Biomarker-specific binding probe and use thereof

Abstract

The present invention relates to a biomarker-specific binding probe and a use thereof. By introducing the probe into a nanopore, it is possible to accurately and rapidly detect the presence and abundance of a specific biomarker in a sample.

Description

Biomarker-specific binding probe and its application

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0194695 filed on December 23, 2024, and all contents disclosed in the document of said Korean Patent Application are incorporated herein as part of this specification.

[0003] Technology field

[0004] The present invention relates to a biomarker-specific binding probe and its application, and more specifically to a technology capable of accurately and rapidly detecting the presence and amount of a specific biomarker in a sample by introducing the probe into a nanopore.

[0005] Liquid biopsy is a representative disease diagnostic method presented as a strong alternative to tissue biopsy, as it eliminates the disadvantages associated with the latter, such as the risks of tissue extraction, tumor heterogeneity, and patient discomfort, while offering advantages in terms of cost-effectiveness and convenience. However, despite the various advantages of liquid biopsy, there are still aspects that need to be considered to establish evidence regarding its clinical utility. In particular, overcoming the limitations of detection sensitivity and accuracy in biomolecular analysis technologies is crucial for the widespread application of liquid biopsy and its replacement of tissue biopsy.

[0006] Meanwhile, nanopores are novel high-precision biosensors capable of detecting subtle morphological changes in biomolecules at the single-molecule level. They can determine the length, size, charge, structure, and morphology of biomolecules based on various electrical signals generated when an analyte passes through the nanopore. Liquid biopsy technology utilizing these nanopores offers advantages such as single-molecule resolution, high sensitivity, label-free operation, and real-time measurement capabilities; consequently, the development of biomolecule analysis technologies using nanopores is actively underway.

[0007] However, biomolecular analysis technologies using such nanopores entail a limitation in that, whenever new biomarkers or proteins are analyzed, newly designed probes or antibody-modified nanopore proteins must be purified and experimental conditions optimized.

[0008] Under this technical background, the inventors manufactured a novel probe that can be easily captured and docked into a nanopore, and confirmed that when the probe is used in conjunction with a nanopore, the presence and amount of trace amounts of biomarkers can be analyzed rapidly and accurately regardless of the type of nanopore and biomarker, thereby completing the present invention.

[0009] One objective of the present invention is to provide a novel probe for detecting specific biomarkers.

[0010] Another objective of the present invention is to provide a nanopore system comprising the probe and nanopore.

[0011] Another objective of the present invention is to provide a method for detecting disease-related biomarkers from a sample using the probe.

[0012] Another objective of the present invention is to provide a method for providing information for disease diagnosis using the probe.

[0013] Another objective of the present invention is to provide a kit for detecting disease-related biomarkers or diagnosing diseases, comprising the probe.

[0014] To achieve the above objective, the present invention provides a Target Catcher (TaC) probe comprising: an adapter protein; a binding domain protein; a linker connecting the adapter protein and the binding domain protein; and an anchor protein.

[0015] In addition, the present invention provides a nanopore system comprising the above-mentioned TaC (Target Catcher) probe; and a nanopore.

[0016] In addition, the present invention provides a method for detecting a disease-related biomarker from a sample, comprising: (1) introducing a TaC (Target Catcher) probe into a chamber containing nanopores; (2) measuring an electrical signal generated from the nanopores after introducing the TaC probe; (3) introducing a sample into the chamber after measuring the electrical signal; (4) measuring an electrical signal generated from the nanopores after introducing the sample; and (5) comparing the electrical signal measured in step (2) with the electrical signal measured in step (4).

[0017] In addition, the present invention provides a method for providing information for disease diagnosis, comprising: (1) introducing a TaC (Target Catcher) probe into a chamber containing nanopores; (2) measuring an electrical signal generated from the nanopores after introducing the TaC probe; (3) introducing a sample into the chamber after measuring the electrical signal; (4) measuring an electrical signal generated from the nanopores after introducing the sample; and (5) comparing the electrical signal measured in step (2) with the electrical signal measured in step (4).

[0018] In addition, the present invention provides a kit for detecting disease-related biomarkers or diagnosing diseases, comprising the above-mentioned TaC (Target Catcher) probe.

[0019] The present invention relates to a biomarker-specific binding probe and a technology capable of detecting the presence and amount of a specific biomarker in a sample by introducing the probe into a nanopore. When using the probe of the present invention, disease-related biomarkers at the single-molecule level can be detected at low cost and high sensitivity without pretreatment such as fluorescent labeling. Therefore, the present invention can be utilized for the early diagnosis and prognosis management of serious diseases that are difficult to diagnose in the early stages, such as pancreatic cancer and biliary tract cancer.

[0020] Figure 1 shows the structure of the TaC probe of the present invention.

[0021] Figure 2 shows the electrical signal pattern according to the presence or absence of PCB (Pancreatic Cancer Biomarker) in a nanopore into which a TaC probe was introduced.

[0022] Figure 3 shows the electrical signal patterns of a TaC probe designed with various linker lengths and a nanopore into which it is introduced.

[0023] Figure 4 shows the electrical signal pattern in a negative control nanopore in which no PCB was introduced.

[0024] Figure 5 shows the electrical signal pattern of a nanopore into which the amino acid from the C-terminal region of the adapter protein in the TaC probe was removed and introduced.

[0025] Figure 6 shows the structure of TaC probes designed with varying lengths and compositions of anchor proteins.

[0026] Figure 7 shows the electrical signal noise values ​​measured in the docking and detection states of the nanopore into which the TaC probe was introduced, and the limit of detection (LOD) values ​​for the Pim-1 antigen.

[0027] Figure 8 shows the results of measuring electrical signals according to the presence of Pim-1 antigen in human serum samples using nanopores with TaC probes introduced.

[0028] Figure 9 shows the results of measuring electrical signals according to the presence of Pim-1 or HMGB-1 antigens using nanopores into which variously designed TaC probes were introduced.

[0029] Figure 10 shows the results of measuring electrical signals according to the presence of Pim-1 or HMGB-1 antigens in human serum samples using nanopores with variously designed TaC probes introduced.

[0030] The present invention will be described in detail below.

[0031]

[0032] 1. TaC (Target Catcher) Probe

[0033] One aspect of the present invention provides a Target Catcher (TaC) probe comprising: an adapter protein; a binding domain protein; a linker having the adapter protein and the binding domain protein; and an anchor protein.

[0034] In one aspect, the anchor protein may be a first anchor protein connected to the binding domain protein.

[0035] In the present invention, the adapter protein refers to a protein that enables the TaC probe to be captured and docked inside a nanopore. The term "docking" refers to a state in which the TaC probe remains inside the nanopore without forming a covalent bond, based on microfluidic flow principles rather than physical or chemical bonding. The microfluidic flow principles may include electroosmosis, electrophoresis, or the principle of equilibrium between electroosmosis and electropsychometry.

