Protein-nucleic acid complex, and target substance detection kit and detection method using same

Abstract

Provided is a highly stable and versatile protein-nucleic acid complex. This protein-nucleic acid complex comprises a nucleic acid molecule having uracil, a uracil DNA glycosylase domain covalently bound to the nucleic acid molecule, and a protein domain covalently bound directly or indirectly to the uracil DNA glycosylase domain.

Description

Protein-nucleic acid complex, substance detection kit and detection method using the same

[0001] The present invention relates to a protein-nucleic acid complex in which a nucleic acid molecule and a protein are covalently bound, a detection kit and method for a substance using the same, and a method for using the protein-nucleic acid complex.

[0002] The covalent bond between nucleic acids and proteins is a major advantage. For example, when using such complexes as sensing elements, noncovalent association raises concerns about dissociation during the washing process of the complex synthesis or in the system in which the complex is used. Currently, reported DNA covalent binding proteins can be broadly classified into HUH endonucleases (histidine-hydrophobic residue-histidine-endonucleases: HUH-endonucleases) and uracil DNA glycosylase X (uracil DNA glycosylase X: UdgX).

[0003] A complex between UdgX and nucleic acid is described in Patent Document 1. Patent Document 1 discloses that fusing UdgX with an antibody-binding protein such as protein A enables the formation of a complex between an antibody, UdgX, and the nucleic acid bound to UdgX. Patent Document 1 also discloses that the complex can be used in immuno-PCR by binding a uracil-containing nucleic acid molecule as a probe to UdgX. UdgX, derived from Mycobacterium smegmatis, is an enzyme belonging to the uracil-DNA glucosidase family and is involved in the mismatch repair of nucleic acid base sequences. UdgX recognizes and removes uracil from ssDNA (singlestranded DNA) or doublestranded DNA (dsDNA), and then forms a glycosidic bond at the abasic site via a histidine residue (Non-Patent Document 1).

[0004] On the other hand, nucleic acid-protein complexes can also be used in biosensing. Biosensing refers to a technology for detecting specific substances in living organisms. One example of biosensing is a technology that quantitatively detects specific molecules by utilizing the property of biochemical molecules, such as enzymes that perform chemical reactions in living organisms and antibodies that perform immune reactions, to specifically react with specific molecules. A substance used in such biosensing and in a system that detects a specific substance to be detected is called a biosensing element.

[0005] Generally, biosensing elements consist of a molecular recognition motif that binds to the target substance and a signal transduction motif that converts the binding signal into a detectable signal. Detecting disease-related substances using biosensing technology is expected to be applied in a variety of situations, such as early detection of diseases such as cancer and health management, such as measuring blood glucose levels in diabetes patients.

[0006] Molecular recognition motifs in conventional biosensing elements include proteins such as antibodies and nucleic acid molecules such as nucleic acid aptamers. Nucleic acid aptamers are nucleic acid molecules that bind with high specificity to various substances such as ions, low-molecular-weight compounds, and proteins. Furthermore, because nucleic acid molecules have the property of binding to complementary base sequences, they can be used as molecular recognition motifs when a target nucleic acid having a specific base sequence is used as the substance to be detected.

[0007] On the other hand, nucleic acid-protein complexes can be immobilized on a substrate by introducing a thiol group to the end of the nucleic acid molecule. In other words, by using nucleic acid-protein complexes, proteins can be immobilized on a substrate via nucleic acid molecules. In this way, by immobilizing proteins that function as signaling motifs on a substrate, it is possible to construct a device equipped with multiple biosensing elements.

[0008] Korean Patent No. 10-2020-0084690

[0009] Sang, PB; Srinath, T.; Patil, AG; Woo,EJ; Varshney, U. A unique uracil-DNA binding protein of the uracil DNAglycosylase superfamily. Nucleic acids research, 2015, 43(17), 8452-8463.

[0010] Patent Document 1 discloses a complex of nucleic acid and protein, but the complex is configured such that UdgX bound to a nucleic acid molecule and an antibody are bound via Protein A. In other words, the complex disclosed in Patent Document 1 is not practical because the target protein is non-covalently associated and cannot be said to be tightly bound, and furthermore, it is specialized for antibody binding via Protein A and is not versatile.

[0011] In view of the above-mentioned circumstances, an object of the present invention is to provide a protein-nucleic acid complex, which is a complex formed by covalently bonding a nucleic acid molecule and a protein and is not limited to a particular type of protein, as well as a detection reagent and method for detecting a substance using the same, and a method for using the protein-nucleic acid complex.

[0012] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they found that by directly or indirectly covalently binding uracil DNA glycosylase, to which a nucleic acid molecule is covalently bound, with a target protein, it is possible to prepare a protein-nucleic acid complex in which the protein and nucleic acid molecule are covalently linked, regardless of the type of target protein, and thus completed the present invention.

[0013] The present invention encompasses the following: (1) A protein-nucleic acid complex comprising a nucleic acid molecule having uracil, a uracil DNA glycosylase domain covalently bound to the nucleic acid molecule, and a protein domain directly or indirectly covalently bound to the uracil DNA glycosylase domain. (2) The protein-nucleic acid complex according to (1), wherein the uracil DNA glycosylase domain and the protein domain are covalently bound via a tag peptide and a tag capture polypeptide. (3) The protein-nucleic acid complex according to (1), wherein the nucleic acid molecule is a nucleic acid aptamer molecule that specifically binds to a predetermined substance or a molecule containing a base sequence complementary to a specific base sequence. (4) The protein-nucleic acid complex according to (1), wherein the nucleic acid molecule is a chemically modified nucleic acid molecule. (5) A biosensing element comprising the protein-nucleic acid complex according to any one of (1) to (4). (6) The biosensing element according to (5), wherein a substance to which the nucleic acid molecule contained in the protein-nucleic acid complex specifically binds is detected. (7) The biosensing element according to (5), characterized in that it detects a substance to which a protein domain contained in the protein-nucleic acid complex specifically binds. (8) The biosensing element according to (5), characterized in that the nucleic acid molecule contained in the protein-nucleic acid complex is an aptamer molecule that increases the peroxidase activity of a hemoprotein, and the protein domain contained in the protein-nucleic acid complex is an enzyme with oxidase activity that catalyzes an oxidation reaction to produce hydrogen peroxide. (9) The biosensing element according to (5), characterized in that the protein-nucleic acid complex is immobilized on a substrate via chemical modification of the nucleic acid molecule contained in the protein-nucleic acid complex. (10) A substance detection kit comprising: a protein-nucleic acid complex according to any one of (1) to (4), in which the protein domain in the protein-nucleic acid complex is an enzyme with oxidase activity that catalyzes an oxidation reaction to produce hydrogen peroxide; a first substrate that is a substrate for the first enzyme; and a second substrate that is a substrate for a substance to be detected to which the nucleic acid molecule in the protein-nucleic acid complex binds and has peroxidase activity.(11) The detection kit according to (10), wherein the enzyme and the first substrate are glucose oxidase and glucose, respectively, and the substance to be detected and the second substrate are hemoglobin and luminol, respectively. (12) The detection kit according to (10), wherein the enzyme is a fusion protein with an antibody against the substance to be detected. (13) A method for detecting a substance, comprising the steps of: mixing the detection kit according to any one of (10) to (12) above with a sample that may contain the substance to be detected; and measuring an enzymatic reaction that consumes the second substrate contained in the detection kit, wherein the method detects the substance to be detected in the sample based on the enzymatic reaction. This specification incorporates the disclosure of Japanese Patent Application No. 2022-138531, from which the present application claims priority.

