RNA probe for protein detection

Abstract

An RNA probe for protein detection according to the present invention includes: a single-stranded oligonucleotide that includes ribonucleotides as constituent units; a fluorescent molecule; and a quenching molecule that absorbs fluorescence emitted by the fluorescent molecule. The single-stranded oligonucleotide includes a loop region, a 3' stem region that is bonded to the 3' end of the loop region, and a 5' stem region that is bonded to the 5' end of the loop region. The fluorescent molecule is bonded to one of the 3' stem region and the 5' stem region, and the quenching molecule is bonded to the other. The loop region is a 3–9-mer, the 3' stem region is a 2–10-mer, and the 5' stem region is a 2–10-mer. The 3' stem region and the 5' stem region have complementary base sequences. The cytosine content fraction of the loop region is at least 0% but less than 35% relative to the number of bases in the loop region.

Description

RNA probes for detecting proteins

[0001] The present invention relates to RNA probes for the detection of proteins.

[0002] BACKGROUND ART From the viewpoint of early detection of infectious diseases and prevention of their spread, methods for detecting pathogens (e.g., viruses, bacteria, etc.) that cause infectious diseases have been developed.

[0003] Known methods for detecting pathogens include, for example, methods for detecting proteins derived from pathogens and methods for detecting nucleic acids derived from pathogens. Known methods for detecting proteins derived from pathogens include immunological detection methods such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), and immunofluorescence assay (IFA) (Patent Document 1). Known methods for detecting nucleic acids derived from pathogens include, for example, RNA detection methods using RT-PCR and nucleic acid detection methods using nucleic acid probes (Patent Document 2).

[0004] JP 2018-506299 A International Publication No. 96 / 015270

[0005] Conventionally used immunological detection methods have had problems such as requiring a long time to detect pathogens and reducing specificity and sensitivity due to non-specific reactions. Furthermore, methods for detecting nucleic acids derived from pathogens require pretreatment such as lysis of the pathogens because the nucleic acids to be detected are usually present inside the pathogens. Therefore, even in methods for detecting such nucleic acids, there is a problem in that it takes a long time to detect the pathogens. Against this background, there is a demand for the development of methods for detecting proteins, nucleic acids, etc. with high sensitivity.

[0006] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an RNA detection probe for detecting proteins with high sensitivity, and a method for detecting proteins using the RNA detection probe.

[0007] As a result of extensive research, the present inventors discovered that target proteins can be detected with high sensitivity by using RNA probes whose degradation efficiency varies greatly depending on the concentration (density) of RNase, and thus completed the present invention.

[0008] a 3'-mer length of the loop region; a 5'-mer length of the 5'-mer length of the loop region; a 3'-mer length of the loop region; a 2'-mer length of the 3'-mer length of the 5'-mer length of the loop region; a ...

[0009] A second aspect of the present invention relates to a kit for detecting a protein, the kit comprising: an RNA probe for detecting the protein according to the first aspect; and RNase.

[0010] A third aspect of the present invention relates to a method for detecting a protein, comprising: an RNase immobilization step of immobilizing RNase on the surface of an object to be tested; an RNA probe application step of applying an RNA probe for detecting the protein according to the first aspect to the surface of the object to be tested; and a fluorescence observation step of observing the fluorescence intensity on the surface of the object to be tested, wherein the RNase is configured to be localized to the protein.

[0011] A fourth aspect of the present invention relates to a protein detection system, comprising an RNase immobilizer, an RNA probe adder, and a detection unit, wherein the RNase immobilizer comprises RNase, the RNA probe adder comprises an RNA probe for detecting the protein according to the first aspect, and the detection unit detects fluorescence emitted from fluorescent molecules derived from the RNA probe for detection.

[0012] According to the present invention, it is possible to provide a detection RNA probe for detecting a protein with high sensitivity, and a method for detecting a protein using the detection RNA probe.

[0013] FIG. 1 is a schematic diagram showing an example of the configuration of a protein detection RNA probe according to the present embodiment. In FIG. 1, circles represent ribonucleotides, diamonds represent quenching molecules, and pentagons represent fluorescent molecules. FIG. 2 is a schematic diagram illustrating an overview of a protein detection system according to the present embodiment. FIG. 3 is a schematic diagram illustrating the configuration of a protein detection system and detection device according to the present embodiment. FIG. 4 is a graph showing the correlation between the elapsed time (horizontal axis) after reaction of a detection RNA probe according to the present embodiment with RNase A (2 ng / ml or 10 μg / ml) and the fluorescence intensity (vertical axis) derived from the detection RNA probe. FIG. 5 is a graph showing the correlation between the cytosine content (horizontal axis) in the loop region of each detection RNA probe and the fluorescence intensity (vertical axis) derived from the detection RNA probe when reacted with RNase A (2 ng / ml). FIG. 6 is a graph showing the correlation between the RNase A concentration (horizontal axis) and the fluorescence intensity (vertical axis) derived from the detection RNA probe when reacted with the detection RNA probe according to the present embodiment. FIG. 7 is a schematic diagram showing the steps of the protein detection method according to the present embodiment. FIG. 8 is a fluorescence microscope image of quartz glass on which SARS-CoV-2 has been immobilized, after the protein detection method according to the present embodiment has been performed. FIG. 9 is a graph showing the correlation between the uracil content (horizontal axis) in the loop region of each detection RNA probe when reacted with RNase A (2 ng / ml) and the fluorescence intensity (vertical axis) derived from the detection RNA probe. FIG. 10 is a graph showing the correlation between the adenine and uracil content (horizontal axis) in the loop region of each detection RNA probe when reacted with RNase A (2 ng / ml) and the fluorescence intensity (vertical axis) derived from the detection RNA probe. 11 is a graph showing the correlation between the number of bases in the stem region (stem length) (horizontal axis) of each RNA detection probe and the fluorescence intensity (vertical axis) derived from the RNA detection probe when reacted with RNase A (2 ng / ml). The fluorescence intensity is relative to that of SEQ ID NO: 7 (stem length: 5 mer).FIG. 12 is a graph showing the correlation between the elapsed time (horizontal axis) after reacting the detection RNA probe according to this embodiment with RNase A (2 ng / ml or 10 μg / ml) and the fluorescence intensity (vertical axis) derived from the detection RNA probe.

