JP2020096564A - RNA detection method, RNA detection nucleic acid, and RNA detection kit - Google Patents

Abstract

To provide a new method of detecting RNA that can also be suitably used for detecting short-chain RNA.SOLUTION: The method of detecting RNA includes: i) forming a complex by hybridizing a first nucleic acid portion 201 of an RNA detection nucleic acid 2 with an RNA 1 to be detected; ii) decomposing a portion of the first nucleic acid portion 201 in the complex with a Cas9 nuclease 3; (iii) making a nucleic acid having a second nucleic acid portion 202 of the RNA detection nucleic acid 2 into a single-stranded nucleic acid to provide a nucleic acid amplification primer 2'; and (iv) hybridizing the nucleic acid amplification primer 2' with a template nucleic acid 6 to perform an amplification reaction of the template nucleic acid 6 using a polymerase 7.SELECTED DRAWING: Figure 1

Description

The present invention relates to an RNA detection method for detecting RNA to be detected by utilizing the principle of nucleic acid amplification such as LAMP method or PCR method. The present invention particularly relates to an RNA detection method that is suitable for detecting short-chain RNA having 10 to 100 bases and can be used for detecting miRNA and the like.
The present invention also relates to an RNA detection primer and an RNA detection kit.

In the cell, a large amount of short-chain RNA that is not translated into protein exists. Although there was a time when these short-chain RNAs were considered to have no function, studies since the 1990s have revealed that they have an important role in the living body.
Among these short RNAs, short RNAs called miRNAs (microRNAs) have been revealed to play an important role in regulating gene expression. miRNA is a single-stranded RNA having 20 to 25 bases, and as a precursor thereof, a pri-miRNA (primary miRNA) containing a hairpin structure is produced by transcription of a miRNA gene on a chromosome with RNA polymerase, Then, pri-miRNA is cleaved by Drosha, which is a nuclear RNase, to produce a 60-70 base pre-miRNA (precursor miRNA) having a hairpin structure. Pre-miRNA is transported from the nucleus to the cytoplasm and cleaved by Dicer, which is an RNase in the cytoplasm, to become a double-stranded RNA (miRNA duplex) having 21 to 24 bases. The double-stranded RNA is incorporated into the Ago protein, and only one RNA strand (mature miRNA) on one side finally forms a stable complex with the Ago protein, which results in RNA-induced silencing complex (RNA-induced silencing complex). : RISC). RISC regulates gene expression by binding to target mRNA having a sequence partially complementary to mature miRNA incorporated into RISC and suppressing translation of target mRNA.

Although miRNA plays an important role in cell function by regulating the expression of many target genes, it has been revealed that it is closely related to diseases such as cancer. Previous studies have reported miRNAs that show abnormal expression in various diseases such as cancer, and miRNAs are expected to be developed as diagnostic tools for various diseases.

However, as a method for detecting miRNA, although the microarray method is often used, there are problems such as complicated operation and high cost.
As a method for detecting miRNA using the nucleic acid amplification method, miRNA is polyadenylated to add a poly A sequence, and then cDNA is synthesized by a reverse transcription reaction using a primer containing a poly T sequence and a base complementary to the miRNA. And a method for detecting miRNA by performing nucleic acid amplification of cDNA using a primer complementary to this cDNA (Patent Document 1).
Further, a primer having a 3'region specific to miRNA and a stem loop is hybridized with miRNA, an extension reaction of the primer is performed, and an amplification reaction is performed using the extension-reacted primer as a template. A detection method has also been developed (Patent Document 2).

By the way, using an RNase (ribonuclease) that has activity at high temperature, when a primer bound with a blocking group that prevents extension of the primer forms a double strand with the template nucleic acid, the blocking group is removed by RNase. , A method of hot-starting a PCR reaction has been developed (Patent Document 3). In addition, a chimeric probe containing RNA as a part is hybridized to the target DNA, the resulting DNA-RNA hybrid chain is degraded with RNase, and the extension reaction by polymerase is performed using the fragment of the chimeric probe released thereby as a primer. Therefore, a method for detecting the target DNA has been developed (Patent Document 4).