[0014] According to the present invention, a protein-nucleic acid complex can be provided in which a nucleic acid molecule and a protein domain are complexed via a covalent bond. The protein-nucleic acid complex according to the present invention is not limited to a particular type of protein, and any protein can be used as the protein domain. Therefore, by using the protein-nucleic acid complex according to the present invention, which is practical and highly versatile, a detection kit and a detection method for a substance with excellent detection sensitivity can be provided.

[0015] FIG. 1 is a schematic diagram showing an example of a protein-nucleic acid complex to which the present invention is applied. FIG. 2 is a schematic diagram showing a system for detecting hemoglobin using a biosensing element that utilizes a protein-nucleic acid complex to which the present invention is applied. FIG. 3 is a schematic diagram showing a system for detecting hemoglobin using another biosensing element that utilizes a protein-nucleic acid complex to which the present invention is applied. FIG. 4 is a schematic diagram showing a system for detecting genomic DNA methylation using a biosensing element that utilizes a protein-nucleic acid complex to which the present invention is applied. FIG. 5 is a schematic diagram showing another biosensing element that utilizes a protein-nucleic acid complex to which the present invention is applied. FIG. 6 is a schematic diagram showing vectors that express various DNA covalently binding proteins constructed in this example. FIG. 7 is a characteristic diagram showing the results of calculating the DNA binding efficiency for various DNA covalently binding proteins synthesized in this example. FIG. 8 is a characteristic diagram showing the nucleotide sequence and amino acid sequence encoding UdgX-SpyTag prepared in this example. FIG. 9 is a characteristic diagram showing the nucleotide sequence and amino acid sequence encoding UdgX-SpyCatcher prepared in this example. FIG. 10 is a characteristic diagram showing the results of evaluating the DNA binding ability of the aptamer-GOx complex prepared in this example. 1 is a characteristic diagram showing the results of evaluating the oxidase activity of the aptamer-GOx complex prepared in this example.

[0034] FIG. 1 is a characteristic diagram showing the results of evaluating the Hb detection sensitivity of the aptamer-GOx complex prepared in this example when a polypropylene plate or a low-protein adsorption polystyrene plate was used.

[0035] FIG. 1 is a characteristic diagram showing the results of detecting Hb contained in human serum using the aptamer-GOx complex prepared in this example.

[0036] FIG. 1 is an electrophoretic photograph showing the results of SDS-PAGE analysis when a SpyTag / SpyCatcher reaction was performed using UdgX-SpyTag and GDH-SpyCatcher.

[0037] FIG. 1 is an electrophoretic photograph showing the results of SDS-PAGE analysis and fluorescence detection when a UdgX-GDH complex was reacted with the TBA15 aptamer.

[0038] FIG. 1 is an electrophoretic photograph showing the results of SDS-PAGE analysis when a SpyTag / SpyCatcher reaction was performed using UdgX-SpyTag and GOx-SpyCatcher.Electrophoretic photographs showing the results of SDS-PAGE analysis and fluorescence detection when a UdgX-GOx complex was reacted with the TBA15 aptamer. Electrophoretic photographs showing the results of SDS-PAGE analysis when a SpyTag / SpyCatcher reaction was performed using UdgX-SpyTag and GDH-SpyCatcher. Electrophoretic photographs showing the results of SDS-PAGE analysis and fluorescence detection when a UdgX-GDH complex was reacted with the PEA3-01 aptamer.

[0016] The present invention will now be described in detail with reference to the drawings.

[0017] [Protein-Nucleic Acid Complex] As shown schematically in Figure 1, the protein-nucleic acid complex according to the present invention comprises a nucleic acid molecule 101 having uracil, a uracil DNA glycosylase domain 102 covalently bound to the nucleic acid molecule 101, and a protein domain 103 directly or indirectly covalently bound to the uracil DNA glycosylase domain 102. Note that Figure 1 exemplifies a structure in which the protein domain 103 forms a dimer. The protein-nucleic acid complex according to the present invention is not limited to one in which the protein domain 103 forms a dimer, and may also be one in which the protein domain 103 exists as a monomer or as a multimer such as a trimer or tetramer.

[0018] In the example shown in FIG. 1 , the uracil DNA glycosylase domain 102 and the protein domain 103 are indirectly covalently bonded. Here, "indirectly covalently bonded" includes covalently bonding proteins via a peptide. For example, the uracil DNA glycosylase domain 102 and the protein domain 103 can be covalently bonded using a so-called tag peptide 104 and a tag capture peptide 105 (also referred to as a tag peptide and a catcher peptide). Specifically, either the tag peptide 104 or the tag capture peptide 105 is introduced into the C-terminus or N-terminus of the uracil DNA glycosylase domain 102, and the other is introduced into the C-terminus or N-terminus of the protein domain 103. Then, by mixing the uracil DNA glycosylase domain 102 having either the tag peptide 104 or the tag capture peptide 105 with the protein domain 103 having the other, a covalent bond is formed between the tag peptide 104 and the tag capture peptide 105.

[0019] Examples of the tag peptide 104 and the tag capture peptide 105 include the SpyTag / SpyCatcher system (Bijan Zakeri et al., PNAS, 109 (12) E690-E697, 2012) and the SnoopCatcher / SnoopTag system (Gianluca Veggianiet al., PNAS, 113 (5) 1202-1207, 2016). In addition, the tag peptide and tag capture peptide can also be an improved version of the SpyTag / SpyCatcher system, the SpyTag2 / SpyCatcher2 system (Keeble et al., Angew. Chem. Int. Ed. 2017, 56, 16521-16525), or the SpyTag3 / SpyCatcher3 system (Keeble et al., PNAS,116 (52) 26523-26533, 2019).

[0020] 1 , the uracil DNA glycosylase domain 102 and the protein domain 103 may be directly covalently linked. Direct covalent linkage between the uracil DNA glycosylase domain 102 and the protein domain 103 includes synthesis of a fusion protein of the uracil DNA glycosylase domain 102 and the protein domain 103. When the uracil DNA glycosylase domain 102 and the protein domain 103 are used as a fusion protein, a linker sequence (e.g., a GSG linker sequence) may be disposed between the uracil DNA glycosylase domain 102 and the protein domain 103, or the uracil DNA glycosylase domain 102 and the protein domain 103 may be linked without a linker sequence.

