Methods for detecting small RNAs

Abstract

A method for rapid and accurate detection of small RNA is provided. [Solution] A method for detecting small RNA is provided, which uses single-stranded nucleic acids having an XYZ structure and consisting of a base sequence capable of complementary binding to all or part of the base sequence of the small RNA to be detected as (i) a forward primer and probe, (ii) a reverse primer and probe, or (iii) a forward primer, a reverse primer, and a probe.

Description

[Technical field]

[0001] The present invention relates to a method for detecting small RNA or a protein associated with a small RNA, and more specifically, to a method for detecting small RNA or a protein associated with a small RNA, using a single-stranded nucleic acid having an XYZ structure and composed of a base sequence capable of complementary binding to all or a part of the base sequence of the small RNA to be detected. [Background technology]

[0002] MicroRNA (miRNA) is one of the small RNAs present in cells, and is known to play a vital role in biological processes such as cell development and differentiation and programmed cell death. It is known that differences in the expression levels of specific miRNAs in cells are related to various diseases including cancer. For this reason, detection of specific microRNAs (miRNAs) has been attracting attention as an important part of recent scientific research. Specifically, by detecting and identifying specific miRNAs and their associated proteins, they can be used as genetic and biological markers and can be used as indicators of human health. The above detection and identification have made it possible to develop kits that easily utilize peripheral blood, hormones, and spinal fluid, as well as diagnostic kits that can be used outside the body (in vitro). These kits can be applied to tests for dementia diseases (Alzheimer's, Parkinson's, etc.), diabetes, cancer, etc. However, miRNAs are extremely short, about 22 nucleotides, making them difficult to detect and detect, and there are limitations to their use as effective biomarkers.

[0003] In general, detection of miRNA is based on nucleic acid amplification methods, examples of which include PCR (polymerase chain reaction), NASBA (nucleic acid sequence based amplification), etc. Such conventional detection methods using nucleic acid amplification reactions are known to those of ordinary skill in the art to which the present invention pertains. These detection methods usually require labor-intensive processing, such as electrophoresis or the use of probe or blotting techniques that can detect the amplified nucleic acid after nucleic acid amplification.

[0004] The most widely known real-time detection for miRNA detection is performed by RT-PCR, qPCR, etc. These methods are based on a fluorescent hybridization probe that forms a secondary structure that loses fluorescence if it does not hybridize with a target substance, a hybridization probe that is cleaved by Taq polymerase after hybridization and increases fluorescence, a dsDNA-binding agent, etc. At each measurement time point, a probe is opened or cleaved while hybridizing to each amplification product, increasing the fluorescence intensity. In this case, amplification is detected at a ratio of one amplification to one probe. In a given cycle, one amplification indicates one probe (e.g., molecular beacon, Taqman probe, MGB probe, etc.) that is detected by hybridization or cleavage of the probe. In addition, among real-time detection methods, a method of detecting NASBA products simultaneously with amplification using a molecular beacon is also known.

[0005] As specific examples of such miRNA detection, a method using a TaqMan probe and a stem-loop primer, and a method using an oligo-dT adaptor primer with a polyA tail, as shown in Figure 1, are widely used.

[0006] However, the above mentioned techniques have several drawbacks.

[0007] First, the above techniques require the extension of additional oligos, as the probe and primer for miRNA analysis are not located in the target miRNA. In the case of Figure 1, the stem-loop primer, oligo-dT adaptor primer, etc. shown are the extended oligos. In this case, a major problem is that the increase in fluorescence due to the amplification of the extended oligo is measured, rather than the increase in fluorescence that is entirely dependent on the amplified miRNA target, which can lead to inaccurate results due to non-specific amplification and binding.

[0008] Secondly, a certain distance is required between the primer and the probe. For example, in the case of Taqman probe and MGB probe, a donor chromophore is labeled at the 5' end, and when DNA polymerase collides with the probe during the process of extending the primer, the exonuclease activity of the polymerase starts from the 5' end and sequentially degrades the probe. At this time, cleavage is possible when the distance between the primer and the probe is at least 7 to 10 nucleotides. In addition, the distance between the primer and the quencher at the 3' end must be at least 2 to 4 nucleotides. Such restrictions act as a limiting factor in the analysis of short RNA or DNA.

[0009] Third, in the method using NASBA, the probe needs to form a secondary structure that quenches the fluorescence of the donor, so the melting temperature of the beacon must be precisely adjusted. Such adjustment is difficult to apply when the reaction is performed at a constant temperature, as in NASBA and RCA. In addition, the beacon must be designed to maintain a hairpin structure when not binding to the target, and to open at the reaction temperature at which it binds to the target. This makes the design of the probe extremely difficult, and also causes problems related to signal noise, since the beacon often emits fluorescence when it opens at the reaction temperature.

[0010] Fourth, it is difficult to distinguish isoforms of small RNAs such as miRNAs. In the case of miRNAs, there are many isoforms that differ by only 1 to 3 units due to their short length. For example, it is known that it is difficult to distinguish similar isoforms of miRNAs such as let-7, miR-18, and miR-30.

[0011] In the course of research to overcome the above-mentioned drawbacks and develop a method for quickly and accurately detecting small RNAs such as miRNA and siRNA, the inventors have confirmed that small RNAs can be quickly and accurately detected from a trace amount of sample by hybridizing a single-stranded nucleic acid capable of accurately and quickly detecting nucleic acids or proteins in real time, as described in Patent Document 1, which has Korean Patent Application No. 10-2016-0017359 as a priority basis, with small RNA or cDNA of small RNA, cleaving the inside of the nucleic acid with a cleavage reagent, and measuring the amount of separated fluorescent fragments, thereby completing the present invention. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Korean Patent Application No. 10-2017-0020238 Summary of the Invention [Problem to be solved by the invention]

[0013] One object of the present invention is to provide a method for detecting small RNA.