[0014] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0015] <RNA Probe for Protein Detection> A first aspect of this embodiment is an RNA probe for protein detection, wherein the detection RNA probe comprises a single-stranded oligonucleotide having ribonucleotides as building blocks, a fluorescent molecule, and a quencher molecule that absorbs fluorescence emitted by the fluorescent molecule, wherein the single-stranded oligonucleotide comprises a loop region, a 3' stem region bound to the 3' end of the loop region, and a 5' stem region bound to the 5' end of the loop region, wherein the fluorescent molecule binds to one of the 3' stem region and the 5' stem region, and the quencher molecule binds to the other, wherein the number of bases in the loop region is 3 to 9 mer, wherein the number of bases in the 3' stem region is 2 to 10 mer, and wherein the number of bases in the 5' stem region is 2 to 10 mer, wherein the 3' stem region and the 5' stem region have complementary base sequences, and wherein the cytosine content in the loop region is 0% or more and less than 35% based on the number of bases in the loop region.

[0016] <Single-stranded oligonucleotide> The single-stranded oligonucleotide according to this embodiment has ribonucleotides as its constituent units. Here, "oligonucleotide" refers to a nucleotide polymer in which 7 to 31 identical or different nucleotides are linked together via phosphodiester bonds. Examples of nucleic acid bases contained in the ribonucleotide include adenine (A), guanine (G), cytosine (C), and uracil (U). The single-stranded oligonucleotide according to this embodiment usually takes the form of a single strand.

[0017] The single-stranded oligonucleotide comprises a loop region, a 3' stem region linked to the 3' end of the loop region, and a 5' stem region linked to the 5' end of the loop region (top panel of Figure 1).

[0018] The 3' stem region and the 5' stem region have complementary base sequences. Therefore, the single-stranded oligonucleotide forms a double helix structure in the 3' stem region and the 5' stem region, forming a so-called hairpin loop structure (also referred to as a hairpin structure or a stem-loop structure) (middle section of Figure 1). In one aspect of this embodiment, the base sequence of the loop region is preferably not complementary to the base sequences of the 3' stem region and the 5' stem region. Whether the single-stranded oligonucleotide forms a hairpin loop structure can be determined, for example, by calculating a generalized centroid estimator using CentroidFold or the like.

[0019] The number of bases in the loop region is 3 to 9 mer, preferably 3 to 6 mer, and more preferably 3 to 5 mer.

[0020] The number of bases in the 3' stem region is 2 to 10 mer, preferably 2 to 7 mer, and more preferably 2 to 5 mer.

[0021] The number of bases in the 5' stem region is 2 to 10 mer, preferably 2 to 7 mer, and more preferably 2 to 5 mer.

[0022] In one aspect of this embodiment, the single-stranded oligonucleotide may further comprise a ribonucleotide bound to the 5'-end of the 5' stem region (upper row of Figure 1). The number of ribonucleotide bases bound to the 5'-end of the 5' stem region may be one or several, one or two, or even one. The single-stranded oligonucleotide may further comprise a ribonucleotide bound to the 3'-end of the 3' stem region. The number of ribonucleotide bases bound to the 3'-end of the 3' stem region may be one or several, one or two, or even one.

[0023] In one aspect of this embodiment, the total number of bases in the single-stranded oligonucleotide is preferably 7 to 31 mer, more preferably 7 to 23 mer, more preferably 7 to 19 mer, and even more preferably 7 to 15 mer.

[0024] The cytosine content in the loop region is 0% or more but less than 35%, preferably 0% or more but 20%, and more preferably 0% or more but 15%, based on the number of bases in the loop region. If the cytosine content in the loop region is within the above range, the detection RNA probe can be degraded only when RNase accumulates in the protein to be detected (lower panel of Figure 1).

[0025] In one aspect of this embodiment, the uracil content in the loop region is preferably 30% or more and 100% or less, and more preferably 33% or more and 100% or less, based on the number of bases in the loop region.

[0026] In another aspect of this embodiment, the total content of adenine and uracil in the loop region is preferably 65% ​​or more and 100% or less, and more preferably 67% or more and 100% or less, based on the number of bases in the loop region.

[0027] In one aspect of this embodiment, the base sequence of the single-stranded oligonucleotide is preferably: (A) a base sequence having 90% to 100% sequence identity with the base sequence set forth in any one of SEQ ID NOs: 1 to 7, 10, and 21 to 24; (B) a base sequence in which one or several bases have been deleted, substituted, inserted, or added with respect to the base sequence set forth in any one of SEQ ID NOs: 1 to 7, 10, and 21 to 24; or (C) a base sequence that hybridizes under stringent conditions to an oligonucleotide having a base sequence complementary to the base sequence set forth in any one of SEQ ID NOs: 1 to 7, 10, and 21 to 24.

[0028] In this embodiment, "sequence identity" refers to the percentage (%) of identical bases relative to the total overlapping base sequence in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art. The algorithm preferably takes into account the introduction of gaps into one or both of the sequences for optimal alignment. The "sequence identity" of a base sequence can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.

[0029] The base sequence of the single-stranded oligonucleotide may have 95% to 100% sequence identity, 97% to 100%, or 100% sequence identity to the base sequence set forth in any one of SEQ ID NOs: 1 to 7, 10, and 21 to 24.

[0030] In this embodiment, examples of "a base sequence in which one or several bases have been deleted, substituted, inserted or added" include a base sequence that, due to the deletion, substitution, insertion or addition, has a sequence identity of 90% to 99%, 95% to 98%, or 96% to 97% to the base sequence before the deletion, substitution, insertion or addition. The specific number of "one or several bases" may be one, two, three, four, or five of the above-mentioned deletions, substitutions, insertions or additions, each independently, or a combination of multiple deletions, substitutions, insertions or additions.

[0031] In this embodiment, "stringent conditions" refers to conditions in which the sample is incubated for 12 hours at room temperature in a solution containing 6xSSC (1xSSC has the composition: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, and 50% (v / v) formamide, followed by washing with 0.5xSSC at a temperature of 50°C or higher. More stringent conditions are also encompassed, such as incubation for 12 hours at 45°C or 60°C, washing with 0.2xSSC or 0.1xSSC, or washing at a temperature of 60°C or 65°C or higher.

[0032] <Fluorescent Molecule> The fluorescent molecule according to this embodiment is bound to either the 3' stem region or the 5' stem region (upper part of FIG. 1).

