JP7743246B2 - Target RNA detection method and probe kit - Google Patents

Description

The present invention relates to a target RNA detection method and a probe kit, and more specifically to a target RNA detection method and a probe kit that can be suitably used for the method.

Cells contain large amounts of short-stranded RNAs that are not translated into proteins. While these short-stranded RNAs were once thought to have no function, research since the 1990s has revealed that they play important roles in living organisms. For example, among these short-stranded RNAs, single-stranded RNAs approximately 19 to 25 bases long known as microRNAs (miRNAs) have been shown to play an important role in cellular function by regulating gene expression.

The miRNA production process is typically as follows: First, the miRNA gene on the genome is transcribed by RNA polymerase to produce a primary miRNA (primary miRNA) containing a hairpin structure. The primary miRNA is then cleaved by the nuclear RNase Drosha to produce a precursor miRNA (precursor miRNA) with a hairpin structure of 60-70 bases. The precursor miRNA is then transported from the nucleus to the cytoplasm, where it is cleaved by the cytoplasmic RNase Dicer to become a 21-24 base double-stranded RNA (miRNA duplex). The miRNA duplex is then incorporated into the Ago protein, and only one of the RNA strands of the duplex ultimately becomes the mature miRNA.

Mature miRNAs bind to Ago proteins to form RNA-induced silencing complexes (RISCs). RISCs bind to target mRNAs that contain sequences partially complementary to the base sequence of the mature miRNAs contained within them, suppressing the translation of the target mRNAs and thereby regulating the expression of numerous genes.

It is also becoming clear that miRNAs are closely related to a variety of diseases. In recent years, it has been reported that miRNAs exhibit abnormal expression in various diseases, including cancer, and there are hopes for the development of technology to detect such miRNAs as diagnostic tools for these diseases.

The microarray method, which uses a microarray pre-loaded with probes that specifically bind to target RNA, is widely used as a method for detecting short-stranded RNAs such as miRNAs (see, for example, Patent Document 1).

Nucleic acid amplification methods are also used to detect short-stranded RNAs such as miRNAs. For example, a method has been developed in which the target miRNA is polyadenylated to add a polyA sequence, cDNA is synthesized by reverse transcription using a primer containing a polyT sequence and a sequence complementary to the base sequence of the target miRNA, and nucleic acid amplification is performed using this cDNA as a template to detect the target miRNA (Patent Document 2).

Furthermore, a method for detecting a target miRNA has also been developed, for example, by hybridizing the target miRNA to a primer having a 3' region and stem-loop that are complementary to the base sequence of the target miRNA, elongating the primer, and then amplifying the nucleic acid using the elongated primer as a template (Patent Document 3).

In addition, as a method for detecting nucleic acids using nucleic acid amplification, for example, a method has been developed in which a chimeric probe containing RNA in part is hybridized to target DNA, and the resulting DNA-RNA hybrid chain is cleaved with ribonuclease to release the chimeric probe fragment, which is then used as a primer for an extension reaction to detect the target DNA (Patent Document 4).

Furthermore, methods for detecting specific polynucleotides have been developed, for example, by hybridizing a non-detectable polynucleotide sequence with a labeled nucleic acid probe containing a complementary base sequence, and then detecting the resulting DNA-RNA hybrid chain using an anti-DNA/RNA antibody (Patent Document 5). BioVendor has also developed a kit for hybridizing a target miRNA with a biotinylated DNA probe containing a complementary base sequence, and then detecting the resulting DNA-RNA-biotin complex using an ELISA method using an anti-DNA/RNA antibody (Non-Patent Document 1).

Japanese Patent Application Laid-Open No. 2007-075095 US Patent Application Publication No. 2009/0220969 U.S. Patent No. 7,575,863 Japanese Patent Application Laid-Open No. 2008-307029 Japanese Patent Application Publication No. 179657/1983

DENIS Pharma Co., Ltd., "miREIA-miRNA (micro RNA) Enzyme Immunoassay Kit," [online], [searched August 5, 2021], Internet <URL: https://research.sceti.co.jp/images/upload/flyer/16/16.pdf>

However, the microarray method has problems such as complicated operations and high cost. Furthermore, with commonly used microarray methods and nucleic acid amplification methods, the target nucleic acid is first amplified and then detected, so the results are often dependent on the accuracy of the amplification reaction. Furthermore, the reverse transcription reaction can cause variations in detection accuracy, resulting in problems with not accurately reflecting the amount of target nucleic acid in the sample. Furthermore, the inventors have newly discovered that the conventional method of detecting DNA-RNA complexes by ELISA using anti-DNA/RNA antibodies has insufficient sensitivity depending on the type of target RNA, and that the applicable target RNA is particularly limited when detecting short-stranded RNA such as miRNA.

The present invention was made in consideration of the problems associated with the prior art described above, and aims to provide a target RNA detection method that can detect even short-stranded RNAs such as miRNAs with high sensitivity and ease, as well as a probe kit that can be suitably used for this method.

The inventors conducted extensive research to achieve the above-mentioned objectives, and in a method for capturing and detecting a complex formed by hybridizing a target RNA with a DNA probe using an anti-DNA/RNA antibody, they designed the DNA probe into two types: a DNA probe to which a labeling substance is bound, and a chimeric probe that forms a stem-loop structure, the stem region of which is a DNA/RNA hybrid. They found that by using these probes in combination, detection sensitivity can be significantly improved, even when the target RNA is a short-stranded RNA such as miRNA. Furthermore, because this method does not require a nucleic acid amplification reaction, highly accurate quantification that reflects the amount of target RNA in a sample is possible, regardless of the accuracy of the amplification reaction.

Furthermore, with this method, the steps from hybridization to detection can be carried out isothermally without the need for temperature control such as heating, making it possible to detect the amount of target RNA with high accuracy. In addition to the fact that the above-mentioned nucleic acid amplification reaction is unnecessary, the inventors have also discovered that target RNA can be detected significantly more easily than conventional methods, leading to the completion of the present invention.