In recent years, the CRISPR/Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR ASsociated proteins 9) has attracted attention as a mechanism applicable to genome editing. The CRISPR/Cas9 system is an adaptive immune system found in bacteria and archaea against foreign DNA such as viruses and plasmids. In the CRISPR/Cas9 system, first, an invading foreign DNA is cleaved, and then it becomes a spacer sequence sandwiched by repeat sequences, is incorporated into the CRISPR locus, and information of the foreign DNA is stored in the cell. Next, when the same foreign DNA invades a cell, CRISPR RNA (crRNA) containing a part of the nucleotide sequence of the foreign DNA is produced through transcription and processing from the CRISPR locus. This crRNA functions as a guide for finding foreign DNA and induces foreign DNA into Cas9 nuclease. Then, Cas9 nuclease cleaves DNA at a position approximately 3 bases away from a mark called a Proto-spacer Adjacent Motif (PAM) in the exogenous DNA to induce the decay of the exogenous DNA.

US Patent Application Publication No. 2009/0220969 U.S. Pat. No. 7,575,863 Patent No. 5539325 Japanese Patent Laid-Open No. 2008-307029

Although a microarray method has been widely used as a conventional method for detecting miRNA, there have been problems such as complicated operation and high cost. Further, although a method for detecting miRNA using nucleic acid amplification has been developed, there was a problem in practicality in terms of specificity and simplicity.
Therefore, in view of the above conventional circumstances, it is an object of the present invention to develop a new method of detecting RNA that can be suitably used for detecting short-chain RNA.

In order to solve the above-mentioned problems, the present inventors have conducted diligent research and as a result, design and use a special RNA-detecting nucleic acid that forms a complex by hybridization with RNA to be detected and is decomposed by Cas9 nuclease. As a result, it was found that the nucleic acid amplification primer occurs from the RNA for detecting the nucleic acid to cause the nucleic acid amplification reaction only when the RNA to be detected is present, and the present invention has been completed.

