding affinity-based screening for peptide drugs bound to proteins, and can effectively select peptide drugs with high binding affinity to target proteins even in a peptide mixed solution. Therefore, the YaxAB nanopore of the present invention can be effectively used as a drug screening platform.

[0260] [4-8] Drug binding selectivity spectrum analysis for multiple proteins Experiments were performed to verify whether protein binding selectivity profiling of drugs for Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1) is possible using the YaxAB nanopore sensor of the present invention.

[0261] 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. Then, 50 nM of each of the Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1) and mTOR protein as a negative control were added to the cis compartment, and then a (+) voltage was applied.

[0262] At this time, it was observed that the nanopore current blockade signal exhibited unique patterns that could be fully distinguished for each protein, and thus, when expressing the current noise (SD) minus the current blockade (ΔI / I o ) , the distribution of signals from various proteins was observed (see Fig. 20 a to e in the figure).

[0263] Furthermore, equal concentrations (50 nM each) of Bcl-2 family proteins (Bcl-xL, Bcl-w, Bcl-2, and Mcl-1) and mTOR protein as a negative control were mixed to prepare a sample solution. The prepared solution was added to the cis compartment of the YaxAB nanopore system. Thereafter, a (+) voltage was applied. As a result, unique current blockade signals respectively derived from Bcl-xL, Bcl-w, Bcl-2, Mcl-1, and mTOR proteins were observed. In addition, as a result of analyzing the nanopore current blockade signals derived from the mixed solution, protein signal distributions corresponding to four types of Bcl-2 family proteins and one type of negative control were also observed on the 2D scatter plot (see Fig. 20 f).

[0264] As a result of adding A1155463, which is a small molecule drug targeting Bcl-xL, to the mixture solution at a molar ratio of 1:1 (50 nM), followed by analyzing the nanopore current blockade signal, it was observed that in the case of Bcl-w, Bcl-2, Mcl-1, and mTOR, unique current blockade signals of the proteins appeared, while only in the case of the target protein Bcl-xL, the unique current blockade signal of the protein disappeared and a new current blockade signal appeared. As a result of analyzing the measured signals, different signal distributions for Bcl-w, Bcl-2, Mcl-1, and mTOR proteins were observed on the 2D scatter plot. In contrast, the signal distribution for the target protein Bcl-xL disappeared, and instead a new distribution representing the Bcl-xL-A1155463 complex appeared as expected (see Fig. 20 g in the figure).

[0265] The above results verify that the YaxAB nanopore sensor can be used to perform spectrum analysis of Bcl-2 family proteins and identify drugs that are selective for the target protein.

[0266] [Example 5] Analysis of ternary complexes (including protein-protein interactions caused by molecular glue) using the YaxAB nanopore sensor Is it possible to analyze the interaction It was verified whether the YaxAB nanopore system of the present invention can be used to analyze the promotion of protein-protein interactions caused by molecular glue.

[0267] First, the mTOR-FRB domain and FKBP12 protein do not interact in the presence of these two proteins alone, but can be mediated by rapamycin, which acts as a molecular glue, to mediate the protein-protein interaction between the two. Specifically, rapamycin first binds to the FKBP12 protein, and then the mTOR-FRB domain binds to the FKBP12-rapamycin complex to form a ternary complex (see Fig.21 a) in the figure.

[0268] Using the above characteristics, a solution containing 10mM Tris-HCl (pH 8.0), 1mM EDTA, and 1M KCl was placed in each compartment of the YaxAB nanopore system. Then, 160nM mTOR-FRB domain protein (hereinafter, referred to as mTOR protein) or FKBP12 protein was added to the cis compartment, and (+) voltage was applied. As a result, the nanopore current blockade signal was observed (see Fig.21 b and c).

[0269] As a result, unique current blockade signals appeared from the mTOR protein and the FKBP12 protein, respectively, and as a result of the scatter plot analysis, it was verified that the distributions of the signals derived from the various proteins were clearly distinguished from each other. In addition, 160 nM of each of the mTOR protein and the FKBP12 protein was added to the cis compartment at a molar ratio of 1:1 as another negative control. Thereupon, (+) voltage was applied, and the nanopore current blockade signal was observed, and the current blockade signals of the mTOR protein alone and the FKBP12 protein alone appeared in a mixed state, and as a result of the scatter plot analysis, it was verified that the unique signal distribution derived from each protein was reproduced (see Fig.21 f).

[0270] Based on the experimental results, when mTOR protein, FKBP12 protein, and rapamycin acting as a molecular glue were added to the cis compartment at a molar ratio of 1:1:10, (+) voltage was applied, and the characteristic current blockade signal was observed, a new current blockade signal was observed, as well as the current blockade signal corresponding to the mTOR protein and the FKBP12 protein (see Fig.21 g in the figure).