[0033] <h2 style=";text-align:left;direction:ltr">6-Carboxyfluorescein(6-FAM)(494nm, 517nm), Alexa Fluor 555(555nm,565nm), HiLyte Plus 555(552nm,567nm), DyLight 549(550nm,568nm), HiLyte Fluor 555(553nm,568nm), Cy3(550nm,570nm), DyLight 547(557nm,570nm), Rhodamine(550nm,570nm), TRITC(550nm,570nm), DY-548(558nm,572nm), DY-554(551nm,572nm), DY-555(547nm,572nm), Alexa Fluor 546(556nm,573nm), DY-556(548nm,573nm), NorthernLights 557(557nm,574nm), Oyster 550(555nm,574nm), 5-TAMRA(547nm,574nm), DY-547(557nm,574nm), Oyster 556(562nm,575nm), DY-549(560nm,575nm), ATTO 550(554nm,576nm), B-PE(545nm,578nm), R-PE(566nm,578nm), DY-560(559nm,578nm), TAMRA(555nm,580nm), MFP555(560nm,585nm), Spectrum Orange(559nm,588nm), ATTO 565(563nm,592nm), Cy3.5(581nm,596nm), ROX(X-Rhodamine,Rhodamine Red X)(587nm,599nm), DY-590(580nm,599nm), 5-ROX(573nm,602nm), Spectrum Red(587nm,612nm), Texas Red(596nm,615nm), DyLight 594(593nm,618nm), Alexa Fluor 594(590nm,619nm), HiLyte Fluor TR(591nm,622nm), ATTO 590(594nm,624nm), MFP590(597nm,624nm),<h2 style=";text-align:left;direction:ltr">DY-610(610nm,630nm), ATTO 610(615nm,634nm), DY-615(621nm,641nm), C-PC(C-Phycocyanin)(616nm,647nm), ATTO620(619nm,643nm), Phycocyanin(620nm,650nm), ATTO 633(629nm,657nm), DY-630(636nm,657nm), DY-632(637nm,657nm), DY-633(637nm,657nm), MFP631(633nm,658nm), DyLight633(638nm,658nm), NorthernLights 637(637nm,658nm), DY-631(637nm,658nm), DY-634(635nm,658nm), APC(Allophycocyanin)(650nm,660nm), APC-XL(650nm,662nm), Alexa Fluor 647(650nm,665nm), Cy5(643nm,667nm) 645(650nm,669nm), DY-635(647nm,671nm), DY-636(645nm,671nm), DY-647(653nm,672nm), DyLight 647(652nm,673nm), HiLyte Fluor 647(653nm,673nm), DyLight 649(646nm,674nm), HiLyte Plus 647(649nm,674nm), Oyster 650(655nm,674nm), DY-648(653nm,674nm), DY-650(653nm,674nm), DY-652(654nm,675nm), DY-649(655nm,676nm), DY-651(656nm,678nm), Oyster 656(662nm,679nm), ATTO 655(663nm,684nm), Cy5.5(675nm,694nm), DY-677(673nm,694nm), DY-678(674nm,698nm), HiLyte Fluor 680(678nm,699nm), DY-675(674nm,699nm), DY-676(674nm,699nm), IRDye700DX(689nm,700nm),DY-681(691nm,708nm), DY-680(690nm,709nm), DY-682(690nm,709nm),DyLight 680(682nm,715nm), Alexa Fluor 700(702nm,723nm), DY-700(707nm,730nm), DY-701(706nm,731nm), PREX710(710nm,740nm), DY-730(732nm,758nm), DY-732(736nm,759nm), DY-734(736nm,759nm), DY-731(736nm,760nm), DY-752(748nm,772nm), DY-750(747nm,776nm), DyLight 750(752nm,778nm), HiLyte Fluor 750(754nm,778nm), DY-749(752nm,778nm), HiLyte Plus 750(751nm,779nm), DY-751(751nm,779nm), DyLight 800(770nm,794nm), IRDye800CW(774nm,800nm), DY-780(782nm,800nm), DY-781(783nm,800nm), DY-782(784nm,800nm), Examples include DY-776 (771nm, 801nm), DY-777 (771nm, 801nm), and IRDye800 (778nm, 806nm). In addition, in the parentheses of the above fluorescent molecules, the first number indicates the peak wavelength of the excitation spectrum, and the second number indicates the peak wavelength of the fluorescence spectrum.

[0034] <Quencher molecule> The quencher molecule according to this embodiment absorbs the fluorescence emitted by the fluorescent molecule. The quencher molecule is bound to one of the 3' stem region and the 5' stem region, to which the fluorescent molecule is not bound (upper part of Figure 1). That is, the fluorescent molecule is bound to either the 3' stem region or the 5' stem region, and the quencher molecule is bound to the other.

[0035] Examples of such quenching molecules include the Black Hole™ quenchers BHQ1, BHQ2, and BHQ3 (Biosearch Technologies, Inc.) and the ATTO series of quenchers (ATTO540Q, ATTO580Q, and ATTO612Q; Atto-Tec GmbH).

[0036] In one aspect of this embodiment, the pair of the fluorescent molecule and the quencher molecule is preferably selected from the group consisting of a pair of 6-FAM and BHQ1, a pair of 6-FAM and TAMRA, and a pair of Cy5 and BHQ2.

[0037] The RNA detection probe according to this embodiment is preferably used to react with the RNase immobilized on the surface of the protein. Specific procedures will be described later.

[0038] The detection RNA probe according to this embodiment is suitable for use in detecting a protein derived from a pathogen. Here, "protein derived from a pathogen" refers to a protein constituting the pathogen or a protein encoded by a gene of the pathogen. The pathogen is preferably at least one selected from the group consisting of viruses, bacteria, protozoa, and fungi.

[0039] Examples of the proteins derived from the pathogens include SARS-CoV-2 S protein, SARS-CoV-2 M protein, influenza virus HA protein, and influenza virus NA protein.

[0040] <<Method for Producing an RNA Probe for Protein Detection>> The RNA probe for detection according to this embodiment can be produced by known methods. The single-stranded oligonucleotide can be produced, for example, by solid-phase synthesis using the phosphoramidite method. Specifically, a single-stranded oligonucleotide having a predetermined base sequence is first synthesized on a solid support using a commercially available automated nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cleaved from the solid support using a basic substance or the like, and deprotected to obtain a crude single-stranded oligonucleotide. The crude single-stranded oligonucleotide obtained is then purified using HPLC or the like.

[0041] The fluorescent molecule and the quencher molecule are then bound to the resulting single-stranded oligonucleotide using a known chemical modification method, such as carboxy-amino cross-linking. In this manner, the detection RNA probe is synthesized.

[0042] <Protein Detection Kit> A second aspect of the present embodiment is a protein detection kit, comprising: an RNA probe for detecting the protein according to the first aspect; and RNase.