The aspects of the present invention obtained based on these findings are as follows.
[1]
1. A method for detecting a target RNA, comprising:
(I) a first region on the 5'-end or 3'-end side of a single-stranded target RNA, and a second region including the other end of the first region and not overlapping with the first region;
a hybridization step of hybridizing a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region of the target RNA and a region SG to which a labeling substance can be bound, and a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure;
(II) a capturing step of capturing a complex of the target RNA, the DNA probe, and the chimeric probe with a capturing body comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier;
(III) a detection step of detecting the complex captured by the capturer;
A method for detecting a target RNA, comprising:
[2]
The target RNA detection method according to [1], wherein the detection step is a step of detecting the complex using a signal derived from a labeling substance bound to the region SG as an indicator.
[3]
The method for detecting a target RNA according to [1] or [2], wherein the target RNA is a single-stranded RNA molecule having a length of 19 to 25 bases.
[4]
The method for detecting a target RNA according to any one of [1] to [3], wherein the linker is a base sequence consisting of DNA and/or RNA.
[5]
The method for detecting a target RNA according to [4], wherein the linker has a length of 3 to 50 bases.
[6]
The method for detecting a target RNA according to any one of [1] to [5], wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long.
[7]
The method for detecting a target RNA according to any one of [1] to [6], wherein the temperature in the hybridization step is 15 to 60°C.
[8]
The method for detecting a target RNA according to any one of [1] to [7], wherein the water-insoluble carrier is a particle carrier.
[9]
a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region on the 5'-end or 3'-end of a single-stranded target RNA, and a region SG to which a labeling substance can be bound;
a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region that includes the other end of the first region side of the target RNA and does not overlap with the first region, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure;
A probe kit comprising:
[10]
The probe kit according to [9], further comprising a labeling substance capable of binding to the region SG of the DNA probe.
[11]
The probe kit according to [9] or [10], wherein the total length of the base sequence D1 and the base sequence D2 is 10 to 100 bases long.
[12]
The probe kit according to any one of [9] to [11], wherein the linker is a base sequence consisting of DNA and/or RNA.
[13]
The probe kit according to [12], wherein the linker has a length of 3 to 50 bases.
[14]
The probe kit according to any one of [9] to [13], wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long.
[15]
The probe kit according to any one of [9] to [14], further comprising a capture body comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier.
[16]
The probe kit according to [15], wherein the water-insoluble carrier is a particle carrier.
[17]
The probe kit according to any one of [9] to [16], which is a kit for use in the target RNA detection method according to any one of [1] to [8].

While the reason why the above-mentioned object is achieved by the configuration of the present invention is not entirely clear, the inventors speculate as follows. Specifically, in the present invention, as described above, DNA probes to be hybridized to target RNA are divided into two types: DNA probes to which a labeling substance is bound, and chimeric probes that form a stem-loop structure and whose stem region is a DNA-RNA hybrid. The base sequences of these probes are designed based on the base sequence of the target RNA, and they hybridize to non-overlapping regions (first region, second region) on the target RNA. The stem-loop structure of the chimeric probe adds RNA with a base length equal to the stem region in addition to the base length of the target RNA. This increases the recognition site for anti-DNA-RNA chimeric antibodies, which is presumably responsible for the increased sensitivity of detection of the target RNA.

Furthermore, in conventional methods in which a DNA probe is hybridized to the entire length of a target RNA, the Tm value of the DNA probe tends to increase with base length. While RNA and DNA hybridization is possible at low temperatures, maintaining sufficient hybridization accuracy typically requires a temperature approximately 5°C lower than the Tm (e.g., 55°C or higher). Hybridization at temperatures lower than this increases nonspecificity and reduces accuracy. In contrast, in the present invention, the probe hybridized to the target RNA is divided into two as described above, allowing the Tm value of each probe to be lowered. It is believed that sufficient hybridization accuracy can be maintained even when the process from hybridization to detection is carried out at room temperature (e.g., approximately 37°C or lower) and isothermal (all processes at the same temperature). Therefore, the inventors believe that, combined with the elimination of the nucleic acid amplification reaction described above, this method makes it possible to detect target RNA with high sensitivity in a significantly simpler manner than before.

The present invention makes it possible to provide a target RNA detection method that can detect even short-stranded RNAs such as miRNAs with high sensitivity and ease, as well as a probe kit that can be suitably used for this method.

1 is a schematic conceptual diagram showing one embodiment of a target RNA, a DNA probe, a chimeric probe, and a complex thereof according to the present invention. FIG. 1 is a schematic conceptual diagram showing another embodiment of the target RNA, DNA probe, chimeric probe, and complex thereof according to the present invention.

The present invention will be described in more detail below using preferred embodiments as examples, but the present invention is not limited to these.

<Method for detecting target RNA>
The method for detecting a target RNA of the present invention comprises:
(I) a first region on the 5'-end or 3'-end side of a single-stranded target RNA, and a second region including the other end of the first region and not overlapping with the first region;
a hybridization step of hybridizing a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region of the target RNA and a region SG to which a labeling substance can be bound, and a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure;
(II) a capturing step of capturing a complex of the target RNA, the DNA probe, and the chimeric probe with a capturing body comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier;
(III) a detection step of detecting the complex captured by the capturer;
The method includes:

Preferred embodiments of the target RNA detection method of the present invention will be described in detail below, with reference to Figures 1 and 2 as examples, but the present invention is not limited thereto. Figures 1 and 2 are schematic conceptual diagrams showing embodiments of the target RNA, DNA probe, chimeric probe, and complex thereof according to the present invention, respectively. In the following description and drawings, identical or corresponding elements are designated by the same reference numerals, and redundant explanations will be omitted.

In the present invention, a "base sequence consisting of DNA" refers to a base sequence consisting of deoxyribonucleotides (oligoDNA), a "base sequence consisting of RNA" refers to a base sequence consisting of ribonucleotides (oligoRNA), and a "base sequence consisting of DNA and/or RNA" refers to a base sequence consisting of deoxyribonucleotides, a base sequence consisting of ribonucleotides, or a base sequence consisting of deoxyribonucleotides and ribonucleotides (oligonucleotide).

Furthermore, the nucleotides constituting the target RNA, DNA probe, and chimeric probe of the present invention do not have to be composed solely of natural nucleotides (deoxyribonucleotides and/or ribonucleotides), and may, for example, be composed partially or entirely of the non-natural nucleotides. Furthermore, the method for obtaining the base sequences constituting the DNA probe and chimeric probe of the present invention is not particularly limited, and conventionally known methods or methods equivalent thereto can be appropriately adopted. For example, they can be chemically synthesized using a commercially available synthesizer to produce synthetic oligonucleotides.

Furthermore, in the present invention, a "complementary base sequence" to a certain base sequence (or region) is a base sequence that can hybridize with another base sequence to form a double-stranded strand; it does not have to be completely complementary. When "base sequence (or region, hereinafter the same) X and base sequence (or region, hereinafter the same) Y are complementary," the hybridization conditions are preferably such that the sequence complementarity between base sequence X and base sequence Y is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Those skilled in the art can appropriately calculate the sequence complementarity using known methods (e.g., BLAST (NCBI)). Furthermore, the hybridization conditions may be such that base sequence X is completely complementary to the entire length of base sequence Y, with 1 to 5 consecutive bases (preferably 1 to 3 bases, e.g., 3, 2, or 1 base) inserted, deleted, or substituted at one or more positions.

(Target RNA)
In the present invention, "target RNA" refers to RNA to be detected by the target RNA detection method of the present invention. In the present invention, the target RNA is not particularly limited as long as it can hybridize with the DNA probe and chimeric probe described below. The target RNA may be single-stranded in the region to which the DNA probe and chimeric probe hybridize, and may be double-stranded partially containing such a single-stranded region, or may have a three-dimensional structure such as a hairpin structure or a hammerhead structure.