[0036] The adapter protein may be a protein composed of a single domain so as to minimize the influence on the vibrational signal of the TaC probe due to structural dynamics, etc. The molecular weight of the adapter protein may be 10 kDa to 1,000 kDa, specifically 12 kDa to 670 kDa, but may be used without limitation as long as it enables the TaC probe to be stably docked inside the nanopore. A stably docked state refers to a state in which the TaC probe vibrates at a constant rate within the nanopore, and whether a stably docked state is achieved can be confirmed by the generation of a constant pattern of electrical signals when the TaC probe is added under specific voltage conditions.

[0037] The adapter protein may be smaller than the diameter of one opening of the nanopore so as to be captured in the nanopore lumen, and may be larger than the diameter of the other opening of the nanopore so as to be docked in the nanopore lumen.

[0038] Specifically, the diameter of the adapter protein may be 1 to 20 nm. For example, the diameter of the adapter protein may be 1 to 18 nm, 1 to 15 nm, 2 to 12 nm, or 2 to 10 nm.

[0039] In one aspect, the adapter protein may be Bcl-xL, FKBP12, MDM2, holo-transferrin, or mTOR, but any protein composed of a single domain that enables the TaC probe to be stably captured and docked inside the nanopore may be used without limitation.

[0040] In one aspect, the adapter protein may be a Bcl-xL protein (SEQ ID NO. 1), and in particular, a Bcl-xL protein from which the amino acid at the C-terminal region has been removed. In this case, the adapter protein may include the amino acid sequence of SEQ ID NO. 9.

[0041] In one embodiment of the present invention, it was confirmed that when a Bcl-xL protein with amino acids removed from the C-terminal region is used as the adapter protein for a TaC probe, the TaC probe maintains a docked state within the nanopore for a longer period and exhibits a more stable electrical signal compared to when a Bcl-xL protein containing amino acids from the C-terminal region is used.

[0042] In the present invention, the binding domain protein may include all proteins capable of specifically binding to the biomarker to be detected.

[0043] The binding domain protein of the present invention can specifically bind to a biomarker to be detected through the principles of non-covalent bonding, covalent bonding, sequence specificity, and structural specificity. The non-covalent bonding includes hydrogen bonding (when an amino group or a carboxyl group, etc., within an amino acid residue of the binding domain protein binds to a polar site of the biomarker), electrostatic interaction (when an amino acid having a positive or negative charge within the binding domain protein binds to a portion of the biomarker having an opposite charge), hydrophobic interaction (when a non-polar amino acid within the binding domain protein binds to a hydrophobic site within the biomarker), and van der Waals forces (when binding occurs through weak physical forces at adjacent non-polar sites of the binding domain protein and the biomarker), and refers to a binding that is reversible and can be controlled by an external environment.

[0044] The above covalent bond may include all covalent bonds that can occur between a specific amino acid in a biomarker and a cysteine ​​residue-based disulfide bond formation within a peptide, or a strong bond formed by modifying a specific amino acid residue using chemical conjugation.

[0045] The above sequence specificity refers to the phenomenon in which a specific amino acid sequence within the binding domain protein specifically binds to a specific binding site within the biomarker. By utilizing a biomarker-related peptide ligand library, peptide ligands that specifically bind to a specific biomarker can be selected, thereby allowing for the appropriate design of the amino acid sequence of the binding domain protein. The above structural specificity refers to the phenomenon in which the structure of a specific peptide within the binding domain protein specifically binds to a specific binding site within the biomarker.

[0046] The binding domain protein of the present invention can be designed to bind to a specific biomarker with high affinity by using phage display, structure-based peptide ligand mutation generation, or artificial intelligence-based peptide ligand design methods.

[0047] Specifically, the binding domain protein may bind to one or more disease-related biomarkers.

[0048] The above "biomarker" refers to an anatomical, physiological, biochemical, or molecular parameter associated with the presence or progression of a specific physiological state or process. The biomarker may include organic biomolecules such as polypeptides, proteins, or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (e.g., monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease within an individual or in a specific sample derived from an individual.

[0049] The above diseases are not limited to those for which an association with specific biomarkers has been established, and may be infectious or non-infectious diseases. The above-mentioned infectious diseases are diseases caused by microorganisms such as bacteria and viruses, 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, pulmonary tuberculosis, tuberculosis, the common cold, influenza, airway infection, rhinitis, nasopharyngitis, otitis media, bronchitis, lymphadenitis, parotitis, 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, urinary tractitis, cystitis, vaginitis, cervicitis, salpingitis, infectious erythema, bacterial dysentery, abscess and ulcer, bacteremia, diarrhea, dysentery, gastroenteritis, gastroenteritis, genitourinary abscess, infection of open wounds or wounds, purulent inflammation, abscess, boil, pyoderma, impetigo, folliculitis, cellulitis, postoperative wound infection, skin laceration syndrome, skin burn syndrome, thrombotic thrombocytopenia, hemolytic uremic syndrome, renal failure, pyelonephritis, glomerulonephritis, nervous system abscess, otitis media, sinusitis, pharyngitis, tonsillitis, mastoiditis, cellulitis, odontogenic infection, dacryocystitis, pleuritis, abdominal abscess, liver abscess, cholecystitis, splenic abscess, pericarditis, Myocarditis, placentaitis, amniotic fluid, mastitis, puerperal fever, toxic shock syndrome, Lyme disease, gas gangrene, atherosclerosis, Mycobacterium avium syndrome (MAC),It may be 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 listerosis.

[0050] The above non-infectious diseases may be cancer, autoimmune diseases, non-infectious inflammatory diseases, neurodegenerative diseases, heart diseases, strokes, diabetes, metabolic diseases, chronic kidney diseases, or chronic respiratory diseases. The above cancers include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, kidney cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, multiple myeloma, mycosis fungoides, Merkel cell carcinoma, classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytocyte-rich B-cell lymphoma, Epstein-Barr virus (EBV)-positive and -negative post-transplant lymphoproliferative disease (PTLD), EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extrinsic NK / T-cell lymphoma, nasopharyngeal carcinoma, and human herpesvirus 8 (HHV8)-associated primary It may be exudative lymphoma, other blood cancers including Hodgkin lymphoma, primary central nervous system (CNS) lymphoma, spinal tumors, and brainstem gliomas.The above-mentioned autoimmune diseases include lupus, systemic lupus erythematosus, Sjögren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, 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, fibrotic alveolitis, Graves' disease, IgA nephropathy, and idiopathic It may be thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, 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, vitiligo, myasthenia gravis and systemic sclerosis, etc.The above neurodegenerative diseases may include cognitive impairment, brain tumor, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain, and memory loss.

[0051] In one aspect, the biomarker may be a PIM-1 antigen or an HMGB-1 antigen, and in this case, the binding domain protein may include any one of the amino acid sequences of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 13, and SEQ ID NO. 14, respectively, but is not limited thereto. Meanwhile, if the binding domain protein includes the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 14, it may bind to both the PIM-1 antigen and the HMGB-1 antigen.