[0043] The RNase is not particularly limited as long as it can degrade the detection RNA probe, and examples thereof include RNase A, RNase B, and RNase C. In one aspect of this embodiment, the RNase is preferably RNase A. The RNase is preferably labeled with streptavidin.

[0044] The protein detection kit preferably further includes a dissolution buffer for the detection RNA probe. The dissolution buffer preferably includes 100 mM to 1000 mM sucrose and 0.0001 mg / ml to 1 mg / ml of a polylactic acid-polysarcosine amphiphilic polymer. The inclusion of sucrose in the dissolution buffer allows the RNA probe to be uniformly dispersed throughout the droplets containing the detection RNA probe during testing. The inclusion of a polylactic acid-polysarcosine amphiphilic polymer in the dissolution buffer tends to facilitate the maintenance of a circular shape in the droplets containing the detection RNA probe, and also suppresses reflection of excitation light. An example of the polylactic acid-polysarcosine amphiphilic polymer is HYDROX (trade name) manufactured by Shimadzu Corporation.

[0045] The protein detection kit may further comprise one or more items selected from the group consisting of a buffer solution, an HRP-labeled antibody, a biotin-labeled tyramide, a sample tube, a spray bottle, and an instruction manual for the user of the kit.

[0046] <Protein Detection Method> A third aspect of this embodiment is a protein detection method, comprising: an RNase immobilization step of immobilizing RNase on the surface of an object to be tested; an RNA probe application step of applying an RNA probe for detecting the protein according to the first aspect to the surface of the object to be tested; and a fluorescence observation step of observing the fluorescence intensity on the surface of the object to be tested, wherein the RNase is configured to be localized to the protein.

[0047] <RNase Immobilization Step> In this step, RNase is immobilized on the surface of the test object. The RNase is configured to localize to the protein. Therefore, when the test object contains the protein to be detected, the RNase will localize to the protein.

[0048] In this embodiment, the term "test object" refers to an object to be tested for the presence or absence of a target protein. The test object is not particularly limited, but examples thereof include a touch panel, a doorknob, tableware, etc.

[0049] In this embodiment, the RNase is not particularly limited as long as it can degrade the detection RNA probe, and examples thereof include RNase A, RNase B, and RNase C. In one aspect of this embodiment, the RNase is preferably RNase A.

[0050] The RNase is configured to localize to the protein, so that in the RNA probe application step described below, the detection RNA probe is specifically degraded at the site where the protein is localized, and thus fluorescence from the fluorescent molecule is detected.

[0051] In one aspect of this embodiment, the RNase immobilization step includes: (1) applying an antibody labeled with peroxidase to the surface of the test object; (2) applying tyramide labeled with biotin to the surface of the test object; and (3) applying the RNase labeled with streptavidin to the surface of the test object, and it is preferable that the antibody specifically binds to the protein.

[0052] The above steps (1) to (3) may be performed sequentially in this order. Alternatively, all or some of steps (1) to (3) may be performed simultaneously. In one aspect of this embodiment, the RNase immobilization step may be performed in this order: (1) applying a peroxidase-labeled antibody to the surface of the test object; (2) applying biotin-labeled tyramide to the surface of the test object; and (3) applying streptavidin-labeled RNase to the surface of the test object.

[0053] In another aspect of this embodiment, the RNase immobilization step includes: (1) applying a peroxidase-labeled antibody to the surface of the test object; (2) applying a biotin-labeled tyramide to the surface of the test object; and (3) applying the streptavidin-labeled RNase to the surface of the test object. After the step (1), the steps (2) and (3) may be performed simultaneously. Here, an example of a method for simultaneously performing the steps (2) and (3) is to mix the biotin-labeled tyramide and the streptavidin-labeled RNase in advance and apply the resulting mixture to the surface of the test object.

[0054] When a target protein is present on the surface of the test object, the peroxidase-labeled antibody specifically recognizes the protein and localizes to the protein by performing step (1). The antibody may be a polyclonal antibody or a monoclonal antibody, but a polyclonal antibody is preferred. Polyclonal antibodies typically bind to a larger number of molecules per protein antigen molecule than monoclonal antibodies. In this embodiment, the peroxidase-labeled antibody may be produced by a known method or may be purchased commercially.

[0055] In one aspect of this embodiment, the method for applying the peroxidase-labeled antibody is not particularly limited, but it is preferable to apply the antibody to the surface of the test object in solution (e.g., dissolved in PBS). The concentration of the antibody is not particularly limited, but may be, for example, 0.0001 ng / ml to 420 ng / ml, or 0.0042 ng / ml to 0.42 ng / ml. The time for applying the antibody is not particularly limited, but may be, for example, 1 to 3 minutes, 1 to 2 minutes, or 2 to 3 minutes. The temperature at which the antibody is applied is not particularly limited, but may be, for example, 20°C to 33°C, 20°C to 25°C, 25°C to 30°C, or 30°C to 33°C.

[0056] Following step (1), step (2) is carried out, whereby the biotin-labeled tyramide is first activated by the peroxidase labeled on the antibody. The activated tyramide then forms a covalent bond with a tyrosine residue on the protein to be detected (tyramide signal amplification method). In other words, biotin is immobilized on the protein to be detected. In this embodiment, the biotin-labeled tyramide may be produced by a known method or may be purchased as a commercially available product.

[0057] In one aspect of this embodiment, the method for applying the biotin-labeled tyramide is not particularly limited, but it is preferably applied to the surface of the test object in solution (e.g., dissolved in PBS). The concentration of the tyramide is not particularly limited, but may be, for example, 0.01 pg / ml to 0.0001 μg / ml, or 1 pg / ml to 1 μg / ml. The time for applying the tyramide is also not particularly limited, but may be, for example, 1 to 3 minutes, 1 to 2 minutes, or 2 to 3 minutes. The temperature at which the tyramide is applied is not particularly limited, but may be, for example, 20°C to 33°C, 20°C to 25°C, 25°C to 30°C, or 30°C to 33°C.