[0271] In addition, to verify that the new signal was a current blocking signal due to the ternary complex in which mTOR, FKBP12, and the molecular glue were all bound, rather than a binary complex formed by the binding of mTOR or FKBP12 protein to the molecular glue, mTOR protein or FKBP12 protein and rapamycin were added to the cis compartment of the YaxAB nanopore system at a ratio of 1:1, and a (+) voltage was applied. Subsequently, a current blocking signal ( Fig.21 d and e in the figure).

[0272] As a result, since the current blockade signal derived from the mTOR protein and the FKBP12 protein did not significantly change depending on the presence or absence of rapamycin, it was verified that the new current blockade signal was a current blockade signal derived from a ternary complex in which the mTOR protein, the FKBP12 protein, and rapamycin were all bound.

[0273] The above results show that the YaxAB nanopore system of the present invention can distinguish between proteins and ternary complexes, and thus, the promotion of protein-protein interaction caused by molecular glue can be analyzed.

[0274] Next, to verify whether the molecular glue present in the mixed solution of various small molecule compounds can be screened, negative small molecule compounds (astemizole, mibefradil, terfenadine, pranlukast, capecitabine, dimethylmantanolamide, sulfonamide, and monobenzone) that do not bind to the mTOR-FRB domain were selected by nuclear magnetic resonance (NMR) spectroscopy, and whether the molecular glue was screened was verified by observing the ternary complex signal according to the presence or absence of rapamycin as a molecular glue in the mixed solution of the negative small molecule compounds.

[0275] Specifically, when equal amounts of mTOR protein and FKBP12 protein were reacted with a mixed solution of 8 types of negative small molecule compounds at a molar ratio of 1:1:10 and the nanopore current blockade signal was observed, only unique current blockade signals of mTOR protein and FKBP12 protein were observed (see Fig.21 At the same time, when rapamycin as a molecular glue was reacted at a molar ratio of 1:1:10:10 under the above experimental conditions, current blocking signals corresponding to the ternary complex as well as the mTOR protein and the FKBP12 protein were observed (see Fig.21 The i in the figure).

[0276] To summarize the above results, the YaxAB nanopore system of the present invention can distinguish between single proteins, protein-small molecule compound binary complexes, and protein-small molecule compound-protein ternary complexes. In addition, the YaxAB nanopore system can even screen the presence or absence of molecular glue in mixed solutions of various small molecule compounds without being interfered by negative small molecule compounds. Therefore, it is verified that the YaxAB nanopore system of the present invention can be used to screen molecular glues that can promote protein-protein interactions.

[0277] [Example 6] Can the YaxAB nanopore be used to selectively detect and quantify biomarker proteins? The YaxAB nanopore system of the present invention was used to verify whether it is possible to selectively detect and quantitatively analyze specific biomarker proteins in the presence of serum.

[0278] The Bcl-xL protein selected as the analyte is a protein that is overexpressed in various types of malignancies and is widely considered to be a target for cancer therapeutics 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)).

[0279] First, after adding 800 μL of 10 mM Tris (pH 7.5), 1 mM EDTA, and 1 M KCl buffer to the two compartments of the YaxAB nanopore system, 50 nM Bcl-xL protein was introduced into the cis compartment to verify the unique electrical signal of the Bcl-xL protein. Then a voltage of 80 mV was applied and the change in the electrical signal was measured. When the results were statistically analyzed and expressed as current noise (SD)-current blockade (ΔI / I o ) of a two-dimensional scatter plot, such as Fig.23 As shown in A, the two-dimensional scatter plot results showing uniform current pattern and dense distribution are verified.

[0280] Next, in order to use the YaxAB nanopore system of the present invention for cancer diagnosis, experiments were performed to confirm whether trace amounts of Bcl-xL proteins present in a mixed solution could be detected. First, it was observed whether Bcl-xL at concentrations of 10nM and 20nM produced specific characteristic electrical signals in the presence of fetal bovine serum (FBS) similar to human serum. An FBS solution after serum proteins (such as albumin and immunoglobulins) were removed using a depletion mini spin column was used. Thereafter, 5 μL of the FBS solution was added to the cis compartment of the YaxAB nanopore system in which 800 μL of 10mM Tris pH 7.5, 1mM EDTA, and 1M KCl buffer were present in each of the two compartments, and the changes in the electrical signals that occurred after applying a voltage of 80mV were measured.

[0281] As a result, various types of electrical signals were observed, and when these electrical signals were statistically analyzed and expressed as a two-dimensional scatter plot, it was shown as follows Fig.23 The distribution results are shown in B. Specifically, when a voltage of 80 mV was applied to the cis compartment of the YaxAB nanopore system in the presence of FBS from which some proteins were removed (deproteinized FBS), a unique characteristic electrical signal of 10 nM Bcl-xL protein was observed in addition to various nanopore electrical signals measured in deproteinized FBS. When these signals were analyzed and represented as a two-dimensional scatter plot, the signal distribution corresponding to the unique electrical signal of deproteinized FBS and Bcl-xL protein was shown among various scattered signals of deproteinized FBS (see Fig.23 C in.