[0021] [Nucleic Acid Molecule] The nucleic acid molecule 101 has a uracil residue in its molecule for covalent bonding with the uracil DNA glycosylase domain 102. The base sequence of the nucleic acid molecule 101 is not particularly limited and can be any base sequence. The nucleic acid molecule 101 can be a nucleic acid aptamer molecule that specifically binds to a predetermined substance or a molecule containing a base sequence complementary to a specific base sequence. When the nucleic acid molecule 101 is a nucleic acid aptamer molecule or a molecule containing a base sequence complementary to a specific base sequence, it can be easily prepared by chemical synthesis once the base sequence is determined.

[0022] A nucleic acid aptamer molecule refers to a molecule that has a G-quadruplex structure and can bind to a specific target substance. Examples of target substances include, but are not limited to, proteins such as growth factors, enzymes, receptors, membrane proteins, and viral proteins. Examples of target substances include cells that display proteins on their surfaces, such as bacteria, microorganisms, cancer cells, iPS cells, and ES cells. As an example, a nucleic acid aptamer molecule can be a nucleic acid molecule composed of a polynucleotide having a specific base sequence and capable of increasing the peroxidase activity of a hemoprotein. Furthermore, the nucleic acid molecule 101 can be configured by linking two nucleic acid aptamer molecules that bind to different target substances.

[0023] Furthermore, the nucleic acid molecule 101 may have one or more nucleotides added to the 5'-end and / or 3'-end of the nucleic acid aptamer molecule. It is generally believed that the reason an aptamer specifically binds to a target substance is because the aptamer exhibits a specific structure (three-dimensional structure or planar structure). Therefore, even if a base sequence that does not exhibit a specific structure is added to one or both ends of the base sequence of the nucleic acid aptamer molecule, the nucleic acid aptamer molecule can maintain its binding activity to the target substance. For example, one or both ends of the nucleic acid aptamer molecule may contain one to three natural bases other than ACGT, such as inosine or methylated cytosine, or highly hydrophobic unnatural bases. Whether a given base sequence exhibits a certain structure can be determined by computer software analysis (for example, RAMAPO COLLEGE's Mapper (Nucleic Acids Research 2006 July; 34 (Web Server issue):W676-W682), available at http: / / bioinformatics.ramapo.edu / QGRS / analyze.php). For example, poly(t) sequences are widely known as base sequences that do not exhibit a specific structure. Therefore, nucleic acid aptamer molecules with poly(t) sequences added to one or both ends can also maintain specific binding activity for target substances.

[0024] The nucleic acid aptamer molecule used as the nucleic acid molecule 101 can be, for example, the aptamer molecule disclosed in Japanese Patent Application Laid-Open No. 2017-200472. This nucleic acid aptamer molecule is a nucleic acid molecule consisting of a polynucleotide having a predetermined base sequence and capable of increasing the peroxidase activity of a hemoprotein. Furthermore, as disclosed in Japanese Patent Application Laid-Open No. 2017-200472, the nucleic acid aptamer molecule capable of increasing the peroxidase activity of a hemoprotein has a guanine quadruplex structure (also referred to as a G4 structure). Generally, aptamers having a G4 structure (also referred to as G4 aptamers) are known to be of parallel type G4 and antiparallel type G4. However, as described in Japanese Patent Application Laid-Open No. 2017-200472, circular dichroism (CD) spectroscopy has confirmed that this nucleic acid aptamer molecule is of parallel type G4. In G4 aptamers, G-quartets formed by four guanines arranged in a plane are arranged in parallel, and it is known that target substances bind by being inserted into these G-quartets. Examples of heme proteins on which this nucleic acid aptamer molecule acts include myoglobin, hemoglobin, metmyoglobin, catalase, and cytochrome P450. Of these, myoglobin and hemoglobin are preferred.

[0025] Furthermore, the nucleic acid aptamer molecule used as the nucleic acid molecule 101 can be, for example, one that has the ability to bind to the receptor binding domain (RBD) of a viral spike protein. A spike protein is a glycoprotein that is present in multiple, almost uniform portions on the surface of a virus and interacts with a cell surface receptor of a host cell. The receptor binding domain (RBD) refers to a region that interacts with a cell surface receptor of a host cell. Examples of viruses that have a spike protein include seasonal influenza viruses, novel influenza viruses, coronaviruses, novel coronaviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, filoviruses, bunyaviruses, arenaviruses, and retroviruses.

[0026] The nucleic acid molecule 101 may also be chemically modified at its end or along the molecular chain. Examples of chemical modifications, which will be described in detail below, include introducing a thiol group into the end for immobilization on a substrate, or biotinylating the end. Furthermore, the nucleic acid molecule 101 may be chemically modified with a fluorescent substance, a drug, or the like.

[0027] Incidentally, the nucleic acid molecule according to the present invention is a DNA having a uracil residue, and therefore can be easily prepared by chemical synthesis using a commercially available DNA synthesizer or the like.

[0028] [Protein Domain] The protein domain 103 is not particularly limited and may be a protein with any amino acid sequence. The protein constituting the protein domain 103 may have a specific affinity for a predetermined substance, may be an enzyme with a specific enzymatic activity, or may be a fusion protein of a protein with a specific affinity for a predetermined substance and an enzyme with a specific enzymatic activity. Examples of proteins with a specific affinity for a predetermined substance include, but are not limited to, antibodies. The antibody may be an antibody against a target substance to which the nucleic acid molecule 101 binds, or an antibody against a growth factor, enzyme, receptor, or membrane protein. The antibody may also be an anti-methylated DNA antibody, or an anti-EGFR antibody, anti-HER2 antibody, or anti-PD-1 antibody, which are molecular targeted therapeutic agents used in cancer treatment.

[0029] When the protein domain 103 is an enzyme having a predetermined enzymatic activity, it is not particularly limited, but examples thereof include enzymes having oxidase activity that catalyze an enzymatic reaction using a predetermined substance as a substrate (first substrate) to generate hydrogen peroxide. Examples of enzymes having oxidase activity include glucose oxidase, aldehyde oxidase, cytochrome oxidase, catechol oxidase, diphenol oxidase, cholesterol oxidase, cytochrome C oxidase, hypoxanthine oxidase, xanthine oxidase, NADPH oxidase, lactate oxidase, galactose oxidase, and alcohol oxidase. Among these, lactate oxidase or glucose oxidase is preferably used as the enzyme having oxidase activity.

[0030] The protein domain 103 may be a protein having a specific affinity for a specific substance, an enzyme having a specific enzymatic activity, or a protein that generates a signal, such as a fluorescent protein. Examples of such proteins include luciferase and circularly permuted fluorescent proteins such as cp luciferase.

[0031] [Biosensing Element] The above-described protein-nucleic acid complex can be used as a biosensing element that has the function of sensing a substance. A biosensing element generally comprises a molecular recognition motif that binds to a substance to be detected and a signal transduction motif that converts the binding signal into a detectable signal. When the above-described protein-nucleic acid complex is used as a biosensing element, one of the nucleic acid molecule 101 and the protein domain 103 serves as the molecular recognition motif, and the other serves as the signal transduction motif. In other words, the protein-nucleic acid complex may be used as a biosensing element that detects a target substance to which the nucleic acid molecule 101 binds, or may be used as a biosensing element that detects a substance to which the protein domain 103 binds.