[0052] In the present invention, the linker refers to a protein that enables the adapter protein and the binding domain protein of a TaC probe to be connected. The linker protein may further include nucleic acids (DNA or RNA) or macromolecules (such as PEG) in addition to peptides, and may have a linear structure. Since the linker protein has a linear structure, it can physically separate the adapter protein, the binding domain protein, and the anchor protein. If the linker binds directly to the binding domain protein, the binding between the target biomarker and the binding domain protein may be hindered; and if the linker binds directly to the adapter protein, the binding between the anchor protein and the binding domain protein may be hindered. Therefore, it is preferable that the linker does not bind directly to the adapter protein and the binding domain protein.

[0053] In one aspect, the linker may be a peptide composed of 30 to 50 amino acid residues. Specifically, the linker may be a peptide composed of 40 amino acid residues, and more specifically, the linker may be a peptide composed of the amino acid sequence of SEQ ID NO. 6.

[0054] In one embodiment of the present invention, when the Pim-1 biomarker was detected using a peptide consisting of 30, 40, or 50 amino acid residues as a linker for a TaC probe, the TaC probe (TaC-P4) containing a linker with a length of 40 amino acids (Sequence No. 6) maintained a stable docking state within the nanopore compared to the probe containing a linker with a length of 30 or 50 amino acids, and the detection signal generated by the binding of the biomarker was observed more clearly, confirming that it is the optimal linker length.

[0055] In the present invention, the anchor protein refers to a protein capable of inducing the binding region protein to pass through the constricted portion of the nanopore and be exposed to the trans side after the TaC probe is captured and docked inside the nanopore. The trans side refers to the side facing the constricted portion of the nanopore.

[0056] In one aspect, the anchor protein may be a polar protein capable of moving by the principles of electrophoresis or electroosmosis. Additionally, since the anchor protein must pass through the constriction of the nanopore, it must have a size smaller than the diameter of the constriction of the nanopore.

[0057] Specifically, under conditions where voltage is applied, the anchor protein moves to the trans side of the nanopore by passing through the constricted portion of the nanopore due to the phenomenon of electrophoresis or electroosmosis, and under conditions where the application of voltage continues as described above, the anchor protein continues to exist on the trans side of the nanopore due to the phenomenon of electrophoresis or electroosmosis, thereby allowing the binding region protein connected to the anchor protein to also be continuously maintained in a state of exposure to the trans side of the nanopore.

[0058] In one aspect, the anchor protein may be positioned at various locations within the TaC probe. Specifically, the anchor protein may be positioned at a terminal location on the side of the binding domain protein of the TaC probe, at a location between the linker and the binding domain protein of the TaC probe, or at both of these locations.

[0059] More specifically, the TaC probe may be composed of any one of the structures of "adapter protein-linker-binding domain protein-anchor protein", "adapter protein-linker-anchor protein-binding domain protein", or "adapter protein-linker-anchor protein-binding domain protein-anchor protein".

[0060] However, the location of the anchor protein is not limited thereto, and various types of anchor proteins can be selected and their locations adjusted by considering the charge characteristics, size, and binding characteristics of the biomarker to be detected.

[0061] In one aspect, the anchor protein may be a protein comprising the amino acid sequence of SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12, but may be used without limitation as long as it docks the adapter protein within the TaC probe and exposes the binding domain protein to the trans side.

[0062] In one aspect, the first anchor protein may include the amino acid sequence of SEQ ID NO. 8.

[0063] In one aspect, the Tac probe may further include a second anchor interposed between the binding region protein and the first anchor protein.

[0064] In this specification, the term "interposition" means being located between two components to physically, chemically, and / or functionally connect them. Specifically, the interposition component may form covalent bonds, preferably peptide bonds, with each of the components on both sides to form a continuous polypeptide chain. More specifically, the interposition component may perform a role in (i) controlling the physical distance between the components on both sides, (ii) imparting structural flexibility or rigidity, (iii) enabling the independent functional expression of each component, and / or (iv) enhancing the stability and functionality of the entire fusion protein. That is, the interposition component may function to optimize the overall structural and functional characteristics of the fusion protein, going beyond a simple physical connection.

[0065] That is, a portion of the second anchor may be connected to the binding region protein, and another portion of the second anchor may be connected to the first anchor protein. Specifically, one end of the second anchor protein may be connected to the N-terminus or C-terminus of the binding region protein, and the other end of the second anchor protein may be connected to the N-terminus or C-terminus of the first anchor protein. Specifically, the second anchor protein may be connected to the C-terminus of the binding region protein and to the N-terminus of the first anchor protein through a peptide bond, thereby forming a fusion protein structure arranged in the order of binding region protein-second anchor protein-first anchor protein.

[0066] In one aspect, the second anchor protein may include the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12.

[0067] In one aspect, the Tac probe may further comprise a third anchor protein interposed between the linker and the binding region protein.

[0068] That is, a portion of the third anchor may be connected to the linker, and another portion of the third anchor may be connected to the binding region protein. Specifically, one end of the third anchor protein may be connected to the N-terminus or C-terminus of the linker, and the other end of the third anchor protein may be connected to the N-terminus or C-terminus of the binding region protein. Specifically, the third anchor protein may be connected to the C-terminus of the linker and to the N-terminus of the binding region protein through peptide bonds, thereby forming a fusion protein structure arranged in the order of linker-third anchor protein-binding region protein.

[0069] In one aspect, the third anchor protein may be the same as the second anchor protein. That is, the third anchor protein may include the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12.

[0070] The TaC probe of the present invention can be captured and docked inside a nanopore via an adapter protein, and under conditions where voltage is applied, a binding domain protein is exposed to the trans side of the nanopore via an anchor protein, and the exposed binding domain protein can bind to a biomarker to be detected on the trans side of the nanopore. At this time, the presence and amount of the target biomarker in the sample can be effectively confirmed by comparing and analyzing the electrical signals generated in the nanopore. Therefore, the TaC probe of the present invention can be effectively used to detect a specific biomarker.

[0071]

[0072] 2. Nanopore system containing TaC probe

[0073] Another aspect of the present invention provides a nanopore system comprising a TaC (Target Catcher) probe and a nanopore.

[0074] The above TaC probe is described by reference to the above and is not explained redundantly.

[0075] In the present invention, the nanopores may include YaxAB nanopores or ClyA nanopores, FraC nanopores, CytK nanopores, alpha-hemolysin nanopores, Aerolysin nanopores, SPP1 nanopores, Phi29 nanopores, MspA nanopores, OmpG nanopores, FhuA nanopores, PA63 nanopores, Neotrap nanopores, CsgG nanopores, or nanopores with mutations thereof, but may be included without limitation as long as the TaC probe of the present invention can be captured and docked to be used to detect a specific biomarker.

[0076] The above YaxAB nanopore is a heterodimer formed by the combination of YaxA protein and YaxB protein, and consists of 4 to 20 heterodimers. The above YaxAB nanopore has a funnel structure with a wide opening and a narrow outlet, and under pH 7.5 conditions, the amino acids constituting the inner wall of the pore have a negative charge, so electroosmosis can be induced by cations flowing along the inner wall of the pore.