Among these, examples of target RNAs according to the present invention include mRNA (messenger RNA), viral RNA, fragmented RNA, miRNA (microRNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), and siRNA (small interfering RNA). From the perspective of particularly effective application of the target RNA detection method of the present invention, RNA molecules having a length of 10 to 100,000 bases are preferred, single-stranded RNA molecules having a length of 15 to 30 bases are more preferred, and miRNA is even more preferred. Note that "miRNA" generally refers to single-stranded RNA molecules having a length of 19 to 25 bases, which are not translated into protein and are said to be primarily involved in regulating gene expression after transcription in eukaryotes.

miRNAs are also known as markers for various diseases. Examples of such miRNAs include miR21-5p, Let7a, miR1185-3p, miR6875-5p, miR17-3p, miR421, miR27-a-3p, miR149-3p, miR-192-5p, miR-15b-5p, miR-125b, miR-155-5p, miR-885-5p, and miR-1306-5p. Among these, miR21-5p, Let7a, miR1185-3p, miR6875-5p, miR17-3p, miR421, miR27-a-3p, miR149-3p, miR-192-5p, miR-15b-5p, etc. can be detected with particularly higher sensitivity than conventional detection methods using the target RNA detection method of the present invention.

Such target RNA is not particularly limited and may be extracted from a sample that may contain the target RNA or artificially synthesized. The sample that may contain the target RNA is also not particularly limited and may include, for example, solutions containing chemically synthesized RNA, various organisms (including cells, tissues, organs, and individuals) and extracts thereof, human and animal bodily fluids (saliva, nasal aspirate, nasal swab, throat swab, gargle, nasal discharge, tears, sweat, urine, sputum, bronchoalveolar lavage fluid, blood, serum, plasma, cerebrospinal fluid, lymph, semen, amniotic fluid, etc.) and feces, plant biological fluids, biological culture solutions, environmental water (rivers, lakes, harbors, waterways, groundwater, purified water, sewage, wastewater, etc.), and suspensions of solids (soil, cinders, etc.). Furthermore, the sample may be diluted or suspended in a diluent, or may have its pH adjusted as appropriate. Examples of the diluent include biochemical buffers such as sodium phosphate buffer, TriS buffer, phosphate buffer, and Good's buffer. Furthermore, any known method can be used as an appropriate method for extracting target RNA from the sample.

To illustrate the correspondence with the DNA probes and chimeric probes described below, the single-stranded portion of the target RNA will hereinafter be referred to for convenience as including a first region and a second region that do not overlap each other. As shown in Figure 1 (a1), when the first region (111) is set at the 5' end of the target RNA (110), the second region (112) is set at the other end of the first region, i.e., the 3' end of the target RNA. As shown in Figure 2 (a2), when the first region (121) is set at the 3' end of the target RNA (120), the second region (122) is set at the other end of the first region, i.e., the 5' end of the target RNA.

However, the configuration of the first region and the second region is not limited to the configuration shown in Figures 1 and 2. For example, the first region (111, 121) does not have to include the terminal portion of the single-stranded portion (110, 120) of the target RNA. In this case, the first region may be located at a distance of one or more bases from the 5' or 3' end of the target RNA. On the other hand, the second region (112, 122) preferably includes the terminal portion of the target RNA (110, 120) (i.e., located one base from the 5' or 3' end of the target RNA). Furthermore, the first and second regions do not have to be adjacent to each other and may be located via a region (spacer) of one or more bases (e.g., preferably 1 to 15 bases in length when the target RNA is the miRNA), but it is more preferable that they are adjacent to each other.

The preferred ranges for the length of the first region and the length of the second region according to the present invention correspond to the preferred ranges for the length of base sequence D1 and the length of base sequence D2, respectively, shown below.

(DNA probe)
The "DNA probe" according to the present invention is an oligonucleotide probe comprising a base sequence D1 consisting of DNA complementary to the first region of the target RNA, and a region SG to which a labeling substance can be bound.

The total length of the DNA probe according to the present invention can be adjusted depending on the length of the target RNA, but is preferably 5 to 100 bases long, more preferably 10 to 60 bases long, and even more preferably 15 to 40 bases long. For example, when the target RNA is the miRNA, it is preferably 15 to 30 bases long.

[Base sequence D1]
The base sequence D1 according to the present invention is a base sequence consisting of DNA, which is complementary to the first region of the target RNA. Such base sequence D1 can be appropriately designed and prepared to match the base sequence of the first region of the target RNA of interest so as to satisfy the above-mentioned hybridization conditions, and more preferably so as not to hybridize to the chimeric probe described below or other regions.

The length of the base sequence D1 according to the present invention is preferably 5 bases or more, more preferably 5 to 50 bases, and, for example, when the target RNA is the miRNA, preferably 5 to 20 bases. If the length of the base sequence D1 is less than the lower limit, it tends to be less likely to hybridize with the first region of the target RNA. On the other hand, if the length exceeds the upper limit, non-specific reactions tend to occur more easily.

[Area SG]
The region SG according to the present invention is a region consisting of a site to which a labeling substance can be bound. The region SG (site to which a labeling substance can be bound) may be a region consisting of multiple nucleotides, or a region consisting of only a portion of a specific nucleotide. When the region SG is a region consisting of multiple nucleotides, the nucleotide contained in the region SG is preferably a deoxyribonucleotide.

The form of such region SG is not particularly limited. In Figures 1 and 2, base sequence D1 (211, 221) and region SG (212, 222) are separate regions, with one region SG located adjacent to the 3'-end (212) of base sequence D1 (211) or the 5'-end (222) of base sequence D1 (221). However, region SG may be included in base sequence D1, or multiple regions may be present. Furthermore, when base sequence D1 and region SG are separate regions, base sequence D1 and region SG may be separated by a region (spacer) 1 to 50 bases in length (preferably 1 to 20 bases in length). Examples of such spacers include poly(A) sequences. Among these, the preferred embodiment of region SG is that when the DNA probe forms a complex (410, 420) with the target RNA and the chimeric probe, it is located, if necessary via a spacer, at the end of base sequence D1 opposite to the end where base sequence D2 below is located.

The site to which the labeling substance can be attached is not particularly limited and can be appropriately selected depending on the method of attachment with the labeling substance described below. For example, when the labeling substance described below is attached to a DNA probe via binding between biotin and streptavidin (or avidin), the site to which the labeling substance can be attached can be a site modified with biotin. Such modification with biotin (biotinylation) can be achieved by any conventionally known method or a method similar thereto, such as, but not limited to, a method in which biotin is introduced to the 5' end of base sequence D1 during its synthesis using biotin phosphoramidite to produce the DNA probe; or a method in which a 5'-amino-modified oligo is synthesized and a biotin residue is added to the amino group of the oligo using a biotin-X-NHS ester reagent.

[Labeling substance]
In the method for detecting a target RNA of the present invention, a labeling substance is bound to a DNA probe via the region SG, and the target RNA is detected using a signal derived from the labeling substance as an indicator. The labeling substance may be bound to the DNA probe in advance before the hybridization step (I) described below, or may be bound to the DNA probe after the hybridization step (I) or after the capture step (II) described below.

The labeling substance can be any substance used as a labeling substance in known immunological assay methods or methods similar thereto, without any particular limitation. Examples include enzymes; particles such as latex particles and gold colloid particles; low-molecular-weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); and radioactive substances such as ^3H , ^32P , ^35S , and ^125I , and these may be used alone or in combination of two or more.