[0032] When the biosensing element detects a target substance to which the nucleic acid molecule 101 binds, the protein domain 103 can be a signal transduction motif by making the protein domain 103 a fluorescent protein or an enzyme that produces a detectable substance. Conversely, when the biosensing element detects a target substance to which the protein domain 103 binds, the nucleic acid molecule 101 can be a signal transduction motif by using an aptamer molecule that binds to a fluorescently labeled ligand as the nucleic acid molecule 101.

[0033] Furthermore, by using a nucleic acid molecule 101 having a thiol group at its end, the above-described protein-nucleic acid complex can be immobilized on a substrate via the thiol group. Note that by biotinylating the end of the nucleic acid molecule 101, the nucleic acid molecule 101 can also be immobilized on a substrate having avidin. In this case, the protein domain 103 in the protein-nucleic acid complex functions as a molecular recognition motif and a signal transduction motif, and the nucleic acid molecule 101 contributes only to immobilization on the substrate.

[0034] Biosensing Element Example 1 As an example of a biosensing element utilizing the above-described protein-nucleic acid complex, a biosensing element for detecting hemoglobin with high accuracy will be described. An outline of this system is shown in Figure 2. As shown in Figure 2, this system includes the protein-nucleic acid complex shown in Figure 1, a substrate 106 (first substrate 106) of protein domain 103, which is an enzyme with oxidase activity, and a second substrate 108 that serves as a substrate for peroxidase activity in hemoglobin 107 to be detected. Here, protein domain 103 catalyzes an enzymatic reaction using a predetermined substance as a substrate (first substrate 106) by its oxidase activity, generating hydrogen peroxide.

[0035] The first substrate 106 can be appropriately selected and used depending on the type of protein domain 103. The pair of the protein domain 103 and the first substrate 106 is preferably glucose oxidase and glucose or lactate oxidase and lactic acid, because glucose and lactic acid, which are the first substrate 106, are stable compounds and inexpensive.

[0036] 2, the present system also includes a second substrate 108 that serves as a substrate for hemoglobin 107 having peroxidase activity. The second substrate 108 is preferably a substance that can optically detect the progress of the peroxidase reaction caused by hemoglobin 107. Specifically, orthophenylenediamine or luminol can be used as the second substrate 108.

[0037] In the present system configured as described above, hemoglobin 107, which is a target substance, can be detected in a reaction system as shown in FIG. 1 , in which a protein-nucleic acid complex containing a nucleic acid molecule 101 capable of binding to hemoglobin 107 and a protein domain 103 having oxidase activity, a first substrate 106, and a second substrate 108 are present. Specifically, when hemoglobin 107 is present in the reaction system, the nucleic acid molecule 101 in the protein-nucleic acid complex binds to the hemoglobin 107, and the protein domain 103 in the protein-nucleic acid complex comes into close proximity to the hemoglobin 107. In this state, an oxidase reaction using the first substrate 106 proceeds due to the protein domain 103, generating hydrogen peroxide. Because hemoglobin 107 is located in close proximity to the protein domain 103, it can consume hydrogen peroxide generated by the enzymatic reaction of the protein domain 103. This allows a peroxidase reaction using the second substrate 108 by the hemoglobin 107 to proceed.

[0038] Therefore, according to this system, hemoglobin 107 can be detected by measuring the peroxidase reaction of hemoglobin 107 using second substrate 108. For example, when an optically detectable substance such as luminol is used as second substrate 108, hemoglobin 107 can be detected by measuring chemiluminescence generated by a so-called luminol reaction. The means for measuring chemiluminescence is not particularly limited, and may be visual observation or an ultra-weak chemiluminescence measurement method.

[0039] On the other hand, when hemoglobin 107 is not present in a reaction system containing a nucleic acid molecule 101 capable of binding to hemoglobin 107, a protein-nucleic acid complex containing a protein domain 103 having oxidase activity, a first substrate 106, and a second substrate 108, an oxidase reaction using the first substrate 106 by the protein domain 103 proceeds, generating hydrogen peroxide, but a peroxidase reaction using the second substrate 108 by the hemoglobin 107 does not proceed.

[0040] Furthermore, in the above-described method for detecting hemoglobin 107 using the present system, a first reagent containing at least the first substrate 106 and a second reagent containing at least the protein-nucleic acid complex can be used. Components other than the first substrate 106 and the protein-nucleic acid complex (e.g., the second substrate 108) may be contained in either the first reagent or the second reagent, or in both. Separating the first and second reagents can suppress the progress of the oxidase reaction using the first substrate 106 and the protein domain 103 in the protein-nucleic acid complex during storage of the reagents. Furthermore, when the first and second reagents are mixed, the oxidase reaction using the first substrate 106 and the protein domain 103 in the protein-nucleic acid complex can proceed, allowing hemoglobin 107 to be detected as described above.

[0041] 2, the biosensing element may be configured as a fusion protein of an antibody 109 against hemoglobin 107 to be detected and an enzyme having oxidase activity, as shown in Fig. 3. In this case, the antibody 109 and the nucleic acid molecule 101 have different recognition interfaces for hemoglobin 107, and therefore, by cooperating with each other, stronger binding that cannot be achieved by combining antibodies together becomes possible, enabling hemoglobin 107 to be detected with higher sensitivity.

[0042] Biosensing Element Example 2: As another example of a biosensing element utilizing the protein-nucleic acid complex described above, we will explain a biosensing element that detects methylation at a specific position in genomic DNA with high accuracy. A schematic diagram of this system is shown in Figure 4. The protein-nucleic acid complex used in this system comprises a nucleic acid molecule 101 having a base sequence that hybridizes near the position in genomic DNA 110 where methylation (M in Figure 4) is to be detected, and a protein domain 103 consisting of a fusion protein of an anti-methylated DNA antibody 111 and a fluorescent protein 112. Here, the fluorescent protein 112 can be a luciferase that has lost its function through circular permutation (cp luciferase).

[0043] According to the system shown in Figure 4, nucleic acid molecule 101 hybridizes to a predetermined region of genomic DNA 110, and if methylation is present at the predetermined position, anti-methylated DNA antibody 111 binds to the methylated DNA. When this state is reached, the enzymatic activity of cp luciferase is restored, and luciferase luminescence can be observed. Therefore, by using the protein-nucleic acid complex configured as described above, methylation at a predetermined position in genomic DNA 110 can be detected with high sensitivity.

[0044] Biosensing Element Example 3 Another example of a biosensing element utilizing the above-described protein-nucleic acid complex is a biosensing element that detects EGFR, HER2, or PD-1 overexpressed in cancer with high accuracy. In this case, the protein-nucleic acid complex comprises a protein domain 103 containing an anti-EGFR antibody, an anti-HER2 antibody, or an anti-PD-1 antibody, and also comprises a nucleic acid molecule 101 modified with a labeled probe or a nucleic acid molecule 101 bound to a fluorescent protein. Thus, by utilizing the protein-nucleic acid complex, cancer cells that overexpress EGFR, HER2, or PD-1 can be detected with high accuracy.