[0077] In one aspect, the nanopore may be inserted into a phospholipid or phospholipid-sterol composite membrane. The membrane may be included without limitation as long as it serves as a support to which the nanopore can be fixed, while also being capable of dividing the space or chamber in which the nanopore exists into two compartments. The membrane serves to block the passage of a fluid or a substance contained in the fluid without additional means. The membrane layer may include a phospholipid and an amphiphilic material, and the YaxAB nanopore may be inserted into the membrane by a known method, for example, through a change in ion current.

[0078] The above phospholipid is an amphiphilic molecule containing at least one phospholipid and serves as a support for constituting the membrane. A phospholipid membrane refers to a structure in which the hydrophobic heads of the phospholipids point in one direction and the hydrophilic tails point in the opposite direction. The above amphiphilic material refers to a material that possesses both water-soluble and lipophilic properties and stabilizes the membrane by maintaining a constant fluidity of the membrane containing the phospholipid. When the above amphiphilic material is included, the stability of the membrane can be increased, the number of nanopores formed can be influenced, and the nanopore lifetime can be controlled as the nanopore disjoining force changes by influencing the elasticity of the membrane.

[0079] In one aspect, the nanopore system may be contained in a chamber. The chamber refers to a structure having an internal space formed to accommodate a certain volume of fluid. The space within the chamber is a fluid-filled compartment, and the fluid may be an electrolyte solution. Additionally, the chamber may include an electrode capable of generating an electrical potential difference across the membrane to facilitate the flow of electrolyte through the nanopores between chambers. By applying one or more electrical potentials to the electrode, a flow of ions can be generated from one compartment to another across the nanopore-containing membrane. The internal space of the chamber may be divided into two compartments, such as a first and second compartment or a cis and trans compartment, by the nanopore-containing membrane.

[0080] The above nanopore system may include a liquid medium, and the liquid medium may be an aqueous solution containing a salt. An ionic current may flow as the salt passes through the nanopore between two compartments separated by an insulating film. If the salt is an ion, it may be a metal salt, a halide salt, such as an alkali metal chloride salt, etc. Specifically, in the salt, the cation NH 4+ , K + , Na + , Li + , Mg 2+ , Ca 2+ , Gdm + It can be etc., and the anion is F - , SO4 2- , HPO4 2- , C2H3O 2- , Cl - , Br - , NO 3- , ClO 3- , I - , ClO 4- , SCN -The salt may be, for example, sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl), calcium chloride (CaCl2), cesium chloride (CsCl), guanidinium chloride (GdmCl), potassium ferrocyanide, potassium ferricyanide, etc. The salt may be an organic salt, for example, tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methylimidazolium chloride, etc. However, it is not limited thereto, and any charged salt may be included without limitation.

[0081] In the present invention, the adapter protein of the TaC probe may be docked inside the nanopore, and the binding domain protein of the TaC probe may be exposed to the outside of the nanopore. By exposing the binding domain protein of the TaC probe to the outside, binding with the biomarker to be detected is possible, and by comparing and analyzing the electrical signals generated at this time, the presence and amount of the target biomarker in the sample can be effectively confirmed. Therefore, the TaC probe and nanopore system of the present invention can be effectively utilized for the diagnosis of biomarker-related diseases and, furthermore, for the prediction of disease prognosis.

[0082] The above sample is defined as a sample that may contain the above biomarker without limitation and may be one or more selected from the group consisting of, for example, cell samples, tissue samples, blood samples, urine samples, saliva samples, lymph fluid samples, cerebrospinal fluid samples, amniotic fluid samples, pleural fluid samples, pericardial fluid samples, ascites samples, aqueous humor samples, bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, plant samples, microbial samples, virus samples, soil samples, marine samples, and freshwater samples.

[0083] The above tissue sample may be derived from one or more tissues selected from the group consisting of the epididymis, eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchus, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, bladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonads, testis, ovary, uterus, fallopian tube, thymus, pituitary gland, thyroid gland, adrenal gland, and parathyroid gland. Additionally, the above tissue sample may be derived from any various organ of a human or other organism.

[0084] The above cell sample may be derived from animal cells, plant cells, fungal cells, bacterial cells, or protozoan cells, specifically from animal cells, and more specifically from human cells.

[0085] For example, the above cell sample includes 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 alpha cells, pancreatic beta cells, pancreatic endothelial cells, bone marrow lymphoblasts, bone marrow B lymphoblasts, bone marrow macrophages, bone marrow erythroblasts, bone marrow dendritic cells, bone marrow adipocytes, bone marrow osteocytes, bone marrow chondrocytes, proosteocytes, bone marrow megakaryocytes, brain B lymphocytes, brain glial cells, neurons, brain astrocytes, neuroectodermal cells, brain macrophages, brain microglia, brain epithelial cells, cortical neurons, brain fibroblasts, breast epithelial cells, colon epithelial cells, colon B lymphocytes, breast myoepithelial cells, breast fibroblasts, colon intestinal cells, and cervical epithelium. From one or more cells selected from the group consisting of cells, ductal epithelial cells, tongue epithelial cells, tonsillar dendritic cells, tonsillar B lymphocytes, peripheral blood lymphoblasts, peripheral blood T lymphoblasts, peripheral blood cutaneous T lymphocytes, peripheral blood natural killer cells, peripheral blood B lymphoblasts, peripheral blood monocytes, peripheral blood myeloblasts, peripheral blood mononuclear cells, peripheral blood proostomyocytes, peripheral blood macrophages, peripheral blood basophils, hepatic 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, alveoli, gastric epithelial cells, gastric fibroblasts, stem cells, fetal cells, tumor cells, suspected cancer cells, cancer cells, and cells that have undergone gene editing procedures It may have originated from.

[0086] The nanopore system comprising the TaC probe and nanopore of the present invention includes a binding domain protein that specifically binds to a biomarker to be detected, thereby enabling accurate and rapid detection of the presence and amount of a specific biomarker in a sample containing various proteins. Accordingly, the TaC probe and nanopore system of the present invention can be effectively utilized for the diagnosis of biomarker-related diseases and, furthermore, for the prediction of disease prognosis.

[0087]

[0088] 3. Application of nanopore systems containing TaC probes

[0089] Another aspect of the present invention provides a method for detecting disease-related biomarkers from a sample.

[0090] The method for detecting the above-mentioned disease-related biomarker comprises: (1) a step of introducing the TaC (Target Catcher) probe of the present invention into a chamber containing nanopores; (2) a step of measuring an electrical signal generated from the nanopores after the introduction of the TaC probe; (3) a step of introducing a sample into the chamber after the measurement of the electrical signal; (4) a step of measuring an electrical signal generated from the nanopores after the introduction of the sample; and (5) a step of comparing the electrical signal measured in step (2) with the electrical signal measured in step (4).

[0091] The above-mentioned TaC probe, nanopore, chamber, sample, and disease-related biomarker are described by reference to the foregoing and are not explained redundantly.