For example, when an enzyme is used as the labeling substance, various detection methods can be performed depending on the substrate by adding a luminescent substrate such as luminol, luciferin, or lucigenin, a chromogenic substrate, or a fluorescent substrate. Examples of the enzyme include peroxidase (POD: horseradish peroxidase (HRP), etc.), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, β-D-glucosidase, and luciferase. Among these, bioluminescent enzymes such as luciferase are preferred due to their particularly high sensitivity.

The method for binding the labeling substance to the region SG may involve directly or indirectly binding the labeling substance to the DNA probe.

When directly binding, it is preferable to bind the DNA probe in advance before the hybridization step (I) described below, and direct binding can be achieved by a conventionally known method or a method similar thereto.

When the labeling substance is indirectly bound, for example, it can be bound to the DNA probe via an intervening molecule that binds to the DNA probe, thereby indirectly binding the labeling substance to the DNA probe. The intervening molecule is not particularly limited, and any conventionally known molecule can be used as appropriate. Alternatively, the region SG of the DNA probe may be modified in some way, and a substance that captures the modified portion may be bound to the labeling substance, thereby indirectly binding the labeling substance to the DNA probe. For example, a typical example of the modified portion is biotin, and typical examples of a substance that captures the modified portion include streptavidin or avidin, but these are not limited thereto.

(Chimeric probe)
The "chimeric probe" according to the present invention is an oligonucleotide probe comprising, in this order, a base sequence D2 consisting of DNA complementary to the second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure.

The total length of the chimeric probe according to the present invention can be adjusted depending on the length of the target RNA, but is preferably 10 to 200 bases long, and if the target RNA is the miRNA, it is preferably 15 to 50 bases long.

[Base sequence D2]
The base sequence D2 according to the present invention is a base sequence that is complementary to the second region of the target RNA and is composed of DNA. Such base sequence D2 can be appropriately designed and prepared to match the base sequence of the second region of the target RNA of interest so as to satisfy the hybridization conditions described above, and more preferably so as not to hybridize to the DNA probe or other regions.

The length of base sequence D2 in the present invention is preferably 5 bases or more, more preferably 5 to 50 bases, and, for example, when the target RNA is the miRNA, preferably 5 to 20 bases. If the length of base sequence D2 is less than the lower limit, it tends to be difficult to hybridize with the second region of the target RNA. On the other hand, if the length exceeds the upper limit, non-specific reactions tend to occur more easily, or when the target RNA is a short-stranded RNA, base sequence D1 tends to bind less easily.

In addition, in the present invention, the total length of base sequence D2 and base sequence D1 is preferably 10 to 100 bases long, and for example, when the target RNA is the miRNA, it is preferably 10 to 25 bases long. If the total length is less than the lower limit, hybridization with the target RNA or recognition by an anti-DNA/RNA chimeric antibody tends to be difficult. On the other hand, if the total length exceeds the upper limit, non-specific reactions tend to occur more easily.

Furthermore, in the present invention, the ratio of the length of base sequence D2 to the length of base sequence D1 is set depending on the base sequence of the target RNA, taking into consideration the length of the target RNA, the sequence complementarity between base sequence D1 and the first region, the sequence complementarity between base sequence D2 and the second region, and the balance between the Tm value obtained from base sequence D1 and the Tm value obtained from base sequence D2, and therefore cannot be generalized. However, for example, the ratio of the base length of base sequence D2 to the base length of base sequence D1 is preferably 1:9 to 9:1. For example, when the target RNA is the miRNA, the ratio is preferably 1:4 to 4:1, and more preferably 1:3 to 4:1.

[Base sequence SD, linker, base sequence SR]
The base sequence SD according to the present invention is a base sequence made of DNA, the base sequence SR is a base sequence made of RNA, and the linker is a region connecting the base sequence SD and the base sequence SR.

When the chimeric probe of the present invention forms a complex (410, 420) with the target RNA and DNA probe, it contains the base sequence SD, a linker, and the base sequence SR, in this order, at the end of base sequence D2 opposite the end where base sequence D1 is located, and the base sequence SD, linker, and base sequence SR form a stem-loop structure. Depending on the positions of the first and second regions set on the target RNA and the DNA probe to be combined, chimeric probes that form such a stem-loop structure may be in the form (310) of base sequence D1 (311) - base sequence SD (312) - linker (313) - base sequence SR (314) - 5' from the 3' end, as shown in Figure 1, or in the form (320) of base sequence D1 (321) - base sequence SD (322) - linker (323) - base sequence SR (324) - 3' from the 5' end, as shown in Figure 2.

The base sequence SD and the base sequence SR are composed of complementary base sequences, and form a stem region that is a DNA-RNA chimera in the stem-loop structure. The lengths of the base sequence SD and the base sequence SR are not particularly limited, but are preferably each independently 3 to 30 bases long. They are also preferably the same length. If the lengths of the base sequence SD and the base sequence SR are less than the lower limit, they tend to be difficult to hybridize with each other. On the other hand, if they exceed the upper limit, non-specific reactions tend to occur more easily, or the anti-DNA-RNA chimera antibody may compete with the target complex, resulting in reduced reactivity.

The linker forms a loop region in the stem-loop structure that connects the base sequence SD and the base sequence SR in a hairpin loop shape. Such a linker is not particularly limited as long as it is a linear molecule that can link the base sequence SD and the base sequence SR. For example, oligonucleotides, oligopeptides, and other well-known linear molecules commonly referred to as linkers can be used. Among these, the linker of the present invention is preferably an oligonucleotide, from the viewpoint that it can be easily produced in conjunction with the chemical synthesis of the oligonucleotides base sequence SD and base sequence SR when producing the chimeric probe. Examples of the oligonucleotide include a base sequence consisting of deoxyribonucleotides, a base sequence consisting of ribonucleotides, and a base sequence consisting of deoxyribonucleotides and ribonucleotides. Because the base sequence SD and the base sequence SR are connected by the linker, the stem structure can be easily formed and RNA consisting of the base sequence SR can be added to the target RNA.

When the linker according to the present invention is an oligonucleotide, it is preferably 3 to 50 bases long, and more preferably 5 to 20 bases long. If the linker length is less than the lower limit, it tends to be difficult to bend into a hairpin loop. On the other hand, if the length exceeds the upper limit, self-annealing does not occur preferentially, making it difficult to form a stem structure.

The sequences of the base sequence SD, linker, and base sequence SR can be appropriately designed and prepared so that the base sequence SD and the base sequence SR meet the above hybridization conditions, and more preferably so that they do not hybridize to the DNA probe or other regions.

(Capture body)
The capture body of the present invention is a complex comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier, and the water-insoluble carrier and the anti-DNA/RNA chimeric antibody are bound directly or indirectly to each other.

[Non-water-soluble carrier]
In the present invention, the "water-insoluble carrier" is not particularly limited as long as it is capable of immobilizing and supporting an anti-DNA/RNA chimeric antibody and is insoluble in water at room temperature and normal pressure, and examples thereof include water-insoluble carriers conventionally known in immunoassays and the like.