[0058] By performing step (3) following step (2), streptavidin labeling RNase binds to the immobilized biotin. That is, RNase is immobilized on the protein to be detected. In this embodiment, the streptavidin-labeled RNase may be produced by a known method or may be purchased commercially. In one aspect of this embodiment, the RNase may be streptavidin labeled with a carbodiimide-based condensing agent, an NHS ester-based condensing agent, or an NHS-pyridyldithiol-based condensing agent. Examples of the carbodiimide-based condensing agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC). Examples of the NHS ester condensing agent include bis(sulfosuccinimidyl)suberate, disodium salt (BS3) and N,N'-disuccinimidyl suberate (DSS). Examples of the NHS-pyridyldithiol condensing agent include N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and N-succinimidyl 3-oxo-1-(pyridin-2-yldisulfanyl)-7,10,13,16-tetraoxa-4-azanonadecane-19-oate (NHS-PEG4-SPDP).

[0059] In one aspect of this embodiment, the method for applying the streptavidin-labeled RNase is not particularly limited, but it is preferable to apply it to the surface of the test object in solution (e.g., dissolved in PBS). The concentration of the RNase is not particularly limited, but may be, for example, 0.01 μg / ml to 5 μg / ml, or 0.1 μg / ml to 2 μg / ml. The time for applying the RNase is also not particularly limited, but may be, for example, 1 to 3 minutes, 1 to 2 minutes, or 2 to 3 minutes. The temperature at which the RNase is applied is not particularly limited, but may be, for example, 20°C to 33°C, 20°C to 25°C, 25°C to 30°C, or 30°C to 33°C.

[0060] In one aspect of this embodiment, instead of performing the above steps (2) and (3), RNase labeled with tyramide may be applied to the surface of the test object.

[0061] <RNA Probe Application Step> In this step, an RNA probe for detecting the protein according to the first aspect is applied to the surface of the test object. The method for applying the RNA probe for detection is not particularly limited, but it is preferable to apply the probe in solution (e.g., dissolved in PBS) to the surface of the test object. The concentration of the RNA probe for detection is not particularly limited, but may be, for example, 1 μM to 5 μM, 1 μM to 3 μM, 3 μM to 5 μM, or 5 μM to 6 μM. The time for applying the RNA probe for detection is not particularly limited, but may be, for example, 1 to 5 minutes, 1 to 2 minutes, 2 to 3 minutes, or 3 to 5 minutes. The temperature at which the RNA probe for detection is applied is not particularly limited, but may be, for example, 20°C to 33°C, 20°C to 25°C, 25°C to 30°C, or 30°C to 33°C.

[0062] By performing the RNase immobilization step, the RNase is localized to the protein. Therefore, when the detection RNA probe is applied in this step, the decomposition reaction of the detection RNA probe by the RNase is promoted in the area where the protein is present. When the detection RNA probe is decomposed, the fluorescent molecules and quenching molecules in the detection RNA probe are separated, and fluorescence from the fluorescent molecules can be observed (lower part of Figure 1). That is, fluorescence from the fluorescent molecules can be observed in the area where the target protein is present in the test object.

[0063] <Fluorescence Observation Step> In this step, the fluorescence intensity on the surface of the test object is observed. By performing the RNase immobilization step and the RNA probe application step as described above, fluorescence from fluorescent molecules is observed in the part of the test object where the target protein is present. Therefore, the presence and amount of the target protein can be estimated in correlation with the fluorescence intensity.

[0064] The method for observing the fluorescence intensity is not particularly limited, but an example thereof is a method in which a bandpass filter is attached to the industrial camera (manufactured by Imaging Source, product name DMK33UX250) described in the examples, and a predetermined excitation light is irradiated onto the object to be inspected and observed.

[0065] Conventionally, RNA probes have been used to detect target nucleic acids (RNA, DNA), but there has been no idea of ​​using them to detect proteins. However, as a result of intensive research, the present inventors have found that target proteins can be detected with high sensitivity by using RNA probes whose degradation efficiency varies greatly depending on the concentration (density) of RNase, and have completed the present invention.

[0066] In one aspect of this embodiment, when the protein is derived from a pathogen, the method for detecting the protein can also be understood as a method for detecting the pathogen. In another aspect of this embodiment, the method for detecting the protein may be performed by a protein detection system or a protein detection device described below.

[0067] 2 is a diagram illustrating an overview of a protein detection system 100 according to this embodiment. The detection system 100 includes an RNase immobilization device 51, an RNA probe adder 52, and a detection unit 6. In addition, a test subject 1 is used as a target for protein detection in the detection system 100.

[0068] The inspection object 1 is not particularly limited, but examples thereof include a touch panel, a doorknob, tableware, and the like.

[0069] The RNase immobilizer 51 is used to immobilize RNase to a protein 3 present on the surface of the test object 1. In one aspect of this embodiment, the RNase immobilizer 51 may be composed of an antibody adder 51a containing an antibody labeled with peroxidase, a tyramide adder 51b containing a tyramide labeled with biotin, and an RNase adder 51c containing RNase labeled with streptavidin. The antibody adder 51a is, for example, a spray bottle that stores a solution 41a containing the antibody. The solution 41a is a liquid suitable for maintaining the antibody stable in the solution 41a, and is, for example, a buffer solution to which the antibody and, if necessary, a component that prevents aggregation of the antibody have been added.

[0070] The tyramide adder 51b is, for example, a spray bottle that stores the tyramide-containing solution 41b. The solution 41b is a liquid suitable for keeping the tyramide stable in the solution 41b, and is, for example, a buffer solution to which the tyramide and, if necessary, a component that stabilizes the tyramide have been added.

[0071] The RNase adder 51c is, for example, a spray bottle that stores the RNase-containing solution 41c. The solution 41c is a liquid suitable for keeping the RNase stable in the solution 41c, and is, for example, a buffer solution to which the RNase and, if necessary, a component that stabilizes the RNase have been added.

[0072] The RNA probe adder 52 is used to detect proteins 3 present on the surface of the test object 1. The RNA probe adder 52 is, for example, a spray bottle that stores a solution 42 containing the detection RNA probes. The solution 42 is a liquid suitable for keeping the detection RNA probes stable in the solution 42, and is, for example, a buffer solution to which the detection RNA probes and, if necessary, a component that prevents aggregation of the detection RNA probes have been added.

[0073] The RNase immobilization device 51 and the RNA probe adder 52 may be combined as a single unit for a protein detection reaction, forming the detection reaction unit 5. The detection reaction unit 5 may also include other devices. The detection reaction unit 5 is, for example, a clean bench or laboratory table that includes the RNase immobilization device 51 and the RNA probe adder 52.

[0074] The detection unit 6 includes an illuminator 61 and a detector 62. The illuminator 61 irradiates the test object 1 housed in the detection unit with excitation light. The detector 62 detects fluorescence emitted from the test object 1 (fluorescent molecules derived from the RNA probe) by the excitation light. The detection unit 6 is, for example, a general fluorescence detection device.