[0092] First, in step (1), a TaC probe is introduced into a chamber containing a nanopore, so that the TaC probe can be captured into the nanopore by an adapter protein within the TaC probe. At this time, the TaC probe can be docked inside the nanopore through electroosmotic force (EOF), electrophoretic force (EPF), or a balance between electroosmotic force and electrophoretic force. Subsequently, in step (2), an electrical signal generated from the nanopore is measured after the introduction of the TaC probe, and in step (3), a sample is introduced into the chamber, and in step (4), an electrical signal generated from the nanopore is measured after the introduction of the sample. At this time, the sample may or may not contain a disease-related biomarker to be detected. Additionally, the measurement of the electrical signal may be a measurement of noise in the electrical signal generated from the nanopore. Afterwards, in step (5), the electrical signal measured in step (2) and the electrical signal measured in step (4) are compared. If there is no change in the two electrical signals, it can be determined that there is no disease-related biomarker in the sample, and if there is a sudden change in the two electrical signals, it can be determined that there is a disease-related biomarker in the sample.

[0093] The above "electrical signal" refers to a signal generated as ions and / or charged low-molecular-weight substances pass from one compartment separated by a membrane to another, for example, from a first compartment to a second compartment, and specifically refers to the current pattern of the ion current, the fundamental current in the open state (open pore current: I o, I open ), magnitude of current drop (△I), degree of current interruption, event duration, frequency of nanopore signal patterns per unit time, current noise (current noise: I N...etc. may include, but are not limited to. The frequency per unit time of the nanopore signal pattern may include, but is not limited to, event frequency and inter-event interval. The above-mentioned open-state fundamental current may refer to the current in a state where only nanopores are present. The magnitude of the above-mentioned current drop (△I) may refer to the magnitude of the decrease in the passage of electrolytes, such as ions and / or charged low-molecular-weight substances, as a sample is trapped inside the nanopore or passes through the nanopore. The above-mentioned current blockade refers to I, which is the fundamental current (open pore current) in the nanopore open state. o The ratio of △I, which is the magnitude of the current drop caused by the sample, to (△I / I o ) may be. The above event duration may be the time during which the event persists when the sample event appears as the magnitude of the current drop (△I). The above current noise may include power spectral density (PSD) or ion current standard deviation (SD), wherein the power spectral density represents the noise power generated by the flow of ion current on the frequency spectrum, indicating the power density distribution according to the signal frequency. The above standard deviation represents the standard deviation value of the current cutoff signal itself that occurs as the ion current drops. In addition, the above current noise (I N ) may represent the square root of the integral value of the power spectrum of the ion current or the standard deviation value of the ion current during current drop, and may mean the quantification of the electrical noise characteristics of the nanopore; it may also include amplifying the difference in current noise through signal filtering in the low-vibration region. Additionally, the electrical signal is the current cutoff (△I / I o ) and current noise (I NIt may include a density contour map, a two-dimensional scatter plot, or a violin plot that represents ) in two dimensions.

[0094] In one embodiment of the present invention, after setting the binding domain protein of the TaC probe to a protein capable of binding to a pancreatic cancer biomarker (PCB), and comparing the electrical signals before and after the addition of PCB, it was confirmed that the current intensity, dwell time, and frequency values ​​changed dramatically after the addition of PCB, and the limit of detection (LOD) was confirmed to be at a significantly low level of about 1.09 pM.

[0095] Therefore, the method for detecting disease-related biomarkers from a sample according to the present invention can confirm the presence and amount of the biomarker with high accuracy, even if the disease-related biomarker to be detected is present in the sample in trace amounts.

[0096] Another aspect of the present invention provides a method for providing information for disease diagnosis.

[0097] The above method for providing information for disease diagnosis comprises: (1) a step of introducing a TaC (Target Catcher) probe of the present invention into a chamber containing nanopores; (2) a step of measuring an electrical signal generated from the nanopores after the introduction of the TaC probe; (3) a step of introducing a sample into the chamber after the measurement of the electrical signal; (4) a step of measuring an electrical signal generated from the nanopores after the introduction of the sample; and (5) a step of comparing the electrical signal measured in step (2) with the electrical signal measured in step (4).

[0098] In the above step (5), the electrical signal measured in the above step (2) and the electrical signal measured in the above step (4) are compared. If there is no change in the two electrical signals, it can be determined that there is no disease-related biomarker in the sample. If there is a sudden change in the two electrical signals, it can be determined that there is a disease-related biomarker in the sample. In this case, if the amount of the disease-related biomarker is greater than a certain value, the individual from which the sample was isolated can be diagnosed as having a disease related to the biomarker.

[0099] In one embodiment of the present invention, after setting the binding domain protein of the TaC probe to a protein capable of binding to the Pim-1 antigen, a pancreatic cancer biomarker, and comparing the electrical signals before and after the addition of Pim-1, it was confirmed that the noise in the electrical signals in the docking state and the detection state was clearly distinguishable.

[0100] Therefore, even if a disease-related biomarker to be detected by the method for providing information for disease diagnosis from a sample of the present invention exists in a trace amount within the sample, the presence and amount of the biomarker can be confirmed with high accuracy, and thereby it can be effectively utilized for the diagnosis of diseases related to said biomarker, and furthermore, for the prediction of the prognosis of the disease.

[0101] Another aspect of the present invention provides a method for analyzing or screening inhibitors or promoters of interactions between biomolecules.

[0102] A method for analyzing or screening an inhibitor or promoter of the interaction between the above biomolecules comprises: (1) introducing the TaC (Target Catcher) probe into a chamber containing a nanopore; (2) measuring an electrical signal generated from the nanopore after the introduction of the TaC probe; (3) reacting a plurality of biomolecules capable of interacting in the chamber after the measurement of the electrical signal; (4) treating a candidate substance of the interaction inhibitor or promoter in the chamber; (5) measuring an electrical signal generated from the nanopore after the introduction of the candidate substance; and (6) comparing the electrical signal measured in step (2) with the electrical signal measured in step (5).

[0103] The method for analyzing or screening interaction inhibitors or promoters of the present invention first involves introducing a TaC probe into a chamber containing nanopores in step (1), thereby allowing the TaC probe to be captured into the nanopores by adapter proteins within the TaC probes. At this time, the TaC probes can be docked inside the nanopores through electroosmotic force (EOF), electrophoretic force (EPF), or a balance between electroosmotic force and electrophoretic force. Subsequently, in step (2), an electrical signal generated from the nanopores after the introduction of the TaC probes is measured, and after measuring the electrical signal, in step (3), a plurality of biomolecules capable of interacting are reacted in the chamber. After reacting the biomolecules, in step (4), a candidate substance of an interaction inhibitor or promoter is treated in the chamber, and in step (5), an electrical signal generated from the nanopores after treatment with the candidate substance is measured. Afterwards, in step (6), the electrical signal measured in step (2) and the electrical signal measured in step (5) are compared. If the change in the electrical signal is significant, the candidate substance can be determined as an inhibitor that inhibits the interaction between the biomolecules or as a promoter that promotes the interaction.

[0104] In one embodiment of the present invention, biomolecules may be first placed in one of the compartments to react, and then the candidate substance may be treated in the reactants or the opposite compartment. In other words, the candidate substance may be treated after the free biomolecules have been placed in one of the compartments. Additionally, the free biomolecules and the candidate substance may be treated simultaneously in one of the compartments. Furthermore, the treatment of the candidate substance may be carried out in a situation where the biomolecules are induced to move to another compartment through the nanopores, for example, where a gradient is formed between the two compartments separated by the membrane due to a difference in salt concentration, or where a voltage is applied, or where both of these are present.