[0014] Another object of the present invention is to provide a method for detecting proteins associated with small RNAs in conjunction with a real-time amplification reaction. [Means for solving the problem]

[0015] In one embodiment, the present invention provides single stranded nucleic acids for use as primers and probes for real-time detection of small RNAs or proteins associated with small RNAs.

[0016] The single-stranded nucleic acid is derived from the single-stranded nucleic acid described in Patent Document 1, which has Korean Patent Application No. 10-2016-0017359 as its priority basis, and the above-mentioned Korean patent applications, etc. are included as part of the present specification in the present invention.

[0017] Specifically, in the present invention, the single-stranded nucleic acid has a structure of XYZ, and is characterized in that one or more detectable markers are attached to both ends or inside of the single-stranded nucleic acid. In this case, the position of the attached detectable marker is not limited to a specific site, and can be any position where the detectable marker is separated when the Y site is cleaved by a specific enzyme.

[0018] The single-stranded nucleic acid is characterized in that it binds to a specific site of a target small RNA for real-time detection or a target protein associated with the target small RNA to form a complex, and when the Y site is cleaved by a specific enzyme, the Y and Z sites are separated from the specific site of the target small RNA or the target protein associated with the target small RNA, but the X site is not separated and is used as a primer to maintain and amplify the complex.

[0019] In the present invention, the single-stranded nucleic acid is referred to as a "promer", and hereinafter, unless otherwise specified, the term "promer" refers to a single-stranded nucleic acid that can be used simultaneously as a primer and a probe.

[0020] A single stranded nucleic acid of the invention has the structure XYZ, where each of X, Y and Z can have a variable number of nucleotides.

[0021] For example, here, X is DNA or RNA composed of a base sequence of 1 to 60, preferably 1 to 30, more preferably 2 to 30, even more preferably 3 to 30, even more preferably 4 to 30, even more preferably 5 to 30, even more preferably 6 to 30, even more preferably 7 to 30, even more preferably 8 to 30, even more preferably 10 to 30. When X has a base sequence of more than 60, a non-specific reaction may occur during real-time detection of a nucleic acid or protein.

[0022] For example, the Y is a DNA or RNA composed of a base sequence of 1 to 10, preferably 1 to 9, more preferably 1 to 8, even more preferably 1 to 7, even more preferably 1 to 6, even more preferably 1 to 5, even more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2. When Z is composed of 0 base, the Y is at least 3 or more, preferably 3 to 10, and more preferably 1 to 2. Preferably, Y is composed of a base sequence of at least 2, preferably 2 to 10, more preferably 2 to 9, even more preferably 2 to 8, even more preferably 2 to 7, even more preferably 3 to 6, even more preferably 3 to 5, and even more preferably 3 to 4 bases when Z is composed of one base. When the base sequence constituting Y is out of the above range, a non-specific reaction may occur during real-time detection of a nucleic acid or protein, resulting in a decrease in sensitivity.

[0023] For example, Z is DNA or RNA composed of a base sequence of 0 to 10, preferably 1 to 10, more preferably 2 to 10. If the base sequence constituting Z is outside the above range, after Y is cleaved, Z bound to the target nucleic acid or protein remains bound without being released from the target nucleic acid or protein, resulting in a problem that the nucleic acid or protein cannot be detected.

[0024] In this case, when any one of X, Y and Z is DNA, at least one of the other two is RNA. Preferably, X, Y and Z are DNA, RNA and DNA, or RNA, DNA and RNA, in that order.

[0025] According to one specific example, when X is DNA, either Y or Z is RNA, for example, Y is RNA and Z is DNA, or Y is DNA and Z is RNA. Here, the DNA and RNA may be 0 or more, and the number of DNA and RNA is defined by X, Y, and Z.

[0026] In another embodiment, when X is RNA, either Y or Z is RNA, for example, Y is RNA and Z is DNA, or Y is DNA and Z is RNA. Here, the DNA and RNA may be 0 or more, and the number of DNA and RNA is defined by X, Y, and Z.

[0027] In another specific example, when Z is 0, one of X and Y is DNA and the other is RNA. Here, the DNA and RNA are one or more, and the number of DNA and RNA is defined by the X and Y.

[0028] Additionally, X, Y and Z may be synthetically prepared to be either globally methylated or partially methylated to prevent non-specific cleavage.

[0029] In the present invention, the nucleic acid refers to DNA or RNA to be detected in real time from a sample.

[0030] In the present invention, the detectable marker may be a fluorescent substance that binds to a single-stranded nucleic acid by a covalent or non-covalent bond, or a fluorescent pair consisting of a fluorescent substance and a quencher.

[0031] Examples of the fluorescent substance include Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630 / 650, Bodipy 650 / 665, Calfluor ORNAge 546, Calfluor red 610, Quasar 670 and biotin, but is not limited thereto. The quenching substance may be, for example, any one selected from the group consisting of DDQ-1, Dabcyl, Eclipase, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2 and MGBNFQ, but is not limited thereto.

[0032] When a fluorescent pair is used as a detectable marker in the present invention, the locations of the fluorophore and quencher may be at site X or Z, or at site Y, but are not limited to any one of these. For example, the fluorophore may be at site X and the quencher at site Y or Z.