More specifically, examples of such carriers include particle carriers such as latex particles such as polystyrene latex particles and polyethylene latex particles, metal colloid particles such as gold colloid particles, gelatin particles, and magnetic particles; plate carriers such as polystyrene well plates; and membrane carriers made of fibers such as nitrocellulose, polyethylene, polyethylene terephthalate, nylons, glass, and cellulose. Generally used materials may be any of artificial materials, natural materials, and mixtures thereof. Among these, particle carriers are preferred as the water-insoluble carrier of the present invention from the viewpoint of ease of automation, and magnetic particles are even more preferred.

In the present invention, "supporting" is synonymous with "solid phase formation" (also referred to as "immobilization"). The supporting method may be a conventionally known method, such as physical adsorption or chemical bonding, or a method based thereon, as appropriate.

[Anti-DNA/RNA chimeric antibody]
In the present invention, the term "anti-DNA/RNA chimera antibody" refers to an antibody that specifically binds to a double-stranded nucleotide consisting of an oligo-RNA composed of ribonucleotides and an oligo-DNA composed of complementary deoxyribonucleotides (sometimes referred to as a "DNA/RNA chimera" in this specification).

In the present invention, the term "antibody" includes not only complete antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab') ₂ , Fv, single-chain antibodies, diabodies, etc.) and minibodies formed by combining antibody variable regions. The anti-DNA/RNA chimeric antibody of the present invention may be either a polyclonal or monoclonal antibody, with monoclonal antibodies being more preferred. The anti-DNA/RNA chimeric antibody of the present invention can be produced by appropriately adopting and improving conventionally known production methods, and commonly available antibodies may also be used as appropriate.

The capture body of the present invention can be produced by binding the anti-DNA/RNA chimeric antibody to the water-insoluble carrier. This production method can be any conventionally known method or a method based thereon, as appropriate. The anti-DNA/RNA chimeric antibody can be bound directly or indirectly to the water-insoluble carrier.

When directly binding is performed, for example, the water-insoluble carrier and/or the anti-DNA/RNA chimeric antibody may have an active group, or the active group may be added as needed, allowing the anti-DNA/RNA chimeric antibody to be directly bound to the water-insoluble carrier via the active group. Furthermore, when the water-insoluble carrier is a plate, the anti-DNA/RNA chimeric antibody can be applied to the plate, blocked as needed, and then dried, thereby directly binding the anti-DNA/RNA chimeric antibody to the water-insoluble carrier.

Indirect binding can be achieved by, for example, binding the anti-DNA/RNA chimeric antibody to the water-insoluble carrier via an intervening molecule that binds to the anti-DNA/RNA chimeric antibody. The intervening molecule is not particularly limited, and examples include a secondary antibody capable of binding to the anti-DNA/RNA chimeric antibody, protein G, protein A, and a molecule having the active group. Alternatively, the anti-DNA/RNA chimeric antibody may be modified in some way, and a substance that captures the modified portion may be immobilized on the water-insoluble carrier, thereby immobilizing the anti-DNA/RNA chimeric antibody on the water-insoluble carrier. For example, a typical example of the modified portion is biotin, and a typical example of a substance that captures the modified portion is streptavidin or avidin, but these examples are not limited thereto. Furthermore, commercially available capture bodies may be used as appropriate.

(complex)
As described above, the base sequence D1 of the DNA probe and the base sequence D2 of the chimeric probe are designed based on the base sequences of the first and second regions of the target RNA, respectively. Therefore, by hybridizing these, a complex consisting of the target RNA, the DNA probe, and the chimeric probe (target RNA-DNA probe-chimeric probe complex (sometimes simply referred to as "complex" in this specification)) is formed. When the first region is set on the 5'-end side of the target RNA, as shown in (b1) of Figure 1, the base sequence D1 (211) complementary to the first region (111) hybridizes, and the base sequence D2 (311) complementary to the second region (112) hybridizes, thereby forming a complex (410) consisting of the target RNA (110), the DNA probe (210), and the chimeric probe (310). In this case, a stem-loop structure is formed at the 5'-end of the chimeric probe (310) by the base sequence SD (312), the linker (313), and the base sequence RD (314), and RNA corresponding to the base sequence RD (314) is arranged at the 3'-end of the target RNA (110). When the first region is arranged at the 3'-end of the target RNA, the 5'-end and 3'-end are reversed from those in Figure 1, as shown in (b2) of Figure 2.

(Hybridization step (I))
The target RNA detection method of the present invention includes, as a first step, a hybridization step in which the DNA probe and the chimeric probe are hybridized to the target RNA.

In the hybridization step, the target RNA, the DNA probe, and the chimeric probe are contacted to form the complex (target RNA-DNA probe-chimeric probe complex). The contacting method is not particularly limited, and the three may be contacted simultaneously, the DNA probe and the chimeric probe may be added to the target RNA simultaneously or separately, or one may be added to the DNA probe or the chimeric probe simultaneously or separately.

A reaction buffer may be added to the reaction system of the hybridization step as appropriate. Examples of the reaction buffer include known buffers with a pH of 5 to 9 (e.g., sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also contain surfactants, salts, preservatives, stabilizers (e.g., Mg), proteins, etc. as appropriate.

In the hybridization step, the amounts of the DNA probe and the chimeric probe used in the reaction system are each independently preferably 10 fM to 10 mM, and more preferably 10 pM to 10 μM.

In the present invention, the temperature for the hybridization step is, for example, in the range of 15 to 60°C. However, the hybridization step according to the present invention can be carried out at a relatively low temperature that does not require heating, such as a temperature in the range of 25 to 40°C. The reaction time is not particularly limited, but can be, for example, in the range of 5 to 60 minutes.

(Capture step (II))
The target RNA detection method of the present invention includes, as a second step, a capture step of capturing the complex (target RNA-DNA probe-chimeric probe complex) with the capture body.

In the capture step, the complex is brought into contact with the capture body, whereby the complex is captured on the water-insoluble carrier through binding between the DNA/RNA chimera of the complex and the anti-DNA/RNA chimera antibody, thereby solidifying the complex. The contact method is not particularly limited, and the hybridized complex (e.g., a reaction solution) may be added to the capture body, or the capture body (e.g., a particle suspension) may be added to the complex. Furthermore, since the hybridization step and capture step can be performed simultaneously as described below, the hybridization step may be performed in the presence of the capture body. An appropriate reaction buffer may be added to the reaction system in the capture step. Examples of the reaction buffer include the same buffers as those listed for the hybridization step.

In the present invention, the temperature of the capture step is not particularly limited and may be, for example, in the range of 25 to 40°C. The reaction time is also not particularly limited and may be, for example, in the range of 5 to 60 minutes. In the target RNA detection method of the present invention, the temperature of the hybridization step and the temperature of the capture step can be made isothermal, which is preferable from the standpoint of simplicity. Furthermore, therefore, the hybridization step and the capture step can be carried out simultaneously under the same conditions.