[0105] In addition, the above biomolecules may be used without limitation as long as they can interact with each other, for example, by binding to form a complex, and may be, for example, peptides, proteins, lipids, carbohydrates, low-molecular-weight compounds, nucleic acids, or inorganic particles composed of nanomaterials. For example, the above biomolecules may be receptors, antigens, antibodies, or aptamers.

[0106] The above-mentioned ligand candidate substances refer to all types of molecules expected to inhibit or promote interactions between the above-mentioned biomolecules, such as single compounds like nucleic acids, amino acids, monosaccharides, etc., polymers like polynucleotides, peptides, proteins, polysaccharides, etc., and molecules such as natural or synthetic organic or inorganic substances or complexes thereof. For example, the above-mentioned ligand candidate substances may be receptors, antigens, antibodies, or aptamers.

[0107] Among the above-mentioned ligand candidate substances, those that inhibit the interaction between the biomolecules, particularly those that inhibit the interaction between the biomolecules by competitively binding to some of the biomolecules, are referred to as inhibitors, and the inhibitor may reduce the interaction between the biomolecules through such competitive binding. In other words, the inhibitor may bind to some of the biomolecules to increase the proportion of the biomolecules existing in a free state where they do not interact with one another, and may also increase the proportion of complexes in which the inhibitor interacts with some of the biomolecules. As the proportion of some of the biomolecules existing in a non-interacting state increases as described above, the electrical signal resulting from the interaction between the biomolecules decreases, and a new electrical signal pattern exhibited by complexes in which the inhibitor interacts with some of the biomolecules may appear, thereby enabling the direct detection or analysis of the inhibitor that inhibits the interaction between the biomolecules.

[0108] In addition, among the above-mentioned ligand candidate substances, those that promote the interaction between the biomolecules are referred to as promoters. These promoters include molecular glues and proteases (PROTACs) and may induce or increase the interaction between the biomolecules. For example, the promoter may act as a molecular glue and bind to the biomolecules to induce or increase the interaction between them. As described above, if the proportion of biomolecules existing in a mutually interacting state increases, the electrical signal resulting from the interaction between the biomolecules increases, and a new electrical signal pattern may appear in the complex formed by the interaction between the biomolecules and the promoter, thereby allowing for the direct detection or analysis of the promoter that facilitates the interaction between the biomolecules.

[0109] Cases where the above change is significant may include instances where a new signal that was not present before or after treatment with a candidate substance of an interaction inhibitor or promoter appears, or where electrical signals resulting from interactions between biomolecules increase or decrease. In particular, current blockage (△I / I o ) and current noise (I N It may include cases where the pattern appearing on the 2D density contour map of ) changes.

[0110] The method for analyzing or screening inhibitors or promoters of interactions between the above biomolecules can be used for drug screening, such as protein-protein interaction inhibitors or promoters, and for the discovery and design of new drugs through the analysis of protein binding sites.

[0111]

[0112] 4. Kit including TaC probe

[0113] Another aspect of the present invention provides a kit for detecting disease-related biomarkers or diagnosing diseases.

[0114] The above kit includes the TaC probe of the present invention. In one aspect, the kit further includes nanopores.

[0115] The above-mentioned TaC probe, nanopore, and disease-related biomarker are described by reference to the foregoing and are not explained redundantly.

[0116] Using the above kit, the presence and amount of disease-related biomarkers in a specific sample can be detected, and thereby, disease diagnosis related to the biomarkers can be performed.

[0117] The above kit may further include an instruction manual. The instruction manual may include information regarding data interpretation and judgment, for example, criteria may be stated such that if a biomarker detection electrical signal appears, it can be determined that the biomarker is present in the sample, and if the amount of the biomarker is above a certain value, the individual from which the sample was isolated can be diagnosed as having a disease related to the biomarker.

[0118] Another aspect of the present invention provides a kit for the analysis or screening of inhibitors or promoters of interactions between biomolecules.

[0119] The above kit includes the TaC probe of the present invention. In one aspect, the kit further includes nanopores.

[0120] The above-mentioned TaC probe, nanopore, and disease-related biomarker are described by reference to the foregoing and are not explained redundantly.

[0121] The above kit can be used for drug screening, such as for protein-protein interaction inhibitors or promoters, and for the discovery and design of new drugs through the analysis of protein binding sites.

[0122]

[0123] The present invention will be explained in detail below through examples.

[0124] However, the following examples are intended to specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0125]

[0126] [Example 1]

[0127] Manufacturing of TaC (Target Catcher) probes

[0128] A TaC probe capable of effectively detecting a specific biomarker was prepared by being introduced into a nanopore and binding highly specifically to the target biomarker. The TaC probe was composed of an adapter portion that is trapped through the entrance of the nanopore and docked inside the pore, a binding domain portion to which the target biomarker can bind, a linker connecting the adapter and the binding domain, and an anchor portion that guides the TaC probe to pass through the constriction region of the nanopore to dock the adapter to the nanopore and expose the binding domain to the trans side (Fig. 1).

[0129] At this time, pancreatic cancer biomarkers Pim-1 and HMGB-1 antigens were selected as target biomarkers for detection. Then, binding domain proteins capable of detecting Pim-1 (SEQ No. 2, SEQ No. 13) and binding domain proteins capable of detecting HMGB-1 (SEQ No. 3) were designed and produced, and TaC-P probes and TaC-H probes were manufactured using these. In addition, binding domain proteins capable of simultaneously detecting the two antigens (SEQ No. 4, SEQ No. 14) were designed and produced, and TaC-HP probes were manufactured using these (Table 1). Bcl-xL protein (SEQ No. 1) was used as the adapter protein.

[0130] Adapter-linked region linker anchor TaC-P3MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER (Sequence No. 1)ARKRRRHPAGPPAA (Sequence No. 2) or ARKRRRHPAGPPTA (Sequence No. 13)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (Sequence No. 5)HHHHHHHHHH(Sequence No. 8)TaC-P4MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)ARKRRRHPAGPPAA(Sequence No. 2)orARKRRRHPAGPPTA(Sequence No. 13)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Sequence No. 8)TaC-P5MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)ARKRRRHPAGPPAA(Sequence No. 2)orARKRRRHPAGPPTA(Sequence No. 13)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 7)HHHHHHHHHH(Sequence No.8)TaC-H3MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 5)HHHHHHHHHH(Sequence No. 8)TaC-H4MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Sequence No. 8)TaC-H5MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 7)HHHHHHHHHH(Sequence No.8)TaC-HP4MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTEDgsgARKRRRHPAGPPAA(Sequence No. 4)orAMDDLMLSPDDIEQWFTEDgsgARKRRRHPAGPPTA(Sequence No. 14)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Sequence No. 8)

[0131]

[0132] [Example 2]

[0133] Detection of biomarkers using a TaC probe-based nanopore system

[0134] YaxAB nanopores were selected as nanopores for detecting specific biomarkers by introducing the TaC probe prepared in Example 1 above. The YaxAB nanopores are heterodimers formed by the combination of YaxA monomers and YaxB monomers, and consist of 4 to 20 such heterodimers, having a funnel structure with a wide opening and a narrow outlet. Among them, the YaxAB-C8 oligomer nanopore with the best sensing efficiency was selected, and a nanopore system was constructed by introducing it into a phospholipid-sterol composite membrane in which the nanopores can be stably maintained.