[0033] The single-stranded nucleic acid of the present invention can be used as i) an RT primer for synthesizing cDNA from a small RNA, ii) a forward primer for amplifying cDNA synthesized from a small RNA, iii) a reverse primer for amplifying cDNA synthesized from a small RNA, iv) a forward primer and a reverse primer for amplifying cDNA synthesized from a small RNA, or v) a probe for confirming the small RNA to be detected or a protein associated therewith in real time when detecting a small RNA or a protein associated with a small RNA.

[0034] In such a series of specific embodiments, the single-stranded nucleic acid of the present invention is capable of synthesizing and amplifying small RNA by using an enzyme that cleaves the Y site of the single-stranded nucleic acid to separate the Y and Z sites, and the X site maintains a complex with a target small RNA or a target protein related thereto and is used as a primer to synthesize and amplify small RNA. Since only the Y portion of the single-stranded nucleic acid is cleaved by the enzyme that cleaves the Y site of the single-stranded nucleic acid, the small RNA to be detected or a protein related to the small RNA can be detected more accurately than a conventional probe that is degraded by DNA polymerase.

[0035] In particular, in the single-stranded nucleic acid of the present invention, when a fluorescent substance or a quencher is bound to the 3' end of Y or Z, a specific nucleic acid of a target nucleic acid or a target protein can be blocked from being amplified by a polymerase unless the Y site of the single-stranded nucleic acid is cleaved, thereby reducing errors due to non-specific amplification.

[0036] Therefore, when the single-stranded nucleic acid of the present invention is used to detect small RNAs or proteins associated with small RNAs, it is possible to reduce additional processes (e.g., forming the RT primer into a loop, forming polyA, etc.) as well as analysis time and cost compared to conventional techniques, and it is possible to detect the small RNA or proteins associated with small RNAs to be detected more accurately and specifically compared to conventional methods, so it can be effectively used as a kit for detecting small RNAs or proteins associated with small RNAs in real time.

[0037] When the single-stranded nucleic acid of the present invention is used as a kit for detecting a small RNA or a protein associated with a small RNA, it is desirable that the kit further contains, in addition to the single-stranded nucleic acid of the present invention, an enzyme capable of cleaving the Y site of the single-stranded nucleic acid.

[0038] In the present invention, the enzyme capable of cleaving the Y site of the single-stranded nucleic acid may be any enzyme capable of specifically cleaving the Y site of the single-stranded nucleic acid. In this case, it is preferable to use a DNA nuclease (DNase), specifically, DNase I, DNase II, S1 nuclease, nuclease P1, AP endonuclease, UvrABSC nuclease, or the like, and when the Y site is RNA, it is preferable to use an RNA hydrolase (ribonuclease, RNase), specifically, RNase II, RNase III, RNase IV, RNase H, RNase T2, or the like.

[0039] The single-stranded nucleic acid of the present invention can be used as a kit for detecting nucleic acids or proteins in real time. When used as a detection kit, the kit can further contain reagents necessary for a DNA amplification reaction in addition to the single-stranded nucleic acid of the present invention and an enzyme capable of cleaving the Y site of the single-stranded nucleic acid.

[0040] The reagents required for the amplification reaction include, for example, an appropriate amount of DNA polymerase (e.g., a thermostable DNA polymerase obtained from Thermus aquatiucus (Taq), Thermus thermophilus (Tth), Thermist filiformis, Thermist flavus, Thermococcus litoralis, or Pyrococcus furiosis (Pfu)), DNA polymerase cofactor (Mg 2+ ), buffer solution, dNTPs (dATP, dCTP, dGTP and dTTP) and water (H2O). The buffer solution may include, but is not limited to, an appropriate amount of Triton X-100, dimethylsulfoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide and bovine serum albumin (BSA).

[0041] In another aspect, the present invention provides a method for detecting small RNA.

[0042] Each step of the method for detecting small RNA according to the present invention will be specifically described below.

[0043] a) obtaining a template nucleic acid from a biological sample;

[0044] In the present invention, the template nucleic acid refers to RNA including small RNA to be detected from a biological sample. The small RNA is expressed to produce a protein or to regulate the production of a protein in a situation where the biological phenomenon of a living organism needs to be maintained or regulated.

[0045] The biological sample may be any type of sample obtained from an individual, specifically solid tissue samples, liquid tissue samples, biological fluids, bronchial aspirates, cells and cell fragments. Specific examples of biological samples include, but are not limited to, solid tissue samples removed from an individual during surgery, pathological specimens, preserved samples or biopsy specimens, tissue cultures or sections prepared from cells derived therefrom and their progeny.

[0046] The preserved biological sample may be derived from tissue that has been preserved for one week or more, one year or more, for example, one to ten years, frozen, or stored at room temperature after being fixed in formalin, according to conventional preservation methods known in the art.

[0047] In the present invention, RNA can be extracted from a sample using various methods known in the art. For example, trizol or Triton X-100 can be used.

[0048] b) A process for synthesizing cDNA from small RNA.

[0049] Specifically, step b) of the present invention is a step of synthesizing cDNA by mixing the small RNA extracted in step a) with another RT primer.

[0050] In the present invention, the RT primer is a nucleic acid capable of complementary binding to a partial base sequence of an RNA to be detected, and may be an RT primer known in the art or a single-stranded nucleic acid of the present invention.