That is, the present invention provides the following first invention relating to an RNA detection method, the following second invention relating to an RNA detection nucleic acid, and the following third invention relating to an RNA detection kit.
(1) The first invention relates to an RNA detection method for detecting RNA to be detected,
Such RNA detection method,
A) A template nucleic acid that is amplified and serves as an index for detection,
B) For RNA detection including a first nucleic acid portion having a base sequence complementary to at least a part of the detection target RNA and a second nucleic acid portion having a base sequence complementary to a part of the template nucleic acid Nucleic acid,
At least C) Cas9 nuclease and D) polymerase,
i) contacting a test sample containing the RNA to be detected with the nucleic acid for RNA detection to hybridize the first nucleic acid portion to the RNA to be detected to form a complex,
ii) decomposing a part of the first nucleic acid portion in the complex with the Cas9 nuclease,
iii) A single-stranded nucleic acid having a second nucleic acid portion in the RNA detecting nucleic acid is used as a nucleic acid amplification primer,
iv) It is characterized by including hybridizing the nucleic acid amplification primer to the template nucleic acid, and performing a reaction of amplifying the template nucleic acid by the polymerase.
(2) In the RNA detection method of the first invention, it is preferable to perform a reaction of ligating an adapter nucleic acid containing a base sequence derived from the repeat sequence of crRNA to the detection target RNA before the step i). ..
(3) In any of the RNA detection methods described above, in the step i), it is preferable that the test sample containing the RNA to be detected is further contacted with transactivated crRNA.
(4) In the case of (2) above, it is preferable that the adapter nucleic acid contains a base sequence derived from trans-activated crRNA.
(5) In any one of the above RNA detection methods, the Cas9 nuclease is preferably a Class 2 Cas9 nuclease.
(6) As the Class 9 Cas9 nuclease, SpyCas9 nuclease can be used.
(7) NmeCas9 nuclease can be used as the Class 2 Cas9 nuclease.
(8) In any one of the above RNA detection methods, it is preferable that the nucleic acid for RNA detection is a double-stranded nucleic acid.
(9) When NmeCas9 nuclease is used as Cas9 nuclease, the RNA detecting nucleic acid can be a single-stranded nucleic acid.
(10) In any of the above RNA detection methods, it is preferable that the RNA to be detected is a short-chain RNA having 10 to 100 bases.
(11) In any one of the above RNA detection methods, the nucleic acid in the 5′-side portion of the first nucleic acid portion is complementary to a part of the RNA to be detected, and at the same time, the template nucleic acid is used. It is also preferred that a part of is also complementary.
(12) In any one of the above RNA detection methods, it is preferable that nucleic acid amplification by the LAMP method is performed in the step of iv).
(13) The second invention relates to a nucleic acid for RNA detection used for detecting RNA to be detected, and the nucleic acid for RNA detection is
A first nucleic acid part having a base sequence complementary to at least a part of the RNA to be detected, and a second nucleic acid part having a base sequence complementary to a part of the template nucleic acid amplified and used as an index for detection It is characterized by including.
(14) The nucleic acid for RNA detection of the second invention is preferably a double-stranded nucleic acid.
(15) The nucleic acid for RNA detection of the second invention may be a single-stranded nucleic acid.
(16) In any one of the nucleic acids for RNA detection, the first nucleic acid portion preferably has 10 to 100 bases.
(17) In any one of the nucleic acids for RNA detection, the nucleic acid at the 5'side portion of the first nucleic acid portion is complementary to a part of the RNA to be detected, and at the same time, the template is used. It is also preferably complementary to part of the nucleic acid.
(18) A third invention relates to an RNA detection kit used for detecting RNA to be detected,
A) A template nucleic acid that is amplified and serves as an index for detection,
B) For RNA detection including a first nucleic acid portion having a base sequence complementary to at least a part of the detection target RNA and a second nucleic acid portion having a base sequence complementary to a part of the template nucleic acid A nucleic acid, and C) Cas9 nuclease.
(19) The RNA detection kit of the third invention preferably further contains an adapter nucleic acid containing a nucleotide sequence derived from the repeat sequence of crRNA.
(20) In any one of the kits for detecting RNA, it is preferable that the kit further contains trans-activated crRNA.
(21) In the RNA detection kit of (19), it is preferable that the adapter nucleic acid contains a base sequence derived from trans-activated crRNA.
(22) It is preferable that any one of the kits for detecting RNA further contains D) a polymerase.
(23) In any one of the kits for detecting RNA, the Cas9 nuclease is preferably a Class 2 Cas9 nuclease.
(24) When the Class 2 Cas9 nuclease is included in the RNA detection kit, SpyCas9 nuclease can be used.
(25) When the Class 2 Cas9 nuclease is included in the RNA detection kit, NmeCas9 nuclease can be used.
(26) In any one of the RNA detection kits, the RNA detection nucleic acid is preferably a double-stranded nucleic acid.
(27) When the NmeCas9 nuclease is included in the RNA detection kit, the RNA detection nucleic acid can be a single-stranded nucleic acid.
(28) In any one of the RNA detection kits described above, the RNA to be detected is preferably a short chain RNA having 10 to 100 bases.
(29) In any one of the kits for detecting RNA, the nucleic acid in the 5′-side portion of the first nucleic acid portion is complementary to a part of the RNA to be detected, and at the same time, the template is used. It is also preferably complementary to part of the nucleic acid.