[0135] Subsequently, TaC trapping and TaC docking modes were implemented by stably capturing the TaC probe in the nanopore through the control of the flow of water molecules using the electroosmotic force (EOF) that occurs dependent on the internal surface charge of the nanopore. Specifically, the TaC trapping mode is a state in which the TaC probe is captured in the nanopore and remains inside the pore, exhibiting an intermediate level of current blockade intensity, whereas the TaC docking mode is a state in which the anchor and binding region of the TaC probe pass through to the trans side and are exposed, exhibiting a high level of current blockade intensity (Fig. 2).

[0136] Next, the binding domain protein of the TaC probe was set as a protein capable of binding to a pancreatic cancer biomarker (PCB), and then the electrical signal was analyzed after adding the PCB to the nanopore system in which the TaC probe was trapped. Specifically, 100 nM of the TaC probe was treated in a solution of pH 7.5, 10 mM Tris, 1 mM EDTA, and 1 M KCl to trap and dock into the nanopore. In addition, the Pim-1 antigen was selected as the PCB and treated at concentrations ranging from 50 femtoM (fM) to 1,000 nM.

[0137] Analysis results showed that the current intensity, dwell time, and frequency values ​​changed dramatically in the docking state signal after the binding region of the TaC probe and Pim-1 bound, and this was identified as a detection signal for the target marker (Fig. 2). In addition, it was confirmed that a short time of less than 5 minutes was required to observe a change in the current signal after the addition of the Pim-1 antigen.

[0138]

[0139] [Example 3]

[0140] Optimization of linker length within the TaC probe

[0141] After the above TaC probe was docked to the nanopore, the linker length within the TaC probe was adjusted so that the structure of the biomarker binding region could be well exposed to the trans side. Specifically, TaC probes were prepared by varying the linker length to 30, 40, or 50 amino acids, and experiments for detecting the Pim-1 pancreatic cancer biomarker were performed using a nanopore system to which each of these was applied.

[0142] As a result, in the case of the TaC probe (TaC-P4) containing a linker with a length of 40 amino acids (Sequence No. 6), the docking state within the nanopore was maintained stably compared to probes (TaC-P3, TaC-P5) containing linkers with a length of 30 or 50 amino acids, and the detection signal generated by the binding of the biomarker was observed more clearly, confirming that it is the optimal linker length (Fig. 3).

[0143] In addition, only trapping and docking signals were observed in both cases: when only the TaC-P4 probe was docked to the nanopore and the Pim-1 biomarker was not added (negative control, left side of Fig. 4), and when other proteins such as Bcl-xL were added instead of the Pim-1 biomarker (negative control, right side of Fig. 4). Through this, it was confirmed that a nanopore system using a TaC probe with a binding region applied for detecting Pim-1 can highly specifically detect only the Pim-1 biomarker and not other proteins.

[0144]

[0145] [Example 4]

[0146] Optimization of adapter proteins within the TaC probe

[0147] In order for the above TaC probe to be effectively captured and docked in the nanopore, the adapter protein within the TaC probe was mutated, and the electrical signals generated in the nanopore system were analyzed. Specifically, a TaC probe containing a mutant adapter protein was prepared by removing 11 amino acids from the C-terminal region of the Bcl-xL protein (1-211 △1-4, 45-84) used as the adapter protein (Table 2). Subsequently, the electrical signals generated in the nanopore system were analyzed.

[0148] Adapter-linked region-linker-anchor-TaC-P4-dcMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNN(Sequence No. 9)ARKRRRHPAGPPAA(Sequence No. 2)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Sequence No. 8)TaC-P5-dcMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNN(Sequence No. 9)ARKRRRHPAGPPAA(Sequence No. 2)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 7)HHHHHHHHHH(Sequence No. 8)

[0149] As a result, compared to TaC probes containing a non-mutated adapter protein (TaC-P4 and TaC-P5), TaC probes containing a mutant adapter protein (SEQ No. 9) (TaC-P4-dc and TaC-P5-dc) were found to be captured and docked more deeply toward the constriction of the nanopore, maintained a docked state for a longer period of time, and exhibited a more stable electrical signal (Figs. 5, 9, and 10).

[0150]

[0151] [Example 5]

[0152] Optimization of anchor protein length and composition within TaC probe

[0153] To detect various biomarkers using a TaC probe-based nanopore system, binding regions with diverse charge characteristics must be introduced. Accordingly, two TaC probes, TaCM1 and TaCM2, were prepared by optimizing the length and composition of anchor proteins considering the charge or physical characteristics of the binding regions (Fig. 6, Table 3).

[0154] Specifically, TaCM1 was designed to induce stable trapping and docking steps of the TaC probe by introducing various anchor sequences (second anchor; SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12) optimized for the posterior side of the binding region (probe end). Additionally, TaCM2 was designed to induce stable trapping and docking steps of the TaC probe by introducing various anchor sequences (second and third anchors; SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12) optimized for both the anterior side of the binding region (between the linker and the binding region) and the posterior side of the binding region (probe end).

[0155] Adapter-linked region linker anchor TaCM1-H4-2RMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Anchor1, Sequence No. 8) and RR(Anchor2, Sequence No. 10)TaCM1-H4-4RMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Anchor1, Sequence No. 8) and RRRR(Anchor2, Sequence No. 11)TaCM1-H4-6RMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Anchor1, Sequence No. 8)andRRRRRR(Anchor2,Sequence No. 12)TaCM1-H5-6RMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 7)HHHHHHHHHHH(Anchor 1, Sequence No. 8) and RRRRRR(Anchor 2, Sequence No. 12)TaCM2-H4-4R-4RMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER(Sequence No. 1)AMDDLMLSPDDIEQWFTED(Sequence No. 3)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(Sequence No. 6)HHHHHHHHHH(Anchor1, Sequence No. 8) and RRRR(Anchor2, Sequence No. 11),

[0156] As a result of optimizing the anchor length and composition for a TaC probe having a binding region that is difficult to capture by nanopores due to its negative net charge characteristics, it was confirmed that not only was the stability of the trapping and docking steps significantly improved, but the detection signal of HMGB-1, a pancreatic cancer biomarker, binding could also be clearly detected (Figs. 9 and 10).

[0157]

[0158] [Example 6]

[0159] Optimization of electrical signal detection parameters

[0160] In an experiment to detect pancreatic cancer biomarkers using a TaC probe-based nanopore system, the average noise value of the electrical signal was measured for the docking state and the biomarker detection state, and it was confirmed that there was a significant difference in the electrical signal noise value between the docking state and the biomarker detection state (Fig. 7).