[0045] In this case, it is preferable that the protein domain 103 is a fusion protein of an antibody such as an anti-EGFR antibody, an anti-HER2 antibody, or an anti-PD-1 antibody with lactate oxidase. By exhibiting lactate oxidase activity, the protein domain 103 can consume lactic acid near cancer cells, thereby inhibiting angiogenesis and suppressing cancer cell proliferation. At the same time, the hydrogen peroxide produced by lactate oxidase can damage cancer cells and promote their death. By utilizing this protein-nucleic acid complex, the complex can function as a so-called theranostic molecule, which can not only detect cancer but also simultaneously treat it.

[0046] In particular, when protein domain 103 is a fusion protein of the above-mentioned antibody and lactate oxidase, lactate oxidase forms a multimer (e.g., a tetramer), which makes it possible to multivalently bind the antibody and to expect high accumulation ability and cancer growth inhibitory effects.

[0047] Biosensing Element Example 4 Another example of a biosensing element utilizing the protein-nucleic acid complex described above is a configuration utilizing a protein-nucleic acid complex having a nucleic acid molecule 101 with a thiol group at its end and a pair of protein domains 103 indirectly bound via a uracil DNA glycosylase domain 102, a tag peptide 104, and a tag capture peptide 105, as shown in FIG. 5 . That is, the protein-nucleic acid complex in this example has two protein domains 103 per molecule. A protein-nucleic acid complex configured in this manner can be immobilized on a substrate 113 via thiol groups. In this case, the protein-nucleic acid complex has two protein domains 103 per molecule, and can form dimers or higher multimers between adjacent protein domains 103. Therefore, a biosensing element utilizing a protein-nucleic acid complex configured in this manner can have the protein domains 103 integrated and arranged on a substrate 113.

[0048] In such a biosensing element, the target substance can be detected by measuring the current value that changes when the target substance binds to the protein domain 103. More specifically, when the target substance is a virus, the protein domain 103 can be a fusion protein of lactate oxidase 115 and an antibody 114 that has the surface protein of the virus as an antigen. With this configuration, when the virus and the antibody 114 are bound to each other, the lactate oxidase 115 produces hydrogen peroxide using lactate, a biomarker for viral infection, as a substrate. By measuring the current generated at this time, the virus contained in the sample can be detected.

[0049] In particular, when protein domain 103 is a fusion protein of the above-mentioned antibody and lactate oxidase, lactate oxidase forms a multimer (e.g., a tetramer), which makes it possible to multivalently characterize the antibody and improve its sensitivity to the substance to be detected.

[0050] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0051] Example 1: Evaluation of DNA-binding proteins In this example, we focused on DNA covalent binding proteins in order to prepare stoichiometric and stable protein-nucleic acid complexes. By searching for and characterizing several DNA covalent binding proteins, we aimed to select a DNA covalent binding protein suitable for protein-nucleic acid complexes. In this example, four DNA covalent binding proteins, TYLCV, RepB, ​​TraI, and UdgX, which differ in binding mode, host, and molecular weight, were selected and characterized.

[0052] First, we obtained fully synthetic genes for TYLCV, RepB, ​​and UdgX based on literature information, and for TraI, we extracted the F plasmid of E. coli to obtain vectors carrying the target genes. Two types of vectors (pET28a and pET30c) were digested with NdeI and HindIII, and two types of target genes (one with a stop codon at the C-terminus and one without) were similarly digested with restriction enzymes. These vectors were then ligated to construct vectors expressing DNA covalently bound proteins with an N-terminus His-tag, DNA covalently bound proteins with a C-terminus His-tag, and DNA covalently bound proteins without a His-tag (Figure 6).

[0053] Next, E. coli LOBSTR was transformed with the constructed vector by heat shock and plated on LB agar medium containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C. After incubation, the transformants were inoculated into 1.5 mL of LB medium containing kanamycin (final concentration 50 μg / mL) and autoinduction reagent. After overnight culture at 28°C and 140 rpm, the cells were harvested and the soluble and insoluble fractions were subjected to SDS-PAGE analysis. The results showed that for TYLCV and RepB, ​​the addition of a His-tag to the C-terminus did not result in a band near the theoretical molecular weight of the DNA-covalently bound protein, suggesting that the addition of a His-tag to the N-terminus is preferable. For TraI, a band near the theoretical molecular weight was observed regardless of whether the His-tag was added to the N- or C-terminus. Since the active center of TraI is located at the N-terminus, the addition of a His-tag to the C-terminus was preferable. On the other hand, for UdgX, the expression level in the soluble fraction was high when a His-tagged C-terminus was added under the specified culture conditions, suggesting that adding a His-tagged C-terminus is suitable.

[0054] Next, to evaluate the DNA-binding ability of the DNA covalent binding proteins, DNA covalent binding proteins (TraI: final concentration 500 nM, TYLCV, RepB, ​​UdgX: final concentration 4000 nM) were mixed with FAM-modified single-stranded DNA containing the recognition sequence of each DNA covalent binding protein in reaction buffer (final concentration 50 mM Tris, final concentration 100 mM NaCl, pH 7.2). The samples were mixed and incubated at 37°C for 1 hour, then immediately mixed with SDS-PAGE Sample Buffer and heated at 95°C for 10 minutes to terminate the reaction. The resulting samples were subjected to SDS-PAGE analysis. After electrophoresis, FAM was detected by fluorescence and stained with Coomassie Brilliant Blue (CBB). All procedures were performed in the dark.

[0055] In SDS-PAGE analysis, a band believed to be the target protein was observed near the theoretical molecular weight in all lanes containing the DNA covalent binding protein (not shown), and a second band was observed at a higher molecular weight in the lane containing the DNA covalent binding protein and ssDNA. Fluorescence observation also revealed that fluorescence from the FAM-modified ssDNA was detected in the higher molecular weight band, suggesting that the DNA covalent binding protein and ssDNA are covalently bound.

[0056] In addition, the same experiment was repeated with the final concentration of all DNA covalent binding proteins set to 4000 nM, and the ratio of DNA covalent binding proteins to ssDNA was varied to calculate the DNA binding efficiency. The results are shown in Figure 7 and Table 1. The DNA binding efficiency was calculated as 100 × [strength of DNA covalent binding proteins bound to ssDNA] / [strength of DNA covalent binding proteins alone].

[0057]

[0058] As shown in Figure 7 and Table 1, UdgX exhibited particularly high DNA binding ability. This is thought to be due to the absence of the reverse reaction characteristic of HUH-endonuclease in uracil-DNA glycosylase. Therefore, this example enabled us to select UdgX as a DNA covalent binding protein suitable for the preparation of DNA-protein complexes.

[0059] Example 2: Preparation and Evaluation of Protein-Nucleic Acid Complexes In this example, we prepared DNA-protein complexes using UdgX and attempted to apply them to sensing elements. Human hemoglobin (Hb) was selected as the target for detection. Hb concentrations in whole blood are considered to be anemia if they are lower than normal, and polycythemia if they are higher. Hb is used as a marker for diseases such as leukemia, heart disease, and dehydration. Hb is also known to have peroxidase-like activity using hydrogen peroxide as a substrate, catalyzing the luminescence reaction of luminol. In this example, we used an anti-Hb aptamer (PEA3-01) that enhances peroxidase activity, as disclosed in JP 2017-200472 A.