The present invention is for RNA detection comprising a first nucleic acid portion having a base sequence complementary to at least a part of RNA to be detected and a second nucleic acid portion having a base sequence complementary to a part of a template nucleic acid. Nucleic acid is used, and when RNA to be detected is present, the RNA to be detected hybridizes with the nucleic acid for RNA detection to form a complex, and the complex is decomposed by Cas9 nuclease, thereby nucleic acid amplification Generate a primer for. Then, the second polynucleotide portion of the nucleic acid amplification primer hybridizes to the template nucleic acid, and the polymerase initiates the amplification reaction of the template nucleic acid. Thus, according to the present invention, since a normal amplification reaction of the template nucleic acid occurs only when the RNA to be detected is present, it is possible to detect the RNA to be detected using the amplification of the template nucleic acid as an index. ..

It is a schematic diagram which shows 1st Embodiment which is an example of the RNA detection method of this invention. FIG. 1(A) shows a state in which a test sample containing RNA to be detected is mixed with a reagent, and FIG. 1(B) shows that a nucleic acid for RNA detection and RNA to be detected hybridize to form a complex. 1C shows that the first nucleic acid portion in the complex is decomposed by Cas9 nuclease, and FIG. 1D shows that the cleaved nucleic acid for RNA detection is dissociated. Shows a single-stranded structure. It is a schematic diagram following FIG. 1 which shows 1st Embodiment of the RNA detection method of this invention. 2(E) shows a state in which the nucleic acid amplification primer is purified, FIG. 2(F) shows a state in which the nucleic acid amplification primer is hybridized with the template nucleic acid, and FIG. 2(G) shows that the template nucleic acid is The state of being amplified by a polymerase is shown. It is a schematic diagram which shows 2nd Embodiment of the RNA detection method of this invention which connects an adapter nucleic acid. FIG. 3A shows a state in which an adapter nucleic acid, RNA to be detected, and RNA ligase are mixed, and FIG. 3B shows a state in which the adapter nucleic acid is linked to the RNA to be detected. It is a schematic diagram which shows 3rd Embodiment of the RNA detection method of this invention including preparation of guide RNA. FIG. 4(A) shows a state in which the adapter nucleic acid, RNA to be detected and RNA ligase are mixed, and FIG. 4(B) shows a state in which the adapter nucleic acid is linked to the RNA to be detected. It is a schematic diagram following FIG. 4 which shows 3rd Embodiment of the RNA detection method of this invention. FIG. 5(A) shows a state in which a test sample containing RNA to be detected is mixed with a reagent, FIG. 5(B) shows a state in which a complex is formed, and FIG. 5(C) shows a complex. The first nucleic acid part in the inside shows a state of being decomposed by Cas9 nuclease. It is a schematic diagram which shows the 4th Embodiment of the RNA detection method of this invention which uses a single stranded nucleic acid as nucleic acid for RNA detection. FIG. 6(A) shows a state in which a test sample containing RNA to be detected and a reagent are mixed, and FIG. 6(B) shows that the nucleic acid for RNA detection and RNA to be detected hybridize to form a complex. FIG. 6C shows a state in which the first nucleic acid portion in the complex is cleaved by Cas9 nuclease. It is a schematic diagram following FIG. 6 which shows 4th Embodiment of the RNA detection method of this invention. FIG. 7(D) shows a state in which the cleaved nucleic acid for RNA detection is dissociated into a single strand, and FIG. 7(E) shows a state in which the nucleic acid amplification primer is hybridized with the template nucleic acid. FIG. 7(F) shows a state in which the template nucleic acid is amplified by the polymerase. It is a schematic diagram which shows the outline of the nucleic acid amplification reaction of the LAMP method. It is a schematic diagram following FIG. 8 which shows the outline of the nucleic acid amplification reaction of the LAMP method. 3 is a process chart showing the outline of the experiment of Example 1. FIG. 3 is a table showing reagents and the like used in Example 1. FIG. 11(A) shows the base sequence of the nucleic acid reagent, FIG. 11(B) shows the other reagents used, and FIG. 11(C) shows the base sequence of the template nucleic acid. It is a reaction diagram showing a ligation reaction. FIG. 12(A) shows a ligation method in which the 5′ end of the adapter is not adenylated, and FIG. 12(B) shows a ligation method in which the 5′ end of the adapter is adenylated. It is a figure which shows the cutting|disconnection experiment of the double stranded DNA by Cas9 nuclease. 13A shows the reagents and the like used in the reaction, and FIG. 13B shows the result of capillary electrophoresis. It is a graph which shows the result of having measured the amplification reaction product of a LAMP method in real time by the fluorescent dye.