[0075] <Protein Detection Apparatus> Fig. 3 is a diagram illustrating the configuration of a protein detection system 100 and a detection apparatus 100A according to this embodiment. The protein detection system 100 outlined in Fig. 1 will be described in more detail, followed by a description of the configuration when the protein detection system 100 is realized as a detection apparatus 100A.

[0076] In the detection reaction unit 5, in FIG. 2, the RNase immobilization device 51 (antibody adder 51a, tyramide adder 51b, RNase adder 51c) and the RNA probe adder 52 are illustrated as spray bottles, but are not limited to this.

[0077] For example, the detection and reaction unit 5 may be a single device including the RNase immobilizer 51, the RNA probe adder 52, and the reagent washer 53. In this case, for example, the detection and reaction unit 5 has a grid-like table for placing the test subject 1 therein. The detection and reaction unit 5 includes, above the table, a solution sprayer that is a form of the RNase immobilizer 51, and a solution sprayer that is a form of the RNA probe adder 52. The sprayer is controlled, for example, by the control unit 7 described below. In this case, for example, when a laboratory technician places the test subject 1 or "a part of the test subject 1" in the detection and reaction unit 5, the sprayer is configured to automatically spray the solution onto the test subject 1.

[0078] The detection unit 6 includes the illuminator 61 and detector 62 described in Fig. 2. The detection unit 6 may further include an alarm 63. The alarm 63 is a device that visually and / or audibly notifies a laboratory technician of the detection of protein 3 in the detection unit 6. The detection of protein 3 is determined, for example, by whether or not the intensity of the detected fluorescence exceeds a threshold.

[0079] The alarm 63 is, for example, a display, a lamp, or a speaker. The alarm 63 is provided, for example, on the outside of the fluorescence detection device including the irradiator 61 and the detector 62. With this configuration, the laboratory technician can easily recognize that the protein 3 has been detected by the detector 62.

[0080] Such a detection system 100 can carry out the above-described protein detection method.

[0081] The detection system 100 may be realized as a detection device 100A including a detection and reaction unit 5 and a detection unit 6. In this case, for example, the detection device 100A includes a control unit 7 in addition to the detection and reaction unit 5 and the detection unit 6. The control unit 7 controls each unit. The control unit 7 includes, for example, a processor 71 and a memory 72, which are the basic components of a computer. The processor 71 and the memory 72 are connected to each other via a common bus 73. The detection device 100A may further include an input unit that allows a laboratory technician or a subject to operate the detection device 100A, and a handling device that inserts / removes the test object 1 or a "part of the test object 1" into / from the detection and reaction unit 5 and the detection unit 6. In the detection device 100A, the detection and reaction unit 5 and the detection unit 6 may be provided as separate entities (e.g., two housings) or as an integrated entity (e.g., one housing).

[0082] In the detection device 100A, for example, when the test subject 1 or a "part of the test subject 1" is stored, the above-described series of protein detection methods are automatically executed. In this case, the storage in the detection device 100A and the operation of the detection device 100A may be performed by the subject himself (the person who holds the test subject 1) rather than by a laboratory technician.

[0083] If the protein 3 is a protein derived from a pathogen, the detection device 100A allows the subject to self-check for contamination or infection by the pathogen. For example, if the detection device 100A is installed at the entrance of the facility to which the subject belongs, the subject can store the test object 1 in the detection device 100A and perform the self-check when entering or leaving the facility. In this case, the device may be configured so that the subject is prevented from entering or leaving the facility when the alarm 63 reports that the protein 3 (pathogen) has been detected in the test object 1. The detection device 100A may also be configured to include a device for identifying the subject (e.g., an optical camera or a subject ID card sensor at the facility) so that subjects infected with the pathogen can be identified. This configuration helps prevent the spread of pathogens both inside and outside the facility.

[0084] The detection device 100A can also be used in epidemiological studies. For example, if the detection device 100A is installed in a facility where an infected person has stayed, the subject (in this case, a researcher conducting an epidemiological study) can place the detection target 1 in the detection device 100A and identify items or locations contaminated with pathogens. This configuration not only improves the efficiency of swab tests but also makes it possible to trace infection routes via items, helping to prevent the spread of pathogens inside and outside the facility.

[0085] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0086] Experimental Example 1 First, various single-stranded oligonucleotides (RNA) with different hairpin sizes (number of bases in the loop region) and cytosine content were synthesized using a standard phosphoramidite protocol. The base sequences of each single-stranded oligonucleotide are shown in Table 1. In Table 1, the columns "C content," "U content," and "A+U content" indicate the "cytosine content in the loop region," "uracil content in the loop region," and "total adenine and uracil content in the loop region," respectively, based on the number of bases in the loop region. Next, the fluorescent molecule 6-FAM (6-carboxyfluorescein) or Cy5 (cyanine 5) was bound to the 5' end of each single-stranded oligonucleotide, and the quencher molecule BHQ1 (black hole quencher 1) or BHQ2 (black hole quencher 2) was bound to the 3' end by the carboxy-amino crosslinking method. The single-stranded oligonucleotides to which the fluorescent molecules and quencher molecules were bound were then purified and recovered by HPLC. Using the above procedure, self-quenching fluorescent RNA probes (RNA probes for protein detection) were synthesized. The RNA probes corresponding to SEQ ID NOs: 1 to 23 used a pair of 6-FAM and BHQ1, and the RNA probe corresponding to SEQ ID NO: 24 used a pair of Cy5 and BHQ2.

[0087]

[0088] The self-quenching fluorescent RNA probes (final concentration: 2 μM) synthesized by the above procedure were mixed with either a low-concentration (final concentration: 2 ng / ml) or a high-concentration (final concentration: 10 μg / ml) RNase A solution, and the fluorescence intensity (wavelength: 520 nm or 667 nm) was measured using a plate reader (PerkinElmer, trade name: EnSpire). The fluorescence intensity of several RNA probes, including ssRNA-5bp-C20, was suppressed when mixed with low concentrations of RNase A compared to when mixed with high concentrations of RNase A (Figure 4). The results were similar even when the fluorescent molecule and quencher pair was changed from "6-FAM and BHQ1" to "Cy5 and BHQ2" (Figure 12). The RNA probes with reduced fluorescence intensity described above had loop regions with 9 mers or less bases, and the cytosine content in the loop region was less than 35% (Figure 5, RNA probes with a hatched background). Furthermore, these RNA probes had uracil content of 30-100% in the loop region (Figure 9, RNA probes with a hatched background), and the total adenine and uracil content in the loop region was 65-100% (Figure 10, RNA probes with a hatched background). Furthermore, RNA probes with the same loop structure as the RNA probe of SEQ ID NO: 7 but with different stem region lengths (SEQ ID NOs: 21-23) were prepared, and their fluorescence intensities were measured using a low-concentration RNase A solution. The results are shown in Figure 11. Although the fluorescence intensity of SEQ ID NO: 23 (with a 10-mer stem region) was slightly reduced compared to SEQ ID NO: 7, it was found that the length of the stem region had almost no effect on fluorescence intensity (Figure 11).