[0060] Therefore, in this example, we aimed to detect Hb by using UdgX to prepare a complex between an anti-Hb aptamer and glucose oxidase (GOx). By conjugating the anti-Hb aptamer and GOx, we hypothesized that binding of the anti-Hb aptamer to Hb would bring GOx and Hb into close proximity, allowing the hydrogen peroxide generated by GOx to be efficiently delivered to Hb (Figure 2). We anticipated that the resulting chemiluminescence would enable highly sensitive Hb detection. Furthermore, to prepare the complex, we used the SpyTag / SpyCatcher system, a protein-protein linkage module, to fuse UdgX and GOx. SpyTag and SpyCatcher can be covalently linked to proteins simply by mixing them. This system enabled the covalent conjugation of the anti-Hb aptamer, UdgX, and GOx, suggesting that this system could be used as a stable sensing element for general applications.

[0061] [Design of Nucleic Acid Molecule] In this example, a nucleic acid molecule covalently binding to UdgX was synthesized by adding a UdgX recognition sequence containing a uracil residue to the anti-Hb aptamer (PEA3-01). The base sequence of the synthesized nucleic acid molecule is 5'-GGGCGGGTTGGGCTGGG-TTT-CTCAAGTGUAGGCATGCAAGAGCT-TTT-TCTACATCTCAGCATTCT-3' (SEQ ID NO: 1). In this sequence, the portion from the 5' end to the hyphen is the anti-Hb aptamer (PEA3-01), and the underlined portion is the UdgX recognition sequence. In addition, UdgX binds to the 29th U (uracil residue) from the 5' end of this sequence.

[0062] [Design of UdgX-SpyCatcher and UdgX-SpyTag] Furthermore, in this example, we found that fusing SpyCatcher or SpyTag to the C-terminus of UdgX maintained higher DNA binding ability than fusing SpyCatcher or SpyTag to the N-terminus (data not shown). Therefore, we synthesized proteins in which SpyCatcher or SpyTag was fused to the C-terminus of UdgX. The nucleotide sequence (SEQ ID NO: 2) encoding a fusion protein in which SpyTag was fused to the C-terminus of UdgX (UdgX-SpyTag) and the amino acid sequence (SEQ ID NO: 3) of the fusion protein are shown in Figure 8, and the nucleotide sequence (SEQ ID NO: 4) encoding a fusion protein in which SpyCatcher was fused to the C-terminus of UdgX (UdgX-SpyCatcher) and the amino acid sequence (SEQ ID NO: 5) of the fusion protein are shown in Figure 9. In Figures 8 and 9, the amino acid sequence of UdgX is underlined, and the amino acid sequences of SpyCatcher and SpyTag are boxed.

[0063] Synthesis of UdgX-SpyCatcher and UdgX-SpyTag E. coli LOBSTR was transformed with the UdgX-SpyTag and UdgX-SpyCatcher expression vectors and plated on LB agar medium containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C. The resulting transformants were inoculated into 100 mL of LB medium containing kanamycin (final concentration 50 μg / mL) and autoinduction reagent. FeCl3 (final concentration 0.01%) and citric acid (FeCl3:citric acid molar ratio 1:2) were also added to the LB medium. After overnight culture at 28°C and 140 rpm, the cells were harvested, and the soluble and insoluble fractions were analyzed by SDS-PAGE. As a result, highly purified UdgX-SpyTag and UdgX-SpyCatcher were isolated from the soluble fraction, with yields calculated to be 28.2 mg protein / L culture for UdgX-SpyTag and 52.5 mg protein / L culture for UdgX-SpyCatcher, respectively.

[0064] [Evaluation of DNA binding ability of UdgX-SpyCatcher and UdgX-SpyTag] The obtained UdgX-SpyTag and UdgX-SpyCatcher were mixed with the UdgX recognition sequence (5'-CTCAAGTGUAGGCATGCAAGAGCT-3', with a uracil residue at the 9th position from the 5' end (SEQ ID NO: 6)) in reaction buffer (final concentration: 50 mM Tris, 100 mM NaCl, pH 7.2) and incubated at 37°C for 1 hour. The reaction was stopped by mixing with SDS-PAGE sample buffer, vortexing, and heating at 95°C for 10 minutes. The resulting sample was loaded onto an SDS-PAGE gel at a total protein amount of 500 ng per lane, and SDS-PAGE analysis was performed. Polyacrylamide gels were electrophoresed at 20 mA for 90 minutes, and then FAM fluorescence was detected. After staining with Coomassie Brilliant Blue (CBB), the gels were imaged. Band quantification was performed using ImageJ on the CBB-stained electrophoretic images. All procedures were performed in the dark. The results indicated that the UdgX-SpyTag and UdgX-SpyCatcher conjugates retained binding activity comparable to that of UdgX fused with only a His-tag (data not shown).

[0065] [Preparation of Protein-Nucleic Acid Complex] A fusion protein (referred to as GOx-SpyCatcher) was prepared, consisting of glucose oxidase (GOx) and SpyCatcher in that order from the N-terminus. The amino acid sequence of GOx-SpyCatcher is shown in SEQ ID NO: 7.

[0066] First, to prepare the UdgX-GOx complex, a SpyTag / SpyCatcher reaction was performed. Five μL of 4 μM UdgX-SpyTag and 2 μM GOx-SpyCatcher diluted in 20 mM potassium phosphate buffer (PPB, pH 6.0) were mixed and incubated at 4°C for 10 min, 30 min, 1 h, 2 h, and 24 h. GOx-SpyCatcher was recombinantly produced in E. coli, refolded from the insoluble fraction, and purified by His-tag purification. Finally, the resulting samples were analyzed by SDS-PAGE.

[0067] Next, protein-nucleic acid complexes were prepared. Equal volumes of 20 μM UdgX-SpyTag and 10 μM GOx-SpyCatcher diluted in 20 mM PPB (pH 6.0) were mixed and incubated at 4°C for 24 hours. To prepare the aptamer-GOx complex, a sequence combining the UdgX recognition sequence with PEA3-01 and the fluorescent dye FAM was folded (95°C for 10 min, then 95°C to 25°C over 30 min) and then used in a DNA binding reaction. 5 μM UdgX-GOx complex and 10 μM PEA3-01 containing the UdgX recognition sequence were mixed in a reaction buffer (10 mM sodium phosphate buffer (pH 6.0) containing 10 mM KCl) and incubated overnight at 4°C. The resulting sample was analyzed by SDS-PAGE.

[0068] Although electrophoresis diagrams from these SDS-PAGE analyses are not shown, bands were confirmed near the theoretical molecular weight of the UdgX-GOx complex, confirming the formation of a UdgX-GOx complex by the SpyTag / SpyCatcher system, which is a linking module. Furthermore, although electrophoresis diagrams are not shown, the binding reaction of the prepared UdgX-GOx with an aptamer was evaluated by SDS-PAGE analysis. A band was confirmed near the theoretical molecular weight of the complex formed by binding a nucleic acid molecule containing PEA3-01 to the UdgX-GOx complex, suggesting that a protein-nucleic acid complex (hereafter referred to as the aptamer-GOx complex) was successfully prepared.