[0051] In the present invention, when the single-stranded nucleic acid of the present invention is used as an RT primer, the single-stranded nucleic acid is characterized in that it is composed of a base sequence capable of complementary binding to a partial base sequence of a small RNA to be detected.

[0052] In the present invention, the synthesis of the cDNA can be carried out by various methods known in the art, for example, it can be carried out by using reverse transcriptase and DNA polymerase with RNA obtained from a sample as a template. In addition, the RT primer can be an oligo dT primer extended with additional nucleic acid after poly A or a roof-form RT primer.

[0053] c) Amplifying the cDNA.

[0054] Specifically, step c) of the present invention is a step of mixing the cDNA synthesized in step b) with a kit containing a single-stranded nucleic acid of the present invention and / or a forward primer or a reverse primer having a base sequence complementary to the cDNA, and amplifying the cDNA by an extension reaction.

[0055] The kit containing the single-stranded nucleic acid comprises the single-stranded nucleic acid of the present invention and an enzyme capable of cleaving the Y site of the single-stranded nucleic acid.

[0056] The enzyme capable of cleaving the Y site of the single-stranded nucleic acid may be any enzyme capable of specifically cleaving the X site of the single-stranded nucleic acid. For example, when the X site is DNA, a DNA nuclease (DNase), specifically, DNase It is preferable to use nuclease I, DNase II, S1 nuclease, nuclease P1, AP endonuclease, UvrABSC nuclease, or the like, and when the X site is RNA, it is preferable to use an RNA hydrolase (ribonuclease, RNase), specifically, RNase II, RNase III, RNase IV, RNase H, RNase T2, or the like.

[0057] In the present invention, the single-stranded nucleic acid is characterized in that it is composed of a base sequence capable of complementary binding to a partial base sequence of the cDNA synthesized in the step b) and is used as i) a forward primer and a probe, ii) a reverse primer and a probe, or iii) a forward primer, a reverse primer and a probe for amplifying cDNA. The single-stranded nucleic acid of the present invention has a structure of XYZ, and one or more detectable markers are attached to both ends or inside of the single-stranded nucleic acid, and when the Y site is cleaved by a specific enzyme, Y and Z are separated and X functions as a primer.

[0058] The detectable marker may be a fluorescent substance that binds to single-stranded nucleic acid by a covalent or non-covalent bond, or a fluorescent pair consisting of a fluorescent substance and a quencher.

[0059] Examples of the fluorescent substance include Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas red, ORNAge green 488X, ORNAge green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630 / 650, Bodipy The quenching substance may be any one selected from the group consisting of, but is not necessarily limited to, Calfluor ORNAge 546, Calfluor red 610, Quasar 670 and biotin. The quenching substance may be any one selected from the group consisting of, but is not necessarily limited to, DDQ-1, Dabcyl, Eclipse, 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, lowa Black RQ-Sp, QSY-7, QSY-2 and MGBNFQ.

[0060] According to one specific example, when the single-stranded nucleic acid of the present invention is used as a forward primer and a probe, or a reverse primer and a probe, the single-stranded nucleic acid is characterized in that it has a base sequence capable of complementary binding to a partial base sequence of the cDNA synthesized in the step b).

[0061] According to another specific example, when the single-stranded nucleic acid of the present invention is used as a forward primer and a probe, or a reverse primer and a probe, another reverse primer or forward primer can be used in addition to the single-stranded nucleic acid. In this case, the other reverse primer or forward primer is capable of complementary binding to the cDNA synthesized in the step b) and is a DNA composed of a base sequence of 5 to 30, preferably 10 to 30, bases. Here, the reverse primer may be the same as the RT primer in the step b).

[0062] In another specific example, when the single-stranded nucleic acid of the present invention is used as a forward primer, a reverse primer, and a probe, the single-stranded nucleic acid used as the reverse primer may be the same as the single-stranded nucleic acid in step b), and the detectable marker attached to both ends or inside of the single-stranded nucleic acid may be the same as or different from the single-stranded nucleic acid in step b).

[0063] In the present invention, the amplification of cDNA is preferably carried out by A) annealing the single-stranded nucleic acid of the present invention used as a forward primer and a probe, or a reverse primer and a probe, with a forward primer or a reverse primer commonly used in the art, or B) annealing the single-stranded nucleic acid of the present invention used as a forward primer, a reverse primer, and a probe, at an isothermal temperature that does not substantially inhibit the activity of the enzyme used. Here, the term "isothermal" means that there is no thermal cycling, and does not necessarily mean that the temperature is the same physically.

[0064] According to one specific example, the isothermal temperature at which the amplification reaction is carried out in the present invention can be 40 to 80°C, preferably 55 to 75°C, and more preferably 60 to 75°C.

[0065] In the present invention, the amplification reaction of the cDNA includes polymerase chain reaction, rolling circle amplification, strand displacement amplification and nucleic acid sequence-based amplification. eic acid sequence based amplification), but is not limited to these.

[0066] In the present invention, the amplification reaction of the cDNA can be carried out using a kit containing the single-stranded nucleic acid of the present invention, as well as reagents necessary for amplification, such as an appropriate amount of DNA polymerase (e.g., a thermostable DNA polymerase obtained from Thermus aquatiucus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermist flavus, Thermococcus litoralis, or Pyrococcus furiosis (Pfu)), a DNA polymerase cofactor (Mg 2+ The buffer may include, but is not limited to, an appropriate amount of Triton X-100, dimethylsulfoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide, and bovine serum albumin (BSA).