[0089] [Experimental Example 2] The RNA probes shown in Table 1 (final concentration 2 μM) were mixed with various concentrations of RNase A, and the fluorescence intensity (wavelength 520 nm) was measured using a plate reader. As representative experimental data, the measurement results when the RNA probe shown in Table 1 as "ssRNA-9bp-C22b" was used are shown in Figure 6. The results of Experiment 2 showed that the fluorescence intensity of the self-quenching fluorescent RNA probe changed depending on the RNase concentration.

[0090] Experimental Example 3: 173 μg (173 mg / ml x 0.5 μl) of SARS-CoV-2 S protein dissolved in UPDW (ultrapure water) was dropped onto the surface of a quartz glass plate and allowed to stand for 2 minutes at 23°C. Then, HRP-labeled anti-SARS-CoV-2 S protein antibody (0.0042 ng / ml x 0.5 μl, allowed to stand for 2 minutes at 23°C), biotin-labeled tyramide (1 x 10 －８ μg / ml x 0.5 μl, left at 23°C for 2 minutes), streptavidin-labeled RNase A (1 x 10 －２ A sample containing SARS-CoV-2 S protein (2 μg / ml x 0.5 μl, incubated at 23°C for 2 minutes) and a self-quenching fluorescent RNA probe (2 μM x 0.5 μl, 23°C) shown in Table 1 were dropped in this order (Figure 7). The self-quenching fluorescent RNA probe was diluted with a solution of sucrose solution (final concentration 200 mM) to which HYDROX (final concentration 1 mg / ml) was added. A negative control was prepared by performing the same procedure as above using PBS without dissolving SARS-CoV-2 S protein. Fluorescence observation was performed using an industrial camera (Imaging Source, product name DMK33UX250) equipped with a bandpass filter under irradiation with excitation light (wavelength 452 nm). Compared to the negative control sample, the sample containing SARS-CoV-2 S protein exhibited stronger fluorescence (Figure 8). Furthermore, in the sample to which SARS-CoV-2 S protein was dropped, strong fluorescence was observed at the location where the protein was dropped, and almost no fluorescence was observed at the location where the protein was not dropped.

[0091] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0092] (Item 1) An RNA probe for detecting a protein according to one embodiment comprises a single-stranded oligonucleotide having ribonucleotides as building blocks, a fluorescent molecule, and a quencher molecule that absorbs fluorescence emitted by the fluorescent molecule, wherein the single-stranded oligonucleotide comprises a loop region, a 3' stem region bound to the 3' end of the loop region, and a 5' stem region bound to the 5' end of the loop region, wherein the fluorescent molecule binds to one of the 3' stem region and the 5' stem region, and the quencher molecule binds to the other, the number of bases in the loop region is 3 to 9 mer, the number of bases in the 3' stem region is 2 to 10 mer, and the number of bases in the 5' stem region is 2 to 10 mer, the 3' stem region and the 5' stem region have complementary base sequences, and the cytosine content in the loop region is 0% or more and less than 35% based on the number of bases in the loop region. According to the RNA probe for detection described in item 1, the target protein can be detected with high sensitivity.

[0093] (Item 2) In the RNA detection probe according to item 1, the pair of the fluorescent molecule and the quencher molecule is selected from the group consisting of a pair of 6-FAM and BHQ1, a pair of 6-FAM and TAMRA, and a pair of Cy5 and BHQ2. The RNA detection probe according to item 2 enables detection of the target protein with even higher sensitivity.

[0094] (Item 3) In the detection RNA probe of item 1 or 2, the protein is a protein derived from a pathogen. The detection RNA probe according to item 3 can detect the pathogen from which the protein is derived.

[0095] (Item 4) In the RNA detection probe according to item 3, the pathogen is at least one selected from the group consisting of viruses, bacteria, protozoa, and fungi. The RNA detection probe according to item 4 can detect pathogens such as viruses, bacteria, protozoa, and fungi.

[0096] (Item 5) In the RNA detection probe according to any one of Items 1 to 4, the RNA detection probe is used to act on RNase immobilized on the surface of the protein. The RNA detection probe according to Item 5 allows the location and amount of the target protein to be quantified.

[0097] (Item 6) A kit for detecting a protein according to one embodiment includes an RNA probe for detecting the protein according to any one of items 1 to 5, and RNase. The kit for detecting a protein according to item 6 enables the detection of a target protein with high sensitivity.

[0098] (Item 7) The protein detection kit according to item 6 further comprises a dissolution buffer for the RNA detection probe, the dissolution buffer comprising 100 mM to 1000 mM sucrose and 0.0001 mg / ml to 1 mg / ml of a polylactic acid-polysarcosine amphiphilic polymer. The protein detection kit according to item 7 allows the RNA probe to be uniformly dispersed throughout the droplets containing the RNA detection probe during testing, and further allows the droplets containing the RNA detection probe to more easily maintain a circular shape, while also suppressing reflection of excitation light.

[0099] (Item 8) A protein detection method according to one embodiment includes an RNase immobilization step of immobilizing RNase on the surface of an object to be tested, an RNA probe application step of applying an RNA probe for detecting the protein described in any one of Items 1 to 5 to the surface of the object to be tested, and a fluorescence observation step of observing the fluorescence intensity on the surface of the object to be tested, wherein the RNase is configured to be localized to the protein. The protein detection method described in Item 8 enables the detection of the target protein with high sensitivity.

[0100] (Item 9) In the protein detection method described in Item 8, the RNase immobilization step includes (1) applying a peroxidase-labeled antibody to the surface of the test object, (2) applying biotin-labeled tyramide to the surface of the test object, and (3) applying streptavidin-labeled RNase to the surface of the test object, wherein the antibody specifically binds to the protein. According to the protein detection method described in Item 9, the target protein can be detected with even higher sensitivity.