[0069] [Evaluation of DNA binding ability of aptamer-GOx complexes] 100 μL of 100 nM Hb was added to a 96-well plate and incubated for 1 hour to allow Hb to adsorb and immobilize on the plate. After washing with wash buffer (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 8.0), the plate was blocked by adding 2% (w / v) skimmed milk and incubating at room temperature for 1 hour. After washing with wash buffer, 100 μL of aptamer-GOx complex was added and incubated at room temperature for 1 hour. After washing three times with wash buffer, the fluorescence of FAM derived from the FAM-modified aptamer was measured using a plate reader.

[0070] The fluorescence measurement results are shown in Figure 10. As shown in Figure 10, a concentration-dependent increase in fluorescence intensity was observed for PEA3-01 bound to the UdgX-GOx complex, suggesting that PEA3-01 bound to UdgX-GOx has the ability to bind to Hb. Furthermore, the Kd values ​​were calculated to be 38 nM for the aptamer alone, 45 nM for the aptamer-UdgX complex, and 34 nM for the aptamer-GOx complex. The slightly improved binding ability of the aptamer-GOx complex is thought to be due to the fact that GOx forms a dimer, resulting in two aptamers binding to Hb per molecule, which slows the dissociation rate compared to when there is a single binding site per molecule.

[0071] [Enzyme activity evaluation of aptamer-GOx complex] The aptamer-GOx complex was mixed with a reaction solution (final concentration: 2 U / mL HRP, final concentration: 1.5 mM TOOS, final concentration: 1.5 mM 4AA, final concentration: 10 mM PPB (pH 7.0)) and reacted with various concentrations of glucose to evaluate the oxidase activity of GOx. Similar evaluations were also performed for the GOx-SpyCatcher and UdgX-GOx complexes.

[0072] The results of the oxidase activity evaluation are shown in Figure 11. As shown in Figure 11, it was suggested that the activity of GOx in the aptamer-GOx complex was maintained compared to GOx fused with SpyCatcher at the C-terminus.

[0073] [Detection of Hb using aptamer-GOx complex] To examine the 96-well plate to be used for Hb detection, Nunc TM The following experiments were performed using 96-well Polypropylene Storage Microplates (ThermoFisher Scientific) and low-binding surface (LBS) OptiPlate, 96-well (white) (PerkinElmer), a polystyrene plate with low protein binding.

[0074] Aptamer-GOx conjugates were prepared in the same manner using an aptamer bearing the recognition sequence of UdgX without FAM modification. The aptamer-GOx conjugates were diluted with 10 mM sodium phosphate buffer (pH 6.0) containing 10 mM KCl to a final concentration of 50 nM. Hb was diluted with the same buffer to a final concentration of 12.5 nM. A total volume of 20 μL was mixed in a 96-well plate. After 1 hour of incubation at room temperature, 80 μL of reaction solution (final concentrations of 100 mM glucose, 100 μM luminol, and 0.1 nM catalase, pH 8.0) was added, and the resulting chemiluminescence was measured using a plate reader.

[0075] Hb-dependent chemiluminescence was observed using a low-protein-binding 96-well polystyrene plate. Compared to the control, in which the aptamer and GOx were mixed, the aptamer-GOx complex using UdgX exhibited higher chemiluminescence intensity (Figure 12, right panel). The difference in chemiluminescence intensity between the two was greater than that observed when using a polypropylene 96-well plate (Figure 12, left panel). This is thought to be due to the reduced chemiluminescence generated by the adsorption of Hb and GOx onto the plate, making it easier to observe the proximity effect between Hb and GOx due to the aptamer. These results suggest that the use of low-protein-binding polystyrene plates is preferable when detecting Hb with the aptamer-GOx complex.

[0076] [Detection of Hb in human serum using aptamer-GOx conjugate] The following experiment was carried out using a polystyrene low-protein binding plate, Low-binding surface (LBS) OptiPlate, 96-well (white) (PerkinElmer). The aptamer-GOx conjugate was prepared in the same manner as above using an aptamer with a recognition sequence for UdgX that was not modified with FAM. The aptamer-GOx conjugate was diluted with 10 mM sodium phosphate buffer (pH 6.0) containing 10 mM KCl to a final concentration of 50 nM. The aptamer-GOx conjugate was then mixed with human serum (1.0x10 4 Hb at various concentrations diluted in a solvent (2x dilution) was mixed in a 96-well plate to a total volume of 20 μL and incubated at room temperature for 1 hour. After adding 80 μL of reaction solution (final concentration 100 mM glucose, final concentration 100 μM luminol, pH 8.0), the resulting chemiluminescence was measured using a plate reader.

[0077] The results of detecting Hb in serum using a polystyrene 96-well plate with low protein binding are shown in Figure 13. Similar to the control (left panel of Figure 13) in which Hb diluted with 10 mM PPB (pH 6.0) was detected, a concentration-dependent increase in chemiluminescence was observed, suggesting that Hb in serum can also be detected. Furthermore, linearity of the chemiluminescence intensity was confirmed in the Hb concentration range of 62.5 to 500 nM. The clinically required range for detecting Hb in blood is 1.1 to 3.1 mM. Therefore, with this detection system, blood was diluted to 1.0 x 10 4 It was suggested that by diluting the sample 2-fold, it is possible to meet the clinically required range of Hb detection in blood.

[0078] Example 3: Example of preparation of protein-nucleic acid complex In this example, several protein-nucleic acid complexes different from those in the above-mentioned examples were prepared, and the versatility of the method for preparing complexes using UdgX was confirmed.

[0079] In this example, glucose dehydrogenase (GDH) was used as the protein domain in the complex, and the thrombin-binding aptamer TBA15 (5'-GGTTGGTGTGGTTGG-3': SEQ ID NO: 8) was used as the nucleic acid. Specifically, we attempted to create an aptamer TBA15-GDH complex. First, we prepared a fusion protein (referred to as GDH-SpyCatcher) consisting of glucose dehydrogenase and SpyCatcher in that order from the N-terminus. The amino acid sequence of GDH-SpyCatcher is shown in SEQ ID NO: 9.

[0080] Next, a SpyTag / SpyCatcher reaction was performed to prepare the UdgX-GDH complex. 2.5 μL each of 20 μM UdgX-SpyTag (Example 2, SEQ ID NO: 2) and 20 μM GDH-SpyCatcher, both diluted in 10 mM sodium phosphate buffer (pH 6.0) containing 10 mM KCl, was mixed and incubated at 4°C for 24 hours. The GDH-SpyCatcher used here was produced using recombinant Escherichia coli and prepared by His-tag purification from the soluble intracellular fraction. The resulting GDH-SpyCatcher was confirmed by SDS-PAGE analysis.