[0282] Furthermore, as a result of performing electrical measurements under the condition of 20 nM Bcl-xL protein in the presence of deproteinized FBS, a larger number of unique electrical signals of Bcl-xL were measured compared to the condition of 10 nM Bcl-xL protein, and the two-dimensional scatter plot results also showed that the Bcl-xL protein signal distribution was obviously densely represented in a concentration-dependent manner (see Fig.23 D in.

[0283] Therefore, it was verified whether trace amounts of Bcl-xL protein present in a mixed solution can be detected, and this verified that the YaxAB nanopore system of the present invention can be used to specifically and highly sensitively detect and quantitatively analyze trace amounts of biomarker proteins in a mixed solution within a short period of time; therefore, it was verified that the system has the possibility of being used for the diagnosis of various diseases including cancer and genetic diseases and then for early diagnosis that requires the detection of trace amounts of biomarkers.

[0284] At the same time, if Fig.24 As shown in, experiments were performed to verify whether mutant Bcl-xL proteins frequently observed in cancer patients can be detected using the YaxAB nanopore system of the present invention. Specifically, Bcl-xL_R100E, Bcl-xL_R103E mutant proteins and Bcl-xL_E31K, Bcl-xL_E36K mutant proteins were used to verify whether changes in electrical signals can be measured even when amino acid residues of the protein are substituted due to mutations (see Fig.24 Figure (A) ), in the Bcl-xL_R100E and Bcl-xL_R103E mutant proteins, the arginine residues at positions 100 and 103 of the Bcl-xL protein were substituted with glutamic acid with (-) charge to change the total net charge from -11.5e to -14.8e, which can be used as a biomarker for cancer diagnosis; in the Bcl-xL_E31K and Bcl-xL_E36K mutant proteins, the glutamic acid residues at positions 31 and 36 were substituted with lysine residues with (+) charge to change the total net charge to -7.4e.

[0285] Using the YaxAB nanopore system, each of the Bcl-xL wild-type protein, Bcl-xL_E31K, Bcl-xL_E36K mutant proteins, and Bcl-xL_R100E, Bcl-xL_R103E mutant proteins were added to a 10 mM Tris pH 7.5, 1 mM EDTA, and 1 M KCl buffer, and then the nanopore electrical signal was measured. As a result, three types of unique nanopore electrical signals were observed for the three types of proteins that were different from each other (see Fig.24This is because the net charge of the Bcl-xL protein affects the oscillation of the analyte protein inside the pore, which is caused by the balance between the electroosmotic force (EOF) of the YaxAB nanopore and the electrophoretic force (EPF) induced by the applied voltage.

[0286] In the case of Bcl-xL_R100E and Bcl-xL_R103E mutant proteins, the signal frequency of L1, which is the minimum current blocking level, increased. It is predicted that Bcl-xL_R100E and Bcl-xL_R103E mutants have a strong repulsive force caused by EPF due to the increase in (-) charge compared to the wild-type Bcl-xL protein, and therefore have a tendency to escape from the YaxAB nanopore without staying in the YaxAB nanopore for a long time, and this tendency is revealed by electrical signal analysis. In addition, in the case of an increase in the signal frequency of L3, which is the deepest current blocking level in Bcl-xL_E31K and Bcl-xL_E36K, since the (+) charge is increased compared to the wild-type Bcl-xL protein, the influence of EPF is small, and there is a tendency to be located deeper in the YaxAB nanopore, which is also shown by the result of the increase in the electrical signal frequency of L3.

[0287] Thus, when the net charge of the protein changes, the oscillatory motion of the protein inside the nanopore is affected, and the generated electrical signal is also affected. Therefore, since significantly different changes in the electrical signal are detectable even in the case of mutants with only two points in the protein, not only Bcl-xL wild-type protein but also trace amounts of mutant proteins can be accurately detected and distinguished using the YaxAB nanopore system of the present invention, which demonstrates the possibility of diagnosing various diseases (such as cancer or genetic diseases) that require detection of mutant proteins observed in cancer patients.

[0288] [Example 7] Is electroosmotic or electrophoretic based detection and analysis of analyte translocation through the YaxAB nanopore possible? It was demonstrated that the YaxAB nanopore of the present invention can be used to detect and analyze single-molecule proteins and nucleic acids that translocate through the nanopore driven by electrophoretic or electroosmotic forces.