[0101] (Item 10) In the protein detection method according to item 9, the RNase is streptavidin labeled with a carbodiimide condensing agent, an NHS ester condensing agent, or an NHS-pyridyldithiol condensing agent. According to the protein detection method according to item 10, the target protein can be detected with even higher sensitivity.

[0102] (Item 11) A protein detection system according to one embodiment includes an RNase immobilizer, an RNA probe adder, and a detection unit, wherein the RNase immobilizer includes RNase, the RNA probe adder includes an RNA probe for detecting the protein according to any one of items 1 to 5, and the detection unit detects fluorescence emitted from fluorescent molecules derived from the RNA probe for detection. The protein detection system according to item 11 enables detection of the target protein with high sensitivity.

[0103] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0104] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

[0105] 1 Test object, 3 Protein, 5 Detection reaction unit, 6 Detection unit, 7 Control unit, 41a Antibody-containing solution, 41b Tyramide-containing solution, 41c RNase-containing solution, 42 Detection RNA probe-containing solution, 51 RNase immobilization device, 51a Antibody adder, 51b Tyramide adder, 51c RNase adder, 52 RNA probe adder, 61 Irradiator, 62 Detector, 63 Alarm, 71 Processor, 72 Memory, 73 Bus, 100 Detection system, 100A Detection device.

Claims

1. A kit for detecting a protein, comprising: an RNA probe for detecting a protein; RNase, and Including, the detection RNA probe comprises a single-stranded oligonucleotide having ribonucleotides as building blocks, a fluorescent molecule, and a quencher molecule that absorbs fluorescence emitted by the fluorescent molecule; the single-stranded oligonucleotide comprises a loop region, a 3' stem region linked to the 3' end of the loop region, and a 5' stem region linked to the 5' end of the loop region; the fluorescent molecule is bound to one of the 3' stem region and the 5' stem region, and the quencher molecule is bound to the other; the number of bases in the loop region is 3 to 9 mer, the number of bases in the 3' stem region is 2 to 10 mer, the number of bases in the 5' stem region is 2 to 10 mer, the 3' stem region and the 5' stem region have complementary base sequences, the content of cytosine in the loop region is 0% or more and less than 35% based on the number of bases in the loop region; The detection kit comprises: an RNase immobilization step of immobilizing the RNase on the surface of an object to be tested; an RNA probe application step of applying the detection RNA probe to the surface of the test object; a fluorescence observation step of observing a fluorescence intensity on the surface of the inspection object, A protein detection kit for use in a protein detection method, wherein the RNase is configured to be localized to the protein.

2. Further comprising a dissolution buffer for the detection RNA probe; 2. The protein detection kit according to claim 1, wherein the dissolution buffer contains 100 mM to 1000 mM sucrose and 0.0001 mg / ml to 1 mg / ml of a polylactic acid-polysarcosine amphiphilic polymer.

3. A kit for detecting a protein as described in claim 1, wherein the pair of fluorescent molecule and quenching molecule is selected from the group consisting of a pair of 6-FAM and BHQ1, a pair of 6-FAM and TAMRA, and a pair of Cy5 and BHQ2.

4. A kit for detecting the protein described in claim 1, wherein the protein is a protein derived from a pathogen.

5. A kit for detecting a protein as described in claim 4, wherein the pathogen is at least one selected from the group consisting of viruses, bacteria, protozoa and fungi.

6. A method for detecting a protein, comprising: an RNase immobilization step of immobilizing RNase on the surface of the test object; an RNA probe application step of applying an RNA probe for detecting a protein to the surface of the test object; a fluorescence observation step of observing a fluorescence intensity on the surface of the inspection object, the detection RNA probe comprises a single-stranded oligonucleotide having ribonucleotides as building blocks, a fluorescent molecule, and a quencher molecule that absorbs fluorescence emitted by the fluorescent molecule; the single-stranded oligonucleotide comprises a loop region, a 3' stem region linked to the 3' end of the loop region, and a 5' stem region linked to the 5' end of the loop region; the fluorescent molecule is bound to one of the 3' stem region and the 5' stem region, and the quencher molecule is bound to the other; the number of bases in the loop region is 3 to 9 mer, the number of bases in the 3' stem region is 2 to 10 mer, the number of bases in the 5' stem region is 2 to 10 mer, the 3' stem region and the 5' stem region have complementary base sequences, the content of cytosine in the loop region is 0% or more and less than 35% based on the number of bases in the loop region; A method for detecting a protein, wherein the RNase is configured to be localized to the protein.

7. The RNase immobilization step comprises: (1) applying a peroxidase-labeled antibody to the surface of the test object; (2) applying a biotin-labeled tyramide to the surface of the test object; (3) applying the RNase labeled with streptavidin to the surface of the test object; Including, The method for detecting a protein according to claim 6 , wherein the antibody specifically binds to the protein.

8. 8. The method for detecting a protein according to claim 7, wherein the RNase is obtained by labeling the streptavidin with a carbodiimide-based condensing agent, an NHS ester-based condensing agent, or an NHS-pyridyldithiol-based condensing agent.

9. A protein detection system comprising: The detection system includes an RNase immobilization device, an RNA probe addition device, and a detection unit; the RNase immobilizer contains RNase; the RNA probe adder contains an RNA probe for detecting a protein; the detection unit detects fluorescence emitted from fluorescent molecules derived from the detection RNA probe; the detection RNA probe comprises a single-stranded oligonucleotide having ribonucleotides as building blocks, a fluorescent molecule, and a quencher molecule that absorbs fluorescence emitted by the fluorescent molecule; the single-stranded oligonucleotide comprises a loop region, a 3' stem region linked to the 3' end of the loop region, and a 5' stem region linked to the 5' end of the loop region; the fluorescent molecule is bound to one of the 3' stem region and the 5' stem region, and the quencher molecule is bound to the other; the number of bases in the loop region is 3 to 9 mer, the number of bases in the 3' stem region is 2 to 10 mer, the number of bases in the 5' stem region is 2 to 10 mer, the 3' stem region and the 5' stem region have complementary base sequences, the content of cytosine in the loop region is 0% or more and less than 35% based on the number of bases in the loop region; The detection system comprises: an RNase immobilization step of immobilizing the RNase on the surface of an object to be tested; an RNA probe application step of applying the detection RNA probe to the surface of the test object; a fluorescence observation step of observing a fluorescence intensity on the surface of the inspection object, A detection system for use in a method for detecting a protein, wherein the RNase is configured to be localized to the protein.