1. RNA detection method 1-1. Outline of RNA Detection Method of the Present Invention The RNA detection method of the present invention is characterized by using at least the following reagents A) to D).
A) A template nucleic acid that is amplified and serves as an index for detection,
B) RNA detection nucleic acid C containing a first nucleic acid part having a base sequence complementary to at least a part of the detection target RNA and a second nucleic acid part having a base sequence complementary to a part of the template nucleic acid ) Cas9 nuclease, and D) polymerase

In the RNA detection method of the present invention, by using the reagents A) to D) for a test sample, when the test sample contains RNA to be detected, the following i) to iv) are performed. Then, the presence of RNA to be detected can be detected using the amplification of the template nucleic acid as an index.
i) contacting a test sample containing RNA to be detected with a nucleic acid for RNA detection to hybridize the first nucleic acid portion to the RNA to be detected to form a complex,
ii) digesting a part of the first nucleic acid part in the complex with Cas9 nuclease,
iii) A nucleic acid having a second nucleic acid portion in the RNA detecting nucleic acid is made into a single-stranded nucleic acid to be a nucleic acid amplification primer,
iv) A nucleic acid amplification primer is hybridized with a template nucleic acid, and a reaction of amplifying the template nucleic acid with a polymerase is performed.

The reactions i) to iv) will be described below with reference to the drawings.
1 and 2 are drawings schematically showing a first embodiment which is an example of the RNA detection method of the present invention.
FIG. 1A shows a state in which a test sample containing RNA to be detected and a reagent are mixed. Detection target RNA 1, RNA detection nucleic acid 2, Cas9 nuclease 3, and trans-activating crRNA (tracrRNA) 4 are present in the mixed reaction solution.
Here, with respect to trans-activated crRNA4, depending on the type of Cas9 nuclease, the reactions i) to iv) can be proceeded without the presence of trans-activated crRNA. Examples of Cas9 nuclease that does not require trans-activated crRNA include Cpf1 (AsCpf1) derived from Acidaminococcus sp. BV3L6, Cpf1 (LbCpf1) derived from Lachnospiraceae bacterium ND2006, and the like.

In the first embodiment, the RNA to be detected is crRNA, and as shown in FIG. 1(A), the RNA to be detected 1 comprises a nucleic acid portion 101 having a base sequence derived from a spacer sequence and a base derived from a repeat sequence. A nucleic acid portion 102 having a sequence.
In the RNA detection method of the present invention, not only crRNA but also RNA having any base sequence can be detected. When RNA other than crRNA is to be detected, as in the second embodiment, an adapter nucleic acid containing a nucleic acid part having a nucleotide sequence derived from the repeat sequence of crRNA is ligated to the 3′ side of the RNA to be detected. Thus, the nucleic acid molecule can be recognized by the CRISPR/Cas9 system.

As shown in FIG. 1(A), the RNA detecting nucleic acid 2 includes a first nucleic acid portion 201 having a base sequence complementary to at least a part of RNA to be detected, and a template amplified and used as an index for detection. And a second nucleic acid portion 202 having a base sequence complementary to a part of the nucleic acid. Here, the second nucleic acid portion 202 is linked to the 5′ side of the first nucleic acid portion 201.
The RNA detecting nucleic acid 2 further includes a third nucleic acid portion 203 and a fourth nucleic acid portion 204. However, the third nucleic acid portion 203 and the fourth nucleic acid portion 204 are not essential, and the reactions i) to iv) described above can proceed even if they are not present.