(Cleaning process)
The target RNA detection method of the present invention preferably further includes a washing step after the capture step, in which the complex captured by the capture body is separated from other contaminants not bound to (captured by) the capture body and the contaminants are removed. The method for removing the contaminants is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, if the capture body is an antibody-immobilized plate, the liquid phase (supernatant) can be removed from the plate. If the capture body is antibody-immobilized particles, the particles can be recovered by centrifugation or magnetic collection, and the liquid phase (supernatant) can be removed. Furthermore, in the washing step, the injection and removal of a washing solution can be repeated as necessary.

Examples of the washing solution include known buffer solutions with a pH of 5 to 9 (e.g., sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also contain surfactants, salts, preservatives, stabilizers (e.g., Mg), proteins, etc., as appropriate.

(Detection step (III))
The target RNA detection method of the present invention includes, as a third step, a detection step of detecting the complex captured by the capturer.

In the detection step, a signal derived from the labeling substance is detected. For example, if the labeling substance is an enzyme, a chromogenic or luminescent substrate corresponding to the enzyme is added and reacted to detect the signal generated. If the DNA probe used does not have the labeling substance pre-bound, a labeling substance capable of binding to region SG of the DNA probe may be added in this detection step. For example, if region SG is a biotinylated site, an example of such a labeling substance is a streptavidin (or avidin)-bound labeling substance.

This allows a signal corresponding to the labeling substance to be detected, and the presence or absence of the complex, i.e., the presence or absence of the target RNA in the sample, can be detected based on the presence or absence of the signal. In the present invention, "signal" includes color development, reflected light, luminescence, quenching, fluorescence, radiation from radioisotopes, etc., and includes signals that can be confirmed with the naked eye as well as signals that can be confirmed using a measurement method or device appropriate for the type of signal. For example, when a bioluminescent enzyme such as luciferase is used as the labeling substance, luminescence generated by the addition of a substrate such as luciferin can be detected as the signal.

Furthermore, if target RNA is present in the sample, the amount of target RNA in the sample can be obtained as a signal amount (e.g., luminescence intensity), and if necessary, the amount of target RNA in the sample can be quantified by comparing it with the signal amount in a standard sample.

<Probe kit>
The present invention also provides a probe kit that can be suitably used in the above-described method for detecting a target RNA of the present invention.
a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region on the 5'-end or 3'-end of a single-stranded target RNA, and a region SG to which a labeling substance can be bound;
a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region that includes the other end of the first region side of the target RNA and does not overlap with the first region, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure;
The kit includes:

The probe kit of the present invention preferably further includes at least one substance selected from the group consisting of a labeling substance capable of binding to the region SG of the DNA probe, and a capture body comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier.

In the probe kit of the present invention, the target RNA, DNA probe, chimeric probe, labeling substance, and capture body, including their preferred embodiments, are each as described in the target RNA detection method of the present invention.

In the probe kit of the present invention, other components such as buffers, blocking agents, preservatives, and antiseptics may be added to the target RNA, DNA probe, chimeric probe, labeling substance, and capture body (e.g., antibody-immobilized particles).

The probe kit of the present invention may further include the diluent, the reaction buffer, the washing solution, reagents for extracting and purifying target RNA, reagents necessary for detecting each labeled substance (e.g., substrates necessary for each enzymatic reaction), other buffer solutions, reagents such as pH adjusters, standard RNA, control reagents, instructions for use, etc.

The present invention will be explained in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. The target RNA, DNA probe, and chimeric probe used in each example and comparative example are as follows:

<miR6875-5p>
(1) As target RNA1, miR6875-5p (21 bases long, SEQ ID NO: 1) shown in Table 1 below was synthesized by a conventional method and used.

(2) Based on the base sequence of target RNA1, the following DNA probes were used: a DNA probe that hybridizes to the entire length of target RNA1 (miR6875-5p-full length, SEQ ID NO: 2); a DNA probe that hybridizes to the 14 bases at the 3' end of target RNA1 (miR6875-5p-short(14nt), SEQ ID NO: 3); a DNA probe that hybridizes to the 13 bases at the 3' end of target RNA1 (miR6875-5p-short(13nt), SEQ ID NO: 4); and a DNA probe that hybridizes to the 12 bases at the 3' end of target RNA (miR6875-5p-short(12nt), SEQ ID NO: 5). Each of these DNA probes was synthesized using standard methods. Poly(A) and biotin were added to the 5' end of each of these DNA probes.

(3) The chimeric probe used was a chimeric probe (miR-6875-5p-Chimera (7 nt) (serving as miR-6875-5p-Chimera (7 nt) A LOOP 5 nt and miR-6875-5p-Chimera (7 nt) stem 8 nt), SEQ ID NO: 6) synthesized by standard methods. This probe hybridizes to the 7 bases at the 5' end of target RNA1 based on its base sequence and forms a stem-loop containing the stem region of the DNA/RNA chimera (i.e., forming the structure of 320 in Figure 2(a2)). Furthermore, chimeric probes were also used, synthesized by standard methods: a chimeric probe (miR-6875-5p-Chimera (8 nt), SEQ ID NO: 7) in which the base length of miR-6875-5p-Chimera (7 nt) that hybridizes with the base sequence at the 5' end of target RNA1 was increased to 8 bases, and a chimeric probe (miR-6875-5p-Chimera (9 nt), SEQ ID NO: 8) in which the base length was increased to 9 bases.

(4) Additionally, chimeric probes synthesized by standard methods were used: a chimeric probe in which the number of A's in the loop portion (A × 5) of miR-6875-5p-Chimera (7 nt) was 10 (miR-6875-5p-Chimera (7 nt) A LOOP 10 nt, SEQ ID NO: 9), and a chimeric probe in which the number of A's was 20 (miR-6875-5p-Chimera (7 nt) A LOOP 20 nt, SEQ ID NO: 10).

(5) Furthermore, a chimeric probe (miR-6875-5p-Chimera (7 nt) stem 10 nt, SEQ ID NO: 19) was also used, synthesized by standard methods, in which the number of Gs and Cs in the stem portion (G × 8, C × 8) of miR-6875-5p-Chimera (7 nt) was increased to 10 bases each.

<miR149-3p>
(1) As target RNA 2, miR149-3p (miR149, 21 bases long, SEQ ID NO: 11) shown in Table 1 below was synthesized by a conventional method and used.

(2) The DNA probes used were synthesized using standard methods based on the base sequence of target RNA2: a DNA probe (miR149-full length, SEQ ID NO: 12) that hybridizes to the entire length of target RNA2, and a DNA probe (miR149-short, SEQ ID NO: 13) that hybridizes to the 14 bases at the 3' end of target RNA2. Each of these DNA probes had polyA and biotin added to the 5' end.

(3) The chimeric probe used was a chimeric probe (miR149-Chimera, SEQ ID NO: 14) synthesized by standard methods. This probe hybridizes to the seven bases at the 5' end of target RNA2 based on its base sequence and forms a stem-loop containing the stem region of the DNA/RNA chimera (i.e., forms the structure of 320 in Figure 2(a2)).