[0067] d) A step of measuring the amount of single-stranded nucleic acid fragments cleaved by an enzyme capable of cleaving the Y site of the single-stranded nucleic acid.

[0068] In the present invention, the amount of single-stranded nucleic acid fragments can be measured using various detection methods. Specifically, the amount of single-stranded nucleic acid fragments separated by the present invention is preferably measured in real time or after the reaction is completed, and can be measured by measuring the change in fluorescence intensity or chemiluminescence.

[0069] The device for measuring the change in fluorescence intensity or chemiluminescence may be any device known in the art that is capable of detecting a fluorescent marker, such as, but not limited to, a real-time PCR device, a TRIAD Multimode Detector, a Wallac / Victor fluorescence, a Perkin-Elmer LB50B luminescence spectrometer, a Light cycler 96, an Applied Biosystemus 7500, or a Boprad CFX96 real-time PCR termo cycler.

[0070] The method for measuring and detecting the amount of single-stranded nucleic acid fragments cleaved according to the present invention may vary depending on the type of single-stranded nucleic acid or label or detectable marker added to the reaction solution.

[0071] For example, when a single-stranded nucleic acid (promer) with a fluorescent donor attached to the X-terminus and a quencher attached to the Z-terminus hybridizes with a target small RNA or cDNA, and before the Y and Z-terminus are separated by an enzyme capable of cleaving the Y-terminus, the fluorescence of the fluorescent donor attached to the X-terminus is maintained in a state in which it is greatly reduced by the quencher attached to the Z-terminus. However, when the X-terminus is not separated by the enzyme capable of cleaving the Y-terminus, and only the Y and Z-terminus are separated from the target small RNA or cDNA and dispersed in the reaction solution, the fluorescence of the fluorescent donor attached to the X-terminus increases because it is no longer affected by the quencher attached to the Z-terminus. At this time, the fluorescence emission of the fluorescent donor attached to the X-terminus increases, and the target small RNA or cDNA can be detected in real time using the above-mentioned device.

[0072] According to one embodiment, a RT that exhibits complementary binding to small RNA is After synthesizing cDNA using a primer, in accordance with the present invention, a single-stranded nucleic acid that exhibits complementary binding to the cDNA and has FAM attached thereto was used as a forward or reverse primer to amplify the cDNA. As a result, it was confirmed that an amplification curve was observed during the process of amplifying the cDNA, unlike cDNA amplified using a conventional forward primer without a detectable marker.

[0073] The method for detecting small RNA according to the present invention is different from the conventional method using Taqman probe, MGB probe, or intercalator-type double-stranded DNA binder such as Sybr Green, and uses nucleic acid having an XYZ structure as a probe and a primer at the same time, so that the small RNA to be detected can be accurately amplified. Also, compared with the conventional technique of amplifying a template nucleic acid and measuring the amount of a probe decomposed by DNA polymerase at the same time, the method has an advantage that small RNA can be accurately detected from a small amount of sample.

[0074] As one specific example, human-derived miRNA was analyzed using the small RNA detection method of the present invention, and accurate analysis was possible even when the miRNA concentration was diluted to 1 / 10,000 times (see Examples 1 and 2).

[0075] As another specific embodiment, let-7 miRNA was analyzed in real time using the small RNA detection method of the present invention. As a result, when let-7 miRNA was present in trace amounts, for example, even at a concentration of 1 aM, it was possible to stably, rapidly and accurately analyze let-7 miRNA (see Examples 3 and 4).

[0076] In still another aspect, the present invention provides a method for detecting a protein molecule associated with a small RNA using the nucleic acid.

[0077] Specifically, the present invention provides a method for detecting a protein molecule associated with a small RNA, comprising the steps of: a) preparing an antibody to which a nucleic acid having a base sequence complementary to a single-stranded nucleic acid having a structure of XYZ is attached; b) binding a protein associated with a small RNA obtained from a biological sample with the antibody prepared in step a) to form a protein-antibody complex; c) hybridizing a single-stranded nucleic acid having a structure of XYZ to the complex to form a protein-antibody-single-stranded nucleic acid complex; and d) treating the protein-antibody-single-stranded nucleic acid complex with a cleavage reagent to separate single-stranded nucleic acid fragments from the antibody, and then measuring the amount of the separated single-stranded nucleic acid fragments to detect a protein associated with the small RNA.

[0078] Hereinafter, the method for detecting a protein molecule associated with a small RNA according to the present invention will be specifically described by each step.

[0079] a) A step of preparing an antibody having a nucleic acid attached thereto, the nucleic acid having a base sequence complementary to a single-stranded nucleic acid having the structure XYZ.

[0080] In the present invention, the structure, characteristics, etc. of the single-stranded nucleic acid having the structure XYZ are as described above, and therefore will not be described below.

[0081] In the present invention, the nucleic acid having a sequence complementary to the single-stranded nucleic acid having the XYZ structure is synthesized by an amplification reaction commonly used in the art using the base sequence of the nucleic acid having the XYZ structure as a template.

[0082] In the present invention, the antibody having a nucleic acid having a sequence complementary to the single-stranded nucleic acid having the XYZ structure attached thereto has a structure in which the nucleic acid having a sequence complementary to the single-stranded nucleic acid having the XYZ structure is linked to the Fc region of the antibody via a linker. The linker is typically a DNA consisting of a sequence of 1 to 10 bases.