[0289] First, due to the strong cation selectivity of the YaxAB nanopore, mTOR, which is small in size and weakly negatively charged at pH 7.5, translocates through the nanopore due to electroosmotic forces, and the resulting ionic current blockade is measured. Fig.25As shown in , it was observed that as the voltage applied to the cis compartment and the trans compartment increased, the residence time of a single protein molecule in the nanopore also increased. However, above a certain threshold voltage, the residence time decreased significantly. Therefore, it was verified that if the analyte has a diameter similar to that of the YaxAB nanopore, the analyte can be effectively detected and analyzed by direct translocation via electroosmotic forces, rather than relying on a method involving oscillations in the nanopore after electroosmotic introduction.

[0290] Next, EGF, which is a single-molecule protein that is small in size and negatively charged at pH 7.5, was added to the trans compartment, and EGF was translocated through the YaxAB nanopore of the present invention due to electrophoretic force, and the result of ion current blockade was measured. Fig.26 As shown in , it was observed that the residence time spent by a single protein molecule translocating through the nanopore decreased as the voltage applied to the cis and trans compartments increased. In addition, p53, an intrinsically disordered protein (IDP) with a small molecular weight of 1.8 kDa, was TAD1 The peptide is added to the trans compartment, and p53 TAD1 The peptide is translocated through the YaxAB nanopore of the present invention due to electrophoretic forces. Fig. 27 As shown in TAD1 Changes in ion current blockade were observed before and after the peptides, and ion current blockade was detected after their translocation. Fig. 27 As shown in Figure , even for p53 with a molecular weight of 8.3 kDa TAD Protein, when adding p53 TAD Blockage of ionic current was also observed before and after the protein, thereby demonstrating that intrinsically disordered protein and peptide analytes can be detected and analyzed by translocation of the analyte through the nanopore via electrophoresis.

[0291] At the same time, if Fig.28 As shown in , λDNA, single-stranded nucleic acid (50mer) and double-stranded nucleic acid (23bp) were independently added to the trans compartment of the YaxAB nanopore, and the electrophoretic translocation of each nucleic acid was observed. As a result, ion current blockade was verified before and after the addition of each nucleic acid. Therefore, it was verified that the YaxAB nanopore of the present invention enables the detection and analysis of intrinsically disordered proteins and nucleic acids not only due to electroosmotic force but also due to electrophoretic force.

[0292] [Example 8] Analysis of purification efficiency of modified YaxAB nanopores In order to verify whether the purification efficiency of the modified monomer in the YaxAB nanopore of the present invention is improved compared with the wild-type monomer, the purification efficiency of various monomer proteins according to the length of the amino acid sequence was compared.

[0293] Specifically, the modified first monomer (YaxA_(45-410)) having the amino acid sequence of SEQ ID NO: 1, the modified second monomer (YaxB_(12-343)) having the amino acid sequence of SEQ ID NO: 2, the wild-type first monomer (YaxA_FL) having the amino acid sequence of SEQ ID NO: 3, and the wild-type second monomer (YaxB_FL) having the amino acid sequence of SEQ ID NO: 4 were purified using the same method as in Example 1-2. In the protein purification, 6 liters of E. coli culture medium were used for the purification of YaxA_FL, 1 liter of E. coli culture medium was used for the purification of YaxB_FL, 2 liters of E. coli culture medium were used for the purification of YaxA_(45-410), and 1 liter of E. coli culture medium was used for the purification of YaxB_(12-343).

[0294] like Fig.29 As shown in , the purified yield per liter of monomeric protein is: 12 mg for YaxA_FL; 66.2 mg for YaxB_FL; 56.1 mg for YaxA_(45-410); and 72.3 mg for YaxB_(12-343), and under the same purification conditions, the yield of the modified first monomer (YaxA_(45-410)) is 4.7 times the yield of the first monomer (YaxA_FL) of the wild-type YaxAB. In addition, under the same purification conditions, the yield of the modified second monomer (YaxB_(12-343)) is 1.1 times the yield of the wild-type second monomer (YaxB_FL). According to the above results, it was found that in the case of each modified monomer of the building block-modified YaxAB_dN nanopore, under the same purification conditions, the purified yield was higher than the yield of the wild-type monomer.

[0295] [Example 9] Analysis of the oligomerization mechanism of the modified YaxAB nanopore The monomers purified in Example 8 were oligomerized in the same manner as in Examples 1-3 to form YaxAB nanopores (wild-type YaxAB and modified YaxAB_dN), and each nanopore was electrophoresed using a gradient blue polyacrylamide gel, and then the bands of different oligomers of the wild-type nanopore and the modified nanopore were verified.