<miR1268b>
(1) As target RNA 3, miR1268b (20 bases long, SEQ ID NO: 15) shown in Table 1 below was synthesized by a conventional method and used.

(2) Based on the base sequence of target RNA3, the DNA probes used were synthesized by standard methods: a DNA probe (miR1268b-full length, SEQ ID NO: 16) that hybridizes to the entire length of target RNA3; and a DNA probe (miR1268b-short, SEQ ID NO: 17) that hybridizes to the 13 bases at the 3' end of target RNA3. Each of these DNA probes had polyA and biotin added to the 5' end.

(3) The chimeric probe used was a chimeric probe (miR1268b-Chimera, SEQ ID NO: 18) synthesized by standard methods. This chimeric probe hybridizes to the seven bases at the 5' end of target RNA3 based on its base sequence and forms a stem-loop containing the stem region of the DNA/RNA chimera (i.e., forms the structure of 320 in Figure 2(a2)).

(Test Example 1) Confirmation of the effect of the chimeric probe by bioluminescent enzyme immunoassay (1) First, an anti-DNA/RNA antibody was immobilized on magnetic particles for immobilizing antibodies by a known method, and this was diluted with phosphate buffer containing sodium azide to prepare a 1.5 mg/mL suspension of antibody-immobilized magnetic particles.

(2) Next, a hybridization solution containing 1 x ¹⁰ or 1 x ¹⁰ cp/μL of each target RNA and 10 nM of each probe was prepared by mixing them at room temperature (approximately 25°C). The target RNAs and probes were used in the combinations shown in Table 2 below, and when a DNA probe and a chimeric probe were combined, the hybridization solution was prepared so that the concentration of each was 10 nM. Next, 200 μL of the hybridization solution, 20 μL of the antibody-immobilized magnetic particle suspension, and 40 μL of a buffer solution (Tris-HCl (pH 8.0) solution containing salt and surfactant) were mixed and reacted at 37°C for 15 minutes.

(3) The target RNA was detected using the fully automated biochemiluminescence immunoassay system "BLEIA (registered trademark)-1200 (manufactured by Eiken Chemical Co., Ltd.; the same applies hereinafter)" in accordance with the protocol included with "BLEIA (registered trademark) 'Eiken' H. pylori Antigen (manufactured by Eiken Chemical Co., Ltd.)."

That is, after the reaction in (2) above, the solid phase containing the magnetic particles was first washed five times with the washing solution for BLEIA (registered trademark)-1200. After removing the washing solution, 80 μL of labeled streptavidin for BLEIA (registered trademark)-1200 was added, stirred, and allowed to react at 37°C for 15 minutes.

Next, the solid phase containing the magnetic particles was washed five times with the washing solution, and the washing solution was then removed. 50 μL of BLEIA®-1200 BL Luminescent Reagent 1 was added to this and stirred, followed by 50 μL of BLEIA®-1200 BL Luminescent Substrate, and the luminescence intensity (PC) of luciferase at wavelengths of 480 to 650 nm (λmax: 560 nm) was measured. Furthermore, for each target RNA and probe combination, the luminescence intensity was measured in the same manner as above, except that no target RNA was added, and this was used as a control (NC).

(4) For each target RNA and probe combination (Examples 1-5, Comparative Examples 1-3), the S/N ratio was calculated using the following formula: S/N ratio = PC/NC. Additionally, the S/N ratio (vs. Comparative Examples) was calculated for the combination of a DNA probe and a chimeric probe (Examples) compared to the S/N ratio (vs. Comparative Examples) when the DNA probe was used alone (Comparative Examples). The results are shown in Table 2 below.

As shown in Table 2, when the DNA probe and chimeric probe were used in combination (Examples 1 to 5), the S/N ratio, which indicates the detection intensity, was significantly higher for each target RNA, approximately 12 times higher for target RNA 1 (miR6875-5p), approximately 96 times higher for target RNA 2 (miR149-3p), and approximately 23 times higher for target RNA 3 (miR1268b), compared to when the DNA probe was used alone (Comparative Examples 1 to 3). This confirmed that the use of a combination of a DNA probe and a chimeric probe enables highly sensitive detection of miRNAs.

(Test Example 2) Confirmation of Quantitativeness Using Chimeric Probes Quantitativeness when using chimeric probes was confirmed by the same method as in Test Example 1. Specifically, each luminescence intensity (PC and NC) was measured and the S/N ratio was calculated in the ^same manner as in Comparative Example 1 and Example 3 of Test Example 1, except that target RNA1 (miR6875-5p) was adjusted to 1 x 10 4 to 1 x 10 ⁹ cp/μL. The results are shown in Table 3 below.

As shown in Table 3, when the DNA probe and chimeric probe were used in combination (Example 3), the S/N ratio increased depending on the concentration of target RNA1, just as when the DNA probe was used alone (Comparative Example 1).

(Test Example 3) Investigation of the Length of the Loop Region of the Chimeric Probe by Bioluminescent Enzyme Immunoassay The length of the loop region (A LOOP) of the chimeric probe was investigated using the same method as in Test Example 1. Specifically, a hybridization solution containing 1 x 10 ⁶ cp/μL of target RNA1 (miR6875-5p) and 10 nM of each probe was prepared, and the luminescence intensities (PC and NC) were measured and the S/N ratios calculated in the same manner as in Test Example 1, except that the probes were combined as shown in Table 4 below. In addition, the S/N ratios (vs. Comparative Examples) were calculated for the combinations of the DNA probe and chimeric probe (Examples 6 to 8) relative to the S/N ratios when the DNA probe was used alone (Comparative Example 4). The results are shown in Table 4 below.

As shown in Table 4, similar to Test Example 1, the S/N ratio was higher in all cases when the DNA probe and chimeric probe were used in combination (Examples 6 to 8) compared to when the DNA probe was used alone (Comparative Example 4). Of these, the highest S/N ratio was achieved when the loop region (A LOOP) of the chimeric probe was 5 bases long (Example 6). This is presumably because a shorter loop region makes it easier to form a stem region.

(Test Example 4) Stem Length Investigation by Bioluminescent Enzyme Immunoassay The length of the stem region (stem) of the chimeric probe was investigated using the same method as in Test Example 1. Specifically, a hybridization solution containing 1 x 10 ⁶ cp/μL of target RNA1 (miR6875-5p) and 10 nM of each probe was prepared, and the luminescence intensities (PC and NC) were measured and the S/N ratios calculated in the same manner as in Test Example 1, except that the probes were combined as shown in Table 5 below. In addition, the S/N ratios (vs. Comparative Examples) were calculated for the combinations of the DNA probe and chimeric probe (Examples 9-10) compared to the S/N ratios when the DNA probe was used alone (Comparative Example 4). The results are shown in Table 5 below.

As shown in Table 5, similar to Test Example 1, when the DNA probe and chimeric probe were used in combination (Examples 9 and 10), the S/N ratio was higher than when the DNA probe was used alone (Comparative Example 4), and was not affected by the length (number of bases) of the stem region.