[0083] Generally, two methods are used to link an antibody to a nucleic acid having a sequence complementary to the single-stranded nucleic acid having the XYZ structure. According to the first method, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is used. Succinimidyl-4-(N-Maleimidomethl)Cyclohexane-1-Carboxylate (Sulfo-SMCC), n-Succinimidyl-3-(2-Pyridylthio)Propionate (SPDP), N-Succinimidyl-6-[3'-(2-pyridyldithio)-propionamido]hexanoate (NHS-Ic-SPDP), or sulfosuccinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate (Sulfo The 5'-thiolated DNA is linked to the free amino group of the antibody using any one of the following reagents: succinimidyl-6-[3'-(2-pyridyldithio)-propionamido]hexanoate; sulfo-NHS-Ic-SPDP, etc. The reagents differ in the length of the spacer that separates them and in the degree of water solubility. If necessary, the linkage site can be cleaved by a thiolation reagent, which releases the DNA for further manipulation.

[0084] According to the second method, the tetrameric protein streptavidin provides the linking site between the antibody and DNA, and the protein forms an almost irreversible bond with biotin. The free amino groups of the antibody are biotinylated by reacting with biotin-n-hydroxysuccinimide. Biotinylation of DNA may be performed by using 5'-biotin phosphoramidite or by labeling the amino groups at the 5' end followed by reaction with biotin-n-hydroxysuccinimide. A conjugate of DNA, streptavidin and antibody can be prepared by adding 1 molar equivalent of antibody to the DNA-streptavidin complex.

[0085] Based on the above-mentioned method, the antibody bound to the nucleic acid is reacted at 4°C for 1 hour, and then the antibody-nucleic acid complex is purified using a Superdex 200 gel filtration column to obtain the antibody-nucleic acid complex.

[0086] The antibodies prepared according to the present invention specifically recognize only the protein to be detected and can easily be used to detect the desired protein in real time since the single-stranded nucleic acid of the present invention can hybridize thereto.

[0087] b) A step of binding a small RNA-associated protein to the antibody prepared in the step a) to form a protein-antibody complex.

[0088] In the present invention, the protein associated with the small RNA may be selected from the group consisting of an antibody, a ligand, a natural compound, an extract, a synthetic peptide, and a new drug candidate substance, but is not limited thereto.

[0089] In the present invention, binding of a small RNA-associated protein with an antibody may be carried out by mixing the target protein with the antibody prepared in the step a).

[0090] c) A step of hybridizing a single-stranded nucleic acid having the structure XYZ to the protein-antibody complex to form a protein-antibody-single-stranded nucleic acid complex.

[0091] In the present invention, hybridization of a protein-antibody complex with a single-stranded nucleic acid may be carried out by adding the single-stranded nucleic acid to the protein-antibody complex.

[0092] d) treating the protein-antibody-single-stranded nucleic acid complex with a cleavage reagent to separate the single-stranded nucleic acid fragments from the antibody, and then measuring the amount of the separated single-stranded nucleic acid fragments to detect the target protein molecule.

[0093] In the present invention, the cleavage reagent may be any one that can specifically cleave only the Y site of the single-stranded nucleic acid via an enzyme. For example, one selected from the group consisting of deoxyribonuclease (DNase), ribonuclease (RNase), helicase, exonuclease, endonuclease, etc., preferably ribonuclease, may be used.

[0094] In the present invention, the amount of single-stranded nucleic acid may be measured using various detection methods. Specifically, the amount of single-stranded nucleic acid fragments separated by the present invention is preferably measured in real time or after the end of the reaction, and may be measured by measuring the change in fluorescence intensity or chemiluminescence.

[0095] The change in fluorescence intensity or chemiluminescence may be measured using any device capable of detecting fluorescent markers known to those skilled in the art, such as, but not limited to, a TRIAD Multimode Detector, a Wallac / Victor Fluorescence, and a Perkin-Elmer LB50B luminescence spectrometer.

[0096] The method for measuring and detecting the amount of the separated single-stranded nucleic acid may vary depending on the form of the single-stranded nucleic acid or the label or detection marker introduced into the reaction solution.

[0097] The method for detecting a target protein according to the present invention has the advantage of improving the speed and accuracy of detecting a protein associated with a small RNA to be detected by using an antibody bound to a nucleic acid having a sequence complementary to a single-stranded nucleic acid having an XYZ structure, and a nucleic acid having an XYZ structure. Effect of the Invention

[0098] In the past, to analyze small RNA, cDNA was synthesized using an arbitrarily extended oligo, and detection was performed depending on the extended oligo. However, the single-stranded nucleic acid having the XYZ structure according to the present invention does not depend on an arbitrarily extended oligo sequence, but is purely small RNA. It is possible to amplify and detect the single-stranded nucleic acid by its sequence (RNA). This can prevent erroneous amplification or detection caused by detection methods that rely on an arbitrarily extended sequence. In addition, since the single-stranded nucleic acid is cleaved only by a cleavage reagent, it is advantageous in that the amount of the single-stranded nucleic acid to be detected can be measured more accurately than with conventional probes that are degraded by DNA polymerase.