[0296] Results, such as Fig.30As shown in, when low concentrations of the first monomer and the second monomer were used, no bands were detected for the wild-type YaxAB nanopore, while the bands for the modified YaxAB_dN nanopore were clearly distinguished as different oligomer types. In addition, when high concentrations of the first monomer and the second monomer were used, the modified YaxAB_dN and wild-type YaxAB nanopores exhibited different banding patterns, indicating differences in oligomerization behaviors. The above results verified that, compared with the wild-type YaxAB nanopore, the first monomer (YaxA_(45-410)) having the amino acid sequence of SEQ ID NO: 1 and the second monomer (YaxB_(12-343)) having the amino acid sequence of SEQ ID NO: 2 not only had a higher yield, but also were able to oligomerize more stably under the same purification conditions. In addition, it was verified that a YaxAB-C composed of 8 subunits that could not be formed using the wild-type monomer could be formed. 8 .

[0297] In addition, if Fig.31 As shown in FIG. 1 , as a result of showing the distribution of the conductivity of the purified YaxAB nanopore by each band, in the wild-type YaxAB nanopore, C 9 , C 10 and C 11 , while in the modified YaxAB_dN nanopores, C 8 , C 9 , C 10 and C 11 Thus, it was found that the oligomerization mechanism of the YaxAB nanopore can be regulated by changing the amino acid sequence of each of the first monomer or the second monomer.

[0298] In addition, YaxA_(X-411) whose sequence length was adjusted based on the sequence of the wild-type first monomer YaxA_FL was used to verify the oligomerization tendency with the second monomer YaxB_(12-343).

[0299] Specifically, YaxA_(X-411) refers to the first monomer of YaxA_FL(1-411) in which the N-terminal residues from position 1 to X are deleted. Variants of YaxA_(X-411) (wherein X is 11, 21, 31, 41, 51, 61, 71 or 81) were prepared and oligomerized with YaxB_(12-343) using the same method as in Example 1-3.

[0300] Results, such as Fig.32 and Fig.33As shown in FIG, it was verified that when each of the wild-type YaxA_(1-411), YaxA_(11-411), YaxA_(21-411), YaxA_(31-411), YaxA_(41-411), YaxA_(51-411) and YaxA_(61-411) reacted with YaxB_(12-343), YaxAB-C was formed. 8 、YaxAB-C 9 and YaxAB-C 10 On the other hand, when YaxA_(71-411) and YaxA_(81-411) react with YaxB_(12-343), YaxAB-C 7 Oligomers.

[0301] In addition, YaxA_(X-410) with adjusted YaxA sequence length was used to verify the oligomerization tendency with the second monomer YaxB_(12-343).

[0302] Specifically, YaxA_(X-410) refers to the first monomer in which the N-terminal residues from position 1 to X are deleted and the C-terminal residue at position 1 is deleted. Variants of YaxA_(X-410) (wherein X is 3, 5, 8 or 9) were prepared and oligomerized with YaxB_(12-343) using the same method as in Example 1-3.

[0303] Results, such as Fig.33 As shown in , it was confirmed that when each of YaxA_(3-410), YaxA_(5-410), YaxA_(8-410) and YaxA_(9-410) reacted with YaxB_(12-343), YaxAB oligomers were formed.

[0304] In addition, if Fig.34 As shown in, it was verified that the YaxAB nanopore system using YaxAB oligomers formed by reacting each of YaxA_(11-411), YaxA_(21-411), YaxA_(31-411), YaxA_(41-411), YaxA_(51-411) and YaxA_(3-410) with YaxB_(12-343) can detect Bcl-xL protein.

[0305] Thereafter, in order to verify the minimum amino acid sequence that forms the YaxAB nanopore, the C-terminus of the YaxA amino acid sequence was fixed, a sequence from YaxA_(52-410) to YaxA_(60-410) was prepared, and the oligomerization trend was analyzed. As a result, it was verified that when YaxA_(57-410) reacted with YaxB_(12-343), YaxAB oligomers were formed, but when YaxA_(58-410) reacted with YaxB_(12-343), no oligomers were formed. In addition, as a result of preparing a sequence from YaxA_(57-409) to YaxA_(57-391) and analyzing the oligomerization trend, it was verified that in the case of YaxA_(57-398), YaxAB oligomers were formed, while in the case of YaxA_(57-397), YaxAB oligomers were not formed.

[0306] In addition, as a result of analyzing the minimum amino acid sequence of YaxB_(12-343) that forms the YaxAB nanopore by the above method, it was verified to be YaxB_(26-342). In addition, when YaxA_(57-398) reacts with YaxB_(26-342), a YaxAB oligomer is formed, and the nanopore-containing membrane of the present invention is successfully realized by the oligomer, thereby verifying that the Bcl-xL protein is successfully captured using the nanopore system.