Test Example 5: Confirmation of the Effect of Chimeric Probes by Enzyme Immunoassay The target RNA was detected according to the protocol included with the "miREIA-miRNA (micro RNA) Enzyme Immunoassay Kit (BioVendor)." Specifically, measurements were performed using the kit according to the protocol included with the kit, except that target RNA3 (miR1268b) was used as the miRNA in the kit, and the DNA probe and chimeric probe were used in the combination shown in Table 6 below as the biotinylated DNA probe. Specifically, the probes were first preheated in a thermal cycler at 85°C for 3 minutes, 4°C for 2 minutes, and 37°C for 5 minutes, and then incubated on an antibody-coated plate. The antibody-immobilized plate was then washed, and streptavidin-HPR (horseradish peroxidase) conjugate was added and incubated, followed by washing again. TMB substrate was added and the luminescence intensity was measured. Each probe was added to a concentration of 0.17 μM when preparing the DNA Probe Working Solution.

The luminescence intensity was measured at wavelengths of 450 nm and 630 nm, and the difference between these values (luminescence intensity at a wavelength of 450 nm - luminescence intensity at a wavelength of 630 nm) was used as the PC. The control (NC) value was obtained in the same manner as above, except that no target RNA was added. Furthermore, the S/N ratio was calculated using the following formula: S/N ratio = PC/NC. The S/N ratio (vs. Comparative Example 11) when the DNA probe and chimeric probe were used in combination was also calculated compared to when the DNA probe was used alone (Comparative Example 5). The results are shown in Table 6 below.

As shown in Table 6, even with enzyme immunoassay, the S/N ratio was lower than with bioluminescent enzyme immunoassay without preheating. However, when the DNA probe was used alone (Comparative Example 5), i.e., compared to the method of detecting DNA-RNA complexes by ELISA using conventional anti-DNA/RNA antibodies, the S/N ratio was higher when the DNA probe and chimeric probe were used in combination (Example 11). This confirms that the use of a DNA probe and chimeric probe in combination with enzyme immunoassay enables highly sensitive detection of miRNA.

As explained above, the present invention makes it possible to provide a target RNA detection method that can detect even short-stranded RNAs such as miRNAs with high sensitivity and ease, as well as a probe kit that can be suitably used for this method.

110, 120...target RNA, 111, 121...first region, 112, 122...second region, 210, 220...DNA probe, 211, 221...base sequence D1, 212, 222...region SG, 310, 320...chimeric probe, 311, 321...base sequence D2, 312, 322...base sequence SD, 313, 323...linker, 314, 324...base sequence RD, 410, 420...target RNA-DNA probe-chimeric probe complex.

SEQ ID NO: 2
<223> miR6875-5p—full length SEQ ID NO: 3
<223> miR6875-5p-short (14nt)
SEQ ID NO: 4
<223> miR6875-5p-short (13nt)
SEQ ID NO:5
<223> miR6875-5p-short (12nt)
SEQ ID NO: 6
<223> miR-6875-5p-Chimera (7nt)
<223> 21 to 28 represent RNA. SEQ ID NO: 7
<223> miR-6875-5p-Chimera (8nt)
<223> 22 to 29 represent RNA. SEQ ID NO: 8
<223> miR-6875-5p-Chimera (9nt)
<223> 23 to 30 represent RNA. SEQ ID NO: 9
<223> miR-6875-5p-Chimera (7nt) A LOOP10nt
<223> 26 to 33 represent RNA. SEQ ID NO: 10
<223> miR-6875-5p-Chimera (7nt) A LOOP20nt
<223> 36 to 43 represent RNA SEQ ID NO: 12
<223> miR149-full length SEQ ID NO: 13
<223> miR149-short
SEQ ID NO: 14
<223> miR149-Chimera
<223> 21 to 28 represent RNA. SEQ ID NO: 16
<223> miR1268b—full length SEQ ID NO: 17
<223> miR1268b-short
SEQ ID NO: 18
<223> miR1268b-Chimera
<223> 21 to 28 represent RNA. SEQ ID NO: 19
<223> miR-6875-5p-Chimera (7nt) stem10nt
<223> 23 to 32 indicate RNA

Claims (17)

1. A method for detecting a target RNA, comprising:
(I) a first region on the 5'-end or 3'-end side of a single-stranded target RNA, and a second region including the other end of the first region and not overlapping with the first region;
a hybridization step of hybridizing a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region of the target RNA and a region SG to which a labeling substance can be bound, and a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure;
(II) a capturing step of capturing a complex of the target RNA, the DNA probe, and the chimeric probe with a capturing body comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier;
(III) a detection step of detecting the complex captured by the capturer;
Including,
the length of the first region is 5 to 50 bases;
the second region is 5 to 50 bases in length;
the first region and the second region are adjacent to each other;
In the DNA probe, when the DNA probe forms a complex with the target RNA and the chimeric probe, the region SG is located at an end of the base sequence D1 opposite to the end where the base sequence D2 is located.
A method for detecting a target RNA, comprising: The target RNA detection method according to claim 1, characterized in that the detection step is a step of detecting the complex using a signal derived from a labeling substance bound to the region SG as an indicator. The target RNA detection method according to claim 1 or 2, wherein the target RNA is a single-stranded RNA molecule having a length of 19 to 25 bases. The target RNA detection method according to any one of claims 1 to 3, characterized in that the linker is a base sequence consisting of DNA and/or RNA. The target RNA detection method described in claim 4, wherein the linker is 3 to 50 bases long. The method for detecting a target RNA according to any one of claims 1 to 5, wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long. The target RNA detection method described in any one of claims 1 to 6, characterized in that the temperature of the hybridization step is 15 to 60°C. The target RNA detection method according to any one of claims 1 to 7, wherein the water-insoluble carrier is a particulate carrier. a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region on the 5'-end or 3'-end of a single-stranded target RNA, and a region SG to which a labeling substance can be bound;
a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region that includes the other end of the first region side of the target RNA and does not overlap with the first region, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure;
Including,
the length of the first region is 5 to 50 bases;
the second region is 5 to 50 bases in length;
the first region and the second region are adjacent to each other;
In the DNA probe, when the DNA probe forms a complex with the target RNA and the chimeric probe, the region SG is located at an end of the base sequence D1 opposite to the end where the base sequence D2 is located.
A probe kit comprising: The probe kit described in claim 9, further comprising a labeling substance capable of binding to region SG of the DNA probe. The probe kit according to claim 9 or 10, wherein the total length of base sequence D1 and base sequence D2 is 10 to 100 bases long. The probe kit described in any one of claims 9 to 11, characterized in that the linker is a base sequence consisting of DNA and/or RNA. The probe kit described in claim 12, characterized in that the linker is 3 to 50 bases long. The probe kit according to any one of claims 9 to 13, wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long. The probe kit according to any one of claims 9 to 14, further comprising a capture body comprising a water-insoluble carrier and an anti-DNA/RNA chimeric antibody immobilized on the water-insoluble carrier. The probe kit described in claim 15, characterized in that the water-insoluble carrier is a particle carrier. The probe kit according to any one of claims 9 to 16, characterized in that it is a kit for use in the target RNA detection method according to any one of claims 1 to 8.