[0099] Therefore, the method of detecting a small RNA or a protein associated with the small RNA using the nucleic acid having the structure XYZ of the present invention can rapidly and accurately detect the small RNA or the protein associated with the small RNA, and therefore can be useful for the diagnosis and prognosis of various diseases. [Brief description of the drawings]

[0100] [Figure 1] FIG. 1 is a schematic diagram showing two conventional methods for detecting small RNAs: a method using a TaqMan probe and a stem-loop primer, and a method using an oligo-dT adaptor primer with a polyA tail. [Diagram 2] FIG. 1 is a schematic diagram showing a method for synthesizing cDNA using an oligo-dT primer extended after the poly(A) tail of miRNA and detecting small RNA using the single-stranded nucleic acid (promer) according to the present invention. [Diagram 3] FIG. 1 is a schematic diagram showing a method for detecting miRNA using the single-stranded nucleic acid (promer) according to the present invention after synthesizing cDNA using an RT primer without poly(A)-tailing miRNA. [Figure 4] 1 is a graph showing the results of observing gene expression of miRNA-21 using the single-stranded nucleic acid (promer) according to the present invention. [Diagram 5] 1 is a graph showing the results of observing gene expression of miRNA-155 using the single-stranded nucleic acid (promer) according to the present invention. [Figure 6] FIG. 1 shows the results of observing expression after diluting miRNA-21 cDNA at various concentrations using the single-stranded nucleic acid (promer) according to the present invention. [Figure 7] FIG. 1 shows the results of observing expression after diluting miRNA-155 cDNA at various concentrations using the single-stranded nucleic acid (promer) according to the present invention. [Figure 8]FIG. 1 shows the results of observing gene expression of let-7a miRNA using the single-stranded nucleic acid (promer) according to the present invention. [Figure 9] FIG. 1 shows the results of observing gene expression of let-7d miRNA using the single-stranded nucleic acid (promer) according to the present invention. [Figure 10] FIG. 13 is a diagram showing the observation of expression after diluting cDNA of let-7a miRNA at various concentrations in the presence of let-7a lpM using the single-stranded nucleic acid (promer) according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] The present invention will be described in more detail below using examples, etc. These examples are merely intended to more specifically explain the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples, etc., in accordance with the gist of the present invention.

[0102] Example 1: Real-time analysis of human miRNA using single-stranded nucleic acid according to the present invention

[0103] miRNA-21(5'-uag cuu auc aga cug aug uug a), miRNA-155(5'-uua aug cua auc gug aua In order to measure the gene expression of ggg gu, the single-stranded nucleic acid (promoter), primer (primer), and RT primer according to the present invention were used in combination with IDT as shown in Table 1 below. The single-stranded nucleic acid (promoter) according to the present invention was prepared by requesting Integrated DNA Technologies, USA (see Table 1). Here, FAM (fluorescein succinimidyl ester) was attached to the 5' end and 3IABkFG was attached to the 3' end, and ribonucleotides (RNA) were indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0104] [Table 1]

[0105] Using the Exosomal miRNA purification Kit from Genolution Co., Ltd., miRNA was extracted from 200 μl of normal blood serum at a concentration of 11.9 ng / μl. 1 μl, 0.1 μl, and 0.01 μl of the extracted miRNA were reacted with Ambion's poly-(A)Tailing kit and Roche's Nxtscript RT kit (total 20 μl) at 45° C. in the presence of 1 μl of each RT primer at a concentration of 10 μM in Table 1 above for 30 minutes to synthesize cDNA.

[0106] In the presence of 0.5 μl of 10 μM concentration of each of the single-stranded nucleic acids and primers in Table 1 above, 1 μl of each of the synthesized cDNAs, 10 m units of heat-resistant RNase H, and 4 μl of AptaTaq DNA Master (prepared by Roche) were added, and the total volume was adjusted to 20 μl with triple distilled water, and then a polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95 ° C for 5 minutes, 63 to 64 ° C for 60 seconds, and 95 ° C for 10 seconds, and 45 cycles were performed. The results are shown in Figures 4 and 5.

[0107] As a result, it was confirmed that a highly reliable analysis was possible with only a trace amount of total miRNA (5.95 pg) using only 1 / 20 of the cDNA synthesized using only 119 pg of total miRNA.

[0108] Example 2: Real-time analysis of human miRNA using single-stranded nucleic acid according to the present invention

[0109] The cDNA synthesized from 1 μl of the miRNA having a concentration of 11.9 ng / μl in Example 1 was diluted 1 / 10, 1 / 100, 1 / 1000, 1 / 10000, and 1 / 100000 times.

[0110] In the presence of 0.5 μl of 10 μM concentration of each of the single-stranded nucleic acids and primers in Table 1 above, 1 μl of each of the diluted cDNAs, 10 m units of heat-resistant RNase H, and 4 μl of AptaTaq DNA Master (prepared by Roche) were added, and the total volume was adjusted to 20 μl with triple distilled water, and then polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95 ° C for 5 minutes, 63 to 64 ° C for 60 seconds, and 95 ° C for 10 seconds, and 55 cycles were performed. The results are shown in Figures 6 and 7.

[0111] As a result, an amplification curve was drawn up to cDNA diluted to 1 / 1000, and no amplification curve was shown for cDNA diluted further than that. This confirmed that there was no nonspecific amplification even for trace amounts of target, and confirmed that it is possible to analyze less than 10 copies of a specific miRNA using only trace amounts of miRNA.