[0307] In addition, contrary to the above method, as a result of fixing the N-terminus of the YaxA amino acid sequence, preparing the sequence from YaxA_(45-390) to YaxA_(45-369), and analyzing the oligomerization tendency, it was verified that in the case of YaxA_(45-383), YaxAB oligomers were formed, while in the case of YaxA_(45-382), YaxAB oligomers were not formed. In addition, as a result of preparing the sequence from YaxA_(46-383) to YaxA_(58-383) and analyzing the oligomerization tendency, it was verified that in the case of YaxA_(52-383), YaxAB oligomers were formed, while in the case of YaxA_(53-383), YaxAB oligomers were not formed.

[0308] In addition, as a result of preparing the sequence from YaxB_(12-325) to YaxB_(12-314) by the above method and analyzing the oligomerization tendency, it was verified that in the case of YaxB_(12-316), YaxAB oligomers were formed, while in the case of YaxB_(12-315), YaxAB oligomers were not formed. In addition, as a result of preparing the sequence from YaxB_(13-316) to YaxB_(18-316) and analyzing the oligomerization tendency, it was verified that in the case of YaxB_(12-316), YaxAB oligomers were formed, while in the case of YaxB_(13-316), YaxAB oligomers were not formed. In addition, when YaxA_(52-383) reacted with YaxB_(12-316), YaxAB oligomers were formed, and the nanopore-containing membrane of the present invention was successfully realized through the oligomers, thereby verifying that the Bcl-xL protein was successfully captured using the nanopore system.

[0309] Therefore, if Fig.32 , Fig.35 , Fig.36 and Fig.37 As shown in, it was verified that the minimum amino acid sequence of YaxA that forms the 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 YaxA_(52-383) having the amino acid sequence of SEQ ID NO: 24, and the minimum amino acid sequence of YaxB is 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 all YaxAB obtained by their combination form oligomers. In addition, it was verified that the nanopore-containing membrane of the present invention was successfully realized in the case of the above-mentioned oligomers formed by the combination, thereby successfully capturing Bcl-xL protein using the nanopore system (see Fig.36 ).

[0310] In addition, it was verified that in the case of a nanopore subunit comprising a first monomer having at least one of an amino acid sequence consisting 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 having an amino acid sequence consisting of SEQ ID NO: 2, a YaxAB oligomer was stably formed (see Fig.32 ).

[0311] According to the above results, it was found that the minimum amino acid sequence of YaxA that forms the 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, 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 sequence of YaxB is 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. According to the above examples, the minimum amino acid sequence that forms the YaxAB nanopore of the present invention was identified (see Figure 32 to Figure 36 ).

[0312] [Example 10] Verification of the analytical precision of analyte-ligand interactions using the YaxAB nanopore [10-1] Is precise interaction analysis possible even when there is a large difference in molecular weight between the analyte and the ligand? The range of protein-small molecule compound complexes that can be analyzed using the YaxAB nanopore of the present invention was demonstrated.

[0313] Specifically, FKBP12 protein (13.0 kDa) was added to the cis compartment of the sensor made in Examples 1-4 alone at 100 nM, or ii) 100 nM FKBP12 protein and 1 uM FK506 were added to the cis compartment of the sensor made in Examples 1-4 at a molar ratio of 1:10; and (+) and (-) voltages were applied to the cis compartment and the trans compartment, respectively.

[0314] Results, such as Fig.38 As shown in , it was observed that the current blockade pattern changed when FK506 was added together, which was different from the case when FKBP12 protein was added alone, thus verifying that it was possible to distinguish between the protein and the protein-small molecule compound complex (FKBP12+FK506).

[0315] Additionally, i) holotransferrin (77.1 kDa) alone was added to the cis compartment in the same manner as above, or ii) oxaliplatin and holotransferrin were added to the cis compartment; and a voltage was applied. Fig.39 As shown in , a clear change in the current blockade pattern was observed by the addition of oxaliplatin, thus verifying that it is possible to distinguish between the case where the protein was added alone and the case where the protein-small molecule compound complex (holo-transferrin + oxaliplatin) was added.

[0316] In the protein-small molecule compound complex described above, the molecular weight ratio of FKBP12 and FK506 is 16: 1, and the molecular weight ratio of holotransferrin and oxaliplatin is 194: 1. Therefore, although the molecular weight ratio between the protein and the small molecule compound has a difference of 16 to 194 times, it is also verified that the nanopore system of the present invention can sensitively distinguish the signal of the protein and the signal of the protein-small molecule compound complex.

[0317] [10-2] Is accurate interaction analysis possible even when there is a large difference in concentration between the analyte and the ligand or under conditions of high drug concentration? To verify whether the YaxAB nanopore system of the present invention can be effectively used for drug screening under conditions in which various concentrations (including high concentrations) of drugs are present, protein-compound complex signals are compared under conditions in which various concentrations of target-specific binding compounds are present and under conditions in which non-binding compounds are included as negative controls.