[0081] Finally, the TBA15-GDH complex was prepared as follows: 20 μM UdgX-SpyTag and 20 μM GDH-SpyCatcher were diluted in 10 mM sodium phosphate buffer (pH 6.0) containing 10 mM KCl and mixed in equal volumes and incubated at 4°C for 24 hours. To prepare the TBA15-GDH complex, a DNA binding reaction was performed using a sequence that combined the UdgX recognition sequence with TBA15 and the fluorescent dye FAM. 10 μM UdgX-GDH complex and 10 μM TBA15 containing the UdgX recognition sequence were mixed in a reaction buffer (10 mM sodium phosphate buffer (pH 6.0) containing 10 mM KCl) and incubated overnight at 4°C. The resulting sample was subjected to SDS-PAGE analysis. Proteins were identified by Coomassie Brilliant Blue (CBB) staining, and nucleic acids were identified by FAM fluorescence.

[0082] In the above example, the results of SDS-PAGE analysis of the SpyTag / SpyCatcher reaction using UdgX-SpyTag and GDH-SpyCatcher are shown in Figure 14. As shown in Figure 14, a band was observed near the theoretical molecular weight of the UdgX-GDH complex (100.5 kDa), confirming the formation of a UdgX-GDH complex using the SpyTag / SpyCatcher system, which is a linking module. Furthermore, the results of SDS-PAGE analysis and fluorescence detection when the prepared UdgX-GDH complex was reacted with the TBA15 aptamer are shown in Figure 15. As shown in Figure 15, a band was observed near the theoretical molecular weight (114.1 kDa) of the complex in which the TBA15 aptamer bound to the UdgX-GDH complex, confirming the formation of a protein-nucleic acid complex using UdgX.

[0083] On the other hand, in this example, as another example of preparing a protein-nucleic acid complex using UdgX, a protein-nucleic acid complex was prepared using the UdgX-GOx complex prepared in Example 2 and the above-mentioned TBA15 aptamer.

[0084] Figure 16 shows the results of SDS-PAGE analysis of the SpyTag / SpyCatcher reaction using UdgX-SpyTag and GOx-SpyCatcher. As shown in Figure 16, a band was observed near the theoretical molecular weight of the UdgX-GOx complex (102.2 kDa), confirming the formation of a UdgX-GOx complex using the SpyTag / SpyCatcher system (linking module). Figure 17 also shows the results of SDS-PAGE analysis and fluorescence detection of the reaction of the prepared UdgX-GOx complex with the TBA15 aptamer. As shown in Figure 17, a band was observed near the theoretical molecular weight of the UdgX-GOx complex bound to the TBA15 aptamer (115.8 kDa), confirming the formation of a protein-nucleic acid complex using UdgX.

[0085] Furthermore, in this example, as another example of preparing a protein-nucleic acid complex using UdgX, a protein-nucleic acid complex was prepared using the UdgX-GDH complex prepared in this example and the PEA3-01 aptamer prepared in Example 2.

[0086] Figure 18 shows the results of SDS-PAGE analysis of the SpyTag / SpyCatcher reaction using UdgX-SpyTag and GDH-SpyCatcher. As shown in Figure 18, a band was observed near the theoretical molecular weight of the UdgX-GDH complex (100.5 kDa), confirming the formation of a UdgX-GDH complex using the SpyTag / SpyCatcher system, which is a linking module. Figure 19 also shows the results of SDS-PAGE analysis and fluorescence detection when the prepared UdgX-GDH complex was reacted with the PEA3-01 aptamer. As shown in Figure 19, a band was observed near the theoretical molecular weight of the complex in which the PEA3-01 aptamer bound to the UdgX-GDH complex (120.6 kDa), confirming the formation of a protein-nucleic acid complex using UdgX.

[0087] As described above, this example demonstrated that three different types of protein-nucleic acid complexes can be formed using UdgX, demonstrating the versatility of the method for preparing protein-nucleic acid complexes using UdgX.

[0088] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A protein-nucleic acid complex comprising a nucleic acid molecule having DNA containing uracil, a uracil DNA glycosylase domain covalently bound to the nucleic acid molecule, and a protein domain indirectly covalently bound to the uracil DNA glycosylase domain, wherein the uracil DNA glycosylase domain and the protein domain are indirectly covalently bound via a tag peptide and a tag capture peptide.

2. The protein-nucleic acid complex according to claim 1, wherein the uracil DNA glycosylase domain is a fusion protein with the tag peptide or the tag capture peptide, and the protein domain is a fusion protein with the tag capture peptide or the tag peptide, whereby the uracil DNA glycosylase domain and the protein domain are indirectly covalently bound via the tag peptide and the tag capture polypeptide.

3. The protein-nucleic acid complex according to claim 1, wherein the nucleic acid molecule is a nucleic acid aptamer molecule that specifically binds to a predetermined substance or a molecule containing a base sequence complementary to a specific base sequence.

4. The protein-nucleic acid complex according to claim 1, wherein the nucleic acid molecule is a chemically modified nucleic acid molecule.

5. A biosensing element comprising the protein-nucleic acid complex according to any one of claims 1 to 4.

6. The biosensing element according to claim 5, wherein a substance specifically bound by the nucleic acid molecule contained in the protein-nucleic acid complex is detected.

7. The biosensing element according to claim 5, wherein a substance specifically bound by the protein domain contained in the protein-nucleic acid complex is detected.

8. The nucleic acid molecule contained in the protein-nucleic acid complex is an aptamer molecule that increases the peroxidase activity of the hemoprotein, and the protein domain contained in the protein-nucleic acid complex is an enzyme having oxidase activity that catalyzes an oxidation reaction to generate hydrogen peroxide. The biosensing element according to claim 5, characterized in that.

9. The biosensing element according to claim 5, characterized in that the protein-nucleic acid complex is immobilized on a substrate through chemical modification of the nucleic acid molecule contained in the protein-nucleic acid complex.

10. A protein-nucleic acid complex in which the protein domain in the protein-nucleic acid complex according to any one of claims 1 to 4 is an enzyme having oxidase activity that catalyzes an oxidation reaction to generate hydrogen peroxide, A first substrate that is a substrate for the enzyme, A second substrate that is a substance to be detected that binds to the nucleic acid molecule in the protein-nucleic acid complex and is a substrate for a substance having peroxidase activity, A detection kit for substances comprising.

11. The detection kit according to claim 10, characterized in that the enzyme and the first substrate are glucose oxidase and glucose, respectively, and the substance to be detected and the second substrate are hemoglobin and luminol, respectively.

12. The detection kit according to claim 10, characterized in that the enzyme is a fusion protein with an antibody against the substance to be detected.

13. A step of mixing the detection kit according to claim 10 and a sample that may contain a substance to be detected, A step of measuring an enzyme reaction that consumes the second substrate contained in the detection kit, and A method for detecting a substance, characterized in that the substance to be detected in the sample is detected based on the enzyme reaction.

14. The uracil DNA glycosylase domain is fused with a tag peptide or a tag capture peptide, A fusion protein used for indirectly covalently linking a nucleic acid molecule having DNA containing uracil and a protein domain covalently bonded to a tag capture peptide or a tag peptide.