[0112] Example 3: Real-time let-7 miRNA analysis using single-stranded nucleic acids according to the present invention

[0113] It is known that let-7 miRNA has the most isoforms among miRNAs. It is known that it is very difficult to distinguish between such let-7 isoforms, and it is particularly difficult to distinguish between isoforms with a specificity of less than 1%. In this example, in order to measure the gene expression of let-7a (5'-uga ggu agu agg uug uau agu u) and let-7d (5'-aga ggu agu agg uug cau agu u), which are the most difficult among them, the single-stranded nucleic acid, primer, and RT primer according to the present invention were prepared by IDT (Integrated DNA Technologies, USA) as shown in Table 2 below (see Table 2). In addition, for accurate quantification, the let-7 miRNA was prepared by IDT. Here, in the case of single-stranded nucleic acids, FAM (fluorescein succinimidyl ester) is attached to the 5' end and 3IABkFG is attached to the 3' end, and ribonucleotides (RNA) are indicated with the subscript "r" before the sequence to distinguish them from deoxyribonucleotides (DNA).

[0114] [Table 2]

[0115] 1 μl of the synthesized miRNA at a concentration of 20 pM and Ambion's poly-(A)T Using the Ailing kit and Roche's Nxtscript RT kit (total 20 μl), cDNA was synthesized by reaction at 45° C. for 30 minutes in the presence of 1 μl of each RT primer at a concentration of 10 μM in Table 2 above.

[0116] The synthesized cDNA was diluted to concentrations ranging from 100 fM to 1 aM.

[0117] After the synthesis was performed in the presence of 0.5 μl of the single-stranded nucleic acid and primer in Table 2 at a concentration of 10 μM, 2 μl of each diluted cDNA, 10 m units of heat-resistant RNase H, and 4 μl of AptaTaq DNA Master (prepared by Roche) were added, and the total volume was adjusted to 20 μl with triple distilled water, and then a polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95 ° C for 5 minutes, 63 to 64 ° C for 60 seconds, and 95 ° C for 10 seconds, and 45 cycles were performed. The results are shown in Figures 8 and 9.

[0118] As a result, it was confirmed that each miRNA could be analyzed using only 2 μl (approximately 1 copy) of diluted cDNA at a miRNA1aM concentration.

[0119] Example 4: Real-time detection of let-7 miRNA specificity using single-stranded nucleic acid according to the present invention

[0120] In the presence of 0.5 μl of 10 μM concentration of single-stranded nucleic acid and primer in Table 2 above, 2 μl of let-7a cDNA diluted 1 / 10 times from 100 fM to 1 aM was added to 2 μl of let-7d cDNA of 1 pM concentration prepared in Example 3, 10 m units of heat-resistant RNase H, 4 μl of AptaTaq DNA Master (prepared by Roche), and adjusted to a total volume of 20 μl with triple distilled water, and then polymerase chain reaction (PCR) was performed. At this time, the PCR reaction conditions were 95 ° C for 5 minutes, 63 to 64 ° C for 60 seconds, and 95 ° C for 10 seconds, and 45 cycles were performed. The results are shown in FIG. 10.

[0121] As a result, in an experimental group in which 2 μl of let-7a cDNA diluted 1 / 10 times from 100 fM to 1 aM was added to 2 μl of 1 pM let-7d cDNA, it was confirmed that stable analysis was possible from 100 fM to 1 fM, but the graph collapsed at 100 aM. This confirmed that it is possible to analyze isoform miRNA while maintaining specificity down to 0.1%.

Claims

1. A method for detecting small RNA using a single-stranded nucleic acid (promer), comprising: the single-stranded nucleic acid comprises a base sequence capable of complementary binding to a part of a base sequence of a cDNA, and can be used as (i) a forward primer and a probe, (ii) a reverse primer and a probe, or (iii) a forward primer, a reverse primer and a probe, The single-stranded nucleic acid has a structure of X-Y-Z, and one or more detectable markers are attached to both ends or inside of the single-stranded nucleic acid. The single-stranded nucleic acid binds to a specific site of a target nucleic acid for real-time detection to form a complex; X is a DNA consisting of 10 to 30 nucleotides, The Y is an RNA consisting of 1 to 10 nucleotides, Z is a DNA consisting of 2 or 3 nucleotides, the Y site can be cleaved by a specific enzyme, and upon cleavage of the Y site by the specific enzyme, the Y site and the Z site are separated from a specific site of the target nucleic acid, and the X site acts as a primer while remaining complexed with the target nucleic acid; The method comprises the steps of: a-1) obtaining RNA containing a small RNA to be detected from a biological sample; b-1) synthesizing cDNA from the RNA obtained in the step a-1); c-1) mixing the cDNA of step b-1) with the single-stranded nucleic acid; the single-stranded nucleic acid binds complementarily to a partial base sequence of the cDNA, the Y site is cleaved by the specific enzyme, the Y site and the Z site of the single-stranded nucleic acid are separated from the specific site of the cDNA, and the X site acts as a primer while maintaining a complex with the cDNA, thereby amplifying the cDNA; d-1) measuring the amount of the single-stranded nucleic acid fragment cleaved by an enzyme capable of cleaving the Y site of the single-stranded nucleic acid in the c-1) step, The step c-1) comprises: (i) providing a single-stranded nucleic acid having a base sequence capable of complementary binding to a part of a base sequence of cDNA when the single-stranded nucleic acid is used as a forward primer and a probe; and (ii) when the single-stranded nucleic acid is used as a reverse primer and a probe, a forward primer having a base sequence capable of complementary binding to a portion of a base sequence of cDNA is further included.

2. The method for detecting small RNA according to claim 1, wherein the detectable marker is a fluorescent substance bound to the single-stranded nucleic acid by a covalent or non-covalent bond, or a fluorescent pair consisting of a fluorescent substance and a quencher.

3. 2. The method for detecting small RNA according to claim 1, wherein the enzyme used is any one of RNase II, RNase III, RNase IV, RNase H, and RNase T2.

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