[0318] Specifically, the interaction between the Bcl-xL protein and the small molecule compound ABT-737 was analyzed in the same manner as in Example 4-1, and it was verified that drug binding activity could be screened even under the conditions where ABT-737 was added at concentrations (binding ratio) of 200 nM (1:2) and 10 μM (1:100).

[0319] Results, such as Fig.40 As shown in , the same results were obtained regardless of the concentration of the ABT-737 compound, thus verifying that the YaxAB nanopore of the present invention can be used to screen drugs by analyzing the binding activity of the drug to proteins, even under conditions where the drug is present at a high concentration (10 μM or higher) or at a low concentration at the single molecule level.

[0320] In addition, to verify whether nonspecific signals are generated by non-target compounds, e.g. Fig.41 As shown in FIG, four types of small molecule compounds (ABT-737, A-1331852, LCL-161, and GDC-0152) were added to the YaxAB nanopore system of the present invention at a concentration of 200 nM or 1 μM, and the ion blockade current signal was measured for 30 minutes. In addition, when the Bcl-xL protein was captured in the YaxAB-C 8 In the state in the nanopore, four types of non-target compounds (LCL-161, GDC-0152, Birinapant, and Phentolamine) were added to the cis compartment at a concentration (concentration ratio) of 10 μM (1:100), and the ion blockade current signal was observed for 30 minutes.

[0321] Results, such as Fig.41As shown in a, it is verified that when only the compound is added, the flow through YaxAB-C 8 The ionic current of the nanopore is not affected. Fig.41 As shown in b and c, the capture signal obtained when Bcl-xL was added alone and the ion current signal obtained when Bcl-xL and the non-target compound were added together were measured to be almost the same, thus verifying that the YaxAB nanopore system of the present invention can specifically detect the signal of the captured analyte (Bcl-xL) even under the condition that the four types of non-target compounds were added at high concentrations.

[0322] [Example 11] Verify whether the nanopore system of the present invention can operate even in a solution containing DMSO In addition, in order to verify whether the YaxAB nanopore system of the present invention operates stably even under the condition of adding DMSO which is necessary for detecting poorly soluble drugs, Bcl-xL protein and each of three types of ligands (Bak-BH3 peptide, ABT-737 and A-1331852) were added in the same manner as in Example 4-1 under the condition of various concentrations (0, 0.01%, 0.1%, 0.5%, 1%, 2%, 5% and 10%) of DMSO, and the complex signal was observed.

[0323] Results, such as Fig.42 As shown in , it was verified that the YaxAB nanopore system of the present invention enables stable and precise analysis by clearly distinguishing between ion current signals derived from free Bcl-xL and ion current signals derived from complexes formed individually between Bcl-xL and each of three different Bcl-xL-ligands under DMSO concentration conditions of 5% or less.

Claims

1. A YaxAB nanopore, comprising a first opening, a middle region and a second opening, wherein the YaxAB nanopore has a funnel shape. in, The outer diameter of the first opening at the lumen is 5 nm or more, The second opening portion includes a narrowed portion, The diameter of the constriction is 0.5 nm or more, The outer diameter of the second opening at the lumen is 1 nm or more, The depth of the lumen is 5 nm or more.

2. A YaxAB nanopore, comprising at least one subunit comprising a first monomer and a second monomer, wherein the first monomer comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24 or SEQ ID NO: 34, and the second monomer comprises an amino acid sequence that is at least 70% identical 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, in, The YaxAB nanopore contains 4 to 20 subunits.

4. The YaxAB nanopore according to claim 1 or 2, in, The YaxAB nanopore has a cation selectivity that is at least 1.1 times greater than its anion selectivity, or an anion selectivity that is at least 1.1 times greater than its cation selectivity.

5. The YaxAB nanopore according to claim 1 or 2, in, Subunits include: a first monomer having any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34; and The second monomer has any 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. A membrane containing nanopores, wherein the membrane containing nanopores include: Membrane layer; as well as YaxAB nanopores, inserted into the membrane layer, The membrane layer includes at least one selected from the group consisting of: 1,2-diphytanoyl-sn-glyceryl-3-phosphocholine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glyceryl-3-phospho-L-serine, 1,2-adipoyl-sn-glyceryl -3-phosphocholine, 1,2-dilauroyl-sn-glyceryl-3-phosphocholine, 1,2-dimyristoyl-sn-glyceryl-3-phosphocholine, 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glyceryl-3-phosphoglycerol, 1,2-dimyristoyl-sn-glyceryl-3-phospho-L-serine, 1,2-dioleoyl-sn-glyceryl-3-phospho-L-serine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-SN-glycero-3-phosphocholine and mycolic acid, and at least one selected from the group consisting of campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesterol 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glyceryl-3-phosphocholine, 1-palmitoyl-2-cholesterylcarbonyl-sn-glyceryl-3-phosphocholine, 1,2-dicholesteryl hemisuccinyl-sn-glyceryl-3-phosphocholine, 10,12-pentacosadiynoic acid, 10,12-tricosadiynoic acid, 5,7-hexadecadiynoic acid, 9,12-octadecadiynoic acid.