[0161] Through the above experiment, it was confirmed that current intensity, current cutoff time, and noise exist as analysis parameters for TaC probe-based nanopore systems, and that detecting and analyzing electrical signal noise values ​​caused by the motility of the TaC probe within the nanopore is effective for biomarker detection signal analysis.

[0162]

[0163] [Example 7]

[0164] Analysis of the detection limit of TaC probe-based nanopore systems

[0165] The limit of detection (LOD) for the pancreatic cancer biomarker Pim-1 in a TaC probe-based nanopore system was measured.

[0166] First, it was confirmed that the ratio of electrical signal noise due to biomarker detection in the nanopore system increased significantly as the concentration of Pim-1 increased (Fig. 7). In addition, when using a TaC probe-based nanopore system, it was found that detection was possible even at significantly low fM levels, with a concentration range of 50 femtoM (fM) to 1 μM. As a result calculated using the LOD calculation formula (3x(Standard Deviation of Blank / Slope of Calibration Curve)), the LOD value of the TaC probe-based nanopore system of the present invention was confirmed to be approximately 2.49 fM, which is a significantly low level (Fig. 7).

[0167] Through this, the TaC probe-based nanopore system of the present invention can accurately detect even trace amounts of biomarkers present in a sample at the single-molecule level without labeling, and thus has significantly superior detection sensitivity. In particular, considering that the concentration of the pancreatic cancer biomarker Pim-1 present in samples of pancreatic cancer patients is 835 pM to 2,171 pM, it can be seen that the TaC probe-based nanopore system of the present invention can be usefully utilized for the early diagnosis of pancreatic cancer.

[0168]

[0169] [Example 8]

[0170] Detection of biomarkers in blood samples using a TaC probe-based nanopore system

[0171] To verify whether actual disease diagnosis is possible using the TaC probe-based nanopore system of the present invention, experiments were conducted to determine the detection of pancreatic cancer biomarkers in human serum samples.

[0172] Specifically, when a TaC probe-based nanopore system was applied to human serum samples with Pim-1 concentrations ranging from 50 fM to 500 nM, it was confirmed that noise in the electrical signal was clearly distinguishable between the docking state and the biomarker detection state (Figs. 8, 10). In addition, the LOD for biomarker detection in the serum samples was measured to be 1.02 fM, confirming a detection limit that is more improved than the LOD (2.49 fM) measured in the buffer (Fig. 8).

[0173] Through the above experiment, it was found that the TaC probe-based nanopore system of the present invention generates a nanopore electrical signal through specific binding with pancreatic cancer biomarkers without non-specific binding, even though various types and large quantities of macromolecules are present in human serum samples. Consequently, it can be seen that the TaC probe-based nanopore system of the present invention can be usefully utilized in the field of diagnosis through the detection of protein biomarkers.

[0174]

[0175] Attached electronic file of sequence list

Claims

1. Adapter protein; binding domain protein; A linker connecting the adapter protein and the binding domain protein; and A TaC (Target Catcher) probe comprising a first anchor protein connected to the binding region protein.

2. In Claim 1, A TaC probe having a diameter of 1 to 20 nm of the adapter protein.

3. In Claim 1, The adapter protein is a TaC probe that is Bcl-xL, FKBP12, MDM2, holo-transferrin, or mTOR.

4. In Claim 3, The above Bcl-xL is a TaC probe in which the amino acid at the C-terminal region has been removed.

5. In Claim 1, The above adapter protein is a TaC probe comprising the amino acid sequence of SEQ ID NO.

9.

6. In Claim 1, The adapter protein is a TaC probe having a molecular weight of 10 to 1,000 kDa.

7. In Claim 1, The above linker is a TaC probe that is a peptide composed of 30 to 50 amino acid residues.

8. In Claim 1, The above linker is a TaC probe comprising the amino acid sequence of SEQ ID NO.

6.

9. In Claim 1, The above anchor protein is a TaC probe that is a polar protein.

10. In Claim 1, The above anchor protein is a TaC probe comprising the amino acid sequence of SEQ ID NO.

8.

11. In Claim 1, The above Tac probe further comprises a second anchor interposed between the binding region protein and the first anchor protein, a TaC probe.

12. In Claim 11, The above-mentioned second anchor protein is a TaC probe comprising the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO.

12.

13. In Claim 11, The above Tac probe further comprises a third anchor protein interposed between the linker and the binding region protein, a TaC probe.

14. In Claim 13, The above third anchor protein is the same as the above second anchor protein, a Tac probe.

15. In Claim 1, A TaC probe in which the binding domain protein binds to one or more disease-related biomarkers.

16. In Claim 15, TaC probe, the above disease is cancer.

17. A nanopore system comprising a TaC (Target Catcher) probe of any one of claims 1 to 16; and a nanopore.

18. In Claim 17, A nanopore system in which the above nanopore is inserted into a phospholipid or phospholipid-sterol complex membrane.

19. In Claim 17, The adapter protein of the above TaC probe is docked inside the nanopore, and A nanopore system in which the binding domain protein of the above TaC probe is exposed to the outside of the nanopore.

20. (1) A step of introducing a TaC (Target Catcher) probe of any one of claims 1 to 16 into a chamber containing nanopores; (2) A step of measuring the electrical signal generated from the nanopore after the introduction of the TaC probe; (3) A step of introducing a sample into the chamber after measuring the electrical signal; (4) A step of measuring the electrical signal generated from the nanopore after the introduction of the sample; and (5) A step of comparing the electrical signal measured in step (2) above with the electrical signal measured in step (4) above; A method for detecting disease-related biomarkers from a sample containing 21. (1) A step of introducing a TaC (Target Catcher) probe of any one of claims 1 to 16 into a chamber containing nanopores; (2) A step of measuring the electrical signal generated from the nanopore after the introduction of the TaC probe; (3) A step of introducing a sample into the chamber after measuring the electrical signal; (4) A step of measuring the electrical signal generated from the nanopore after the introduction of the sample; and (5) A step of comparing the electrical signal measured in step (2) above with the electrical signal measured in step (4) above; A method for providing information for disease diagnosis including 22. (1) A step of introducing a TaC (Target Catcher) probe of any one of claims 1 to 16 into a chamber containing nanopores; (2) A step of measuring the electrical signal generated from the nanopore after the introduction of the TaC probe; (3) A step of reacting a plurality of biomolecules capable of interacting in the chamber after measuring the electrical signal; (4) A step of treating the chamber with a candidate substance of an interaction inhibitor or promoter; (5) A step of measuring the electrical signal generated from the nanopore after the introduction of the above candidate substance; and (6) A step of comparing the electrical signal measured in step (2) above with the electrical signal measured in step (5) above; a method for analyzing or screening an inhibitor or promoter of interactions between biomolecules.

23. A kit for detecting disease-related biomarkers or diagnosing diseases, comprising a TaC (Target Catcher) probe according to any one of claims 1 to 16.

24. A kit for the analysis or screening of an inhibitor or promoter of interactions between biomolecules comprising a TaC (Target Catcher) probe of any one of claims 1 to 16.

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