7. The nanopore-containing membrane according to claim 6, in, The YaxAB nanopore comprises at least one subunit, the at least one subunit comprising: A first monomer having an amino acid sequence having 70% or higher homology to the amino acid sequence of any one of SEQ ID NO: 7, SEQ ID NO: 24, SEQ ID NO: 34, or SEQ ID NO: 3; and The second monomer has an amino acid sequence having 70% or higher homology to the amino acid sequence of any one of SEQ ID NO: 18, SEQ ID NO: 25 or SEQ ID NO:

4.

8. A nanopore system, the nanopore system include: Chamber; as well as Membranes containing YaxAB nanopores, The space in the chamber is divided into two compartments by a membrane containing nanopores.

9. The nanopore system according to claim 8, in, The nanopore system operates to cause an analyte to translocate through the pore, become trapped in the pore, or induce gating of the pore, Translocation, capture or gating of the analyte is caused by electrophoresis or electroosmosis, The properties of the analyte are analyzed by detecting oscillatory or rotational motion of the analyte, clogging caused by the analyte, pore translocation of the analyte, or pore gating of the analyte that occurs when the analyte is trapped inside the pore, and The characteristic of the analyte is at least one selected from the group consisting of surface charge, isoelectric point, stability, isomers, length, size, molecular weight, structure, dynamics, strength and orientation of dipole moment, orientation, flexibility, conformational heterogeneity, shape and amount of the analyte and changes therein.

10. The nanopore system according to claim 9, in, The molecular weight of the analytes ranged from 1 kDa to 1200 kDa.

11. The nanopore system according to claim 8, in, The nanopore system is used for at least one selected from the group consisting of: analysis of interaction between analyte and ligand, drug screening, nucleic acid sequencing, protein sequencing, peptide sequencing, protein identification, disease diagnosis, protein post-translational modification analysis, proteomic analysis and structural analysis of biomolecules.

12. A single molecule analysis method for a single analyte or a plurality of analytes, the single molecule analysis method The following steps are involved: placing at least one analyte in a compartment of the nanopore system of claim 8; as well as The changes in the electrical signals in the two compartments are measured before and after placement of the analyte.

13. The single molecule analysis method according to claim 12, in, The analyte is a peptide, protein, lipid, carbohydrate, inorganic substance, small molecule compound, nucleic acid or inorganic particle formed by nanomaterials. The electrical signal is the pattern of ionic current, baseline current in the open state, amplitude of current drop, current blockade, event duration, frequency per unit time of the nanopore signal pattern, or current noise, and The assays are used to detect or identify a single analyte or a plurality of analytes.

14. A method for analyzing the interaction between an analyte and a ligand or for ligand screening for an analyte, the method comprising: The following steps are involved: performing a treatment in one compartment or both compartments of the nanopore system according to claim 8 with at least one analyte and at least one ligand candidate for the analyte; as well as Changes in electrical signals in the two compartments before and after treatment with the candidate are measured.

15. The method of claim 14, in, The analysis is a quantitative analysis that calculates the binding affinity between the analyte and the ligand.

16. The method of claim 14, in, The interaction is the competitive binding between an analyte and multiple ligands of multiple analytes, or the competitive binding between multiple analytes and one ligand of one analyte, the ligand binding site map of the analytes, or the structure-activity relationship.

17. The method of claim 14, in, The analyte is a peptide, protein, lipid, carbohydrate, inorganic substance, small molecule compound, nucleic acid or inorganic particle formed by nanomaterial, and The ligand is a peptide, protein, lipid, carbohydrate, small molecule compound or nucleic acid.

18. A method for analyzing or screening for inhibitors or promoters of interactions between biomolecules, the method comprising: The following steps are involved: allowing a plurality of biomolecules capable of interacting to react in one compartment of the nanopore system of claim 8; Treatments were performed in one or both compartments with candidates for inhibitors or promoters of the interaction; as well as Changes in electrical signals in the two compartments before and after treatment with the candidate are measured.

19. A method for identifying and quantifying an analyte in a sample, the method comprising: The following steps are involved: placing a sample in a compartment of the nanopore system as claimed in claim 8; as well as The changes in the characteristic electrical signal caused by the multiple analytes in the sample are compared to a database of electrical signals for each substance.

20. A method for providing information on diagnosing a biomarker-related disease, the method The following steps are involved: placing a sample in a compartment of the nanopore system as claimed in claim 8; as well as Changes in characteristic electrical signals caused by biomarkers for a specific disease among changes in electrical signals induced by the sample are measured.

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