The first nucleic acid portion 201 can be divided into a 5'side portion 201a and a 3'side portion 201b based on the characteristics of its base sequence. The 5′-side portion 201a is a portion designed not only to be complementary to the RNA to be detected but also to have a base sequence complementary to the template nucleic acid. The 5'side portion 201a is preferably provided with one or more bases, more preferably with 2 to 10 bases. The 3'side portion 201b is a portion designed to have a base sequence complementary to the RNA to be detected.
As will be described later, the 5'side portion 201a of the first nucleic acid portion is not designed to be complementary to both the RNA to be detected and the template nucleic acid, and may be complementary to only the RNA to be detected. Even if it is designed, it is possible to proceed the above reactions i) to iv).

The second nucleic acid portion 202 can be divided into a 5'side portion 202a and a 3'side portion 202b based on the characteristics of its base sequence. The 5'side portion 202a is a portion designed to have a base sequence complementary to the template nucleic acid. The 3'side portion 202b has a base sequence complementary to the template nucleic acid, and at the same time, its complementary strand 202b' has a proto-spacer adjacent motif (PAM) recognized by the CRISPR/Cas9 system. The part that is designed to be coded.
Here, regarding the protospacer adjacent motif, under the conditions as in the fourth embodiment, the reactions i) to iv) described above proceed even if the nucleic acid for RNA detection does not have the protospacer adjacent motif. Can be made.

As shown in FIG. 1(A), the RNA for detecting RNA 2 has a chain composed of a first nucleic acid portion 201, a second nucleic acid portion 202, a third nucleic acid portion 203, and a fourth nucleic acid portion 204. , A double-stranded nucleic acid consisting of a strand complementary thereto. The complementary strand comprises a nucleic acid portion 201' complementary to the first nucleic acid portion, a nucleic acid portion 202' complementary to the second nucleic acid portion, a nucleic acid portion 203' complementary to the third nucleic acid portion, and a third nucleic acid portion 203'. 4 has a nucleic acid portion 204' complementary to the nucleic acid portion of 4. In the first embodiment, the RNA detecting nucleic acid 2 is a double-stranded DNA.

At the 5'end of the third nucleic acid portion 203 of the nucleic acid 2 for detecting RNA, a tag 5 that enables purification of the nucleic acid is linked. Although the tag is not limited to these, for example, biotin, fluoroscein isothiocyanate (FITC), digoxigenin (DIG), histidine tag and the like can be used.
As will be described later, when the nucleic acid is not purified, it is not necessary to link the tag 5 to the RNA detecting nucleic acid 2.

FIG. 1(B) shows a state in which a nucleic acid for RNA detection and an RNA to be detected hybridize to form a complex.
As shown in FIG. 1B, the first nucleic acid portion 201 of the RNA detection nucleic acid 2 has a base sequence complementary to a part of the nucleic acid portion 101 of the RNA 1 to be detected. The nucleic acid portion 201 of 1 can hybridize to RNA1 to be detected to form a complex.
According to the knowledge about CRISPR/Cas9 thus far, in the actual reaction, the detection target RNA1, Cas9 nuclease 3, and trans-activated crRNA4 form a complex, and the complex forms a nucleic acid 2 for RNA detection. After recognizing the protospacer adjacent motif (PAM) 202b', it is considered that the first nucleic acid portion 201 hybridizes with the RNA detecting nucleic acid 1 to form a new complex. However, the RNA detection method of the present invention is not limited to this embodiment.

FIG. 1(C) shows a state in which the first nucleic acid portion in the complex is decomposed by Cas9 nuclease.
As shown in FIG. 1(C), when the complex is formed, it is separated from the protospacer-adjacent motif 202b' by about 3 bases on the 5'side (in 201'a) and the complementary position. In the 5'side portion 201a of a certain first nucleic acid portion, DNA cleavage by Cas9 nuclease occurs. Therefore, the cleavage is performed in a form in which the nucleic acid having about 3 bases of the 5'side portion 201a of the first nucleic acid portion is linked to the 3'side portion 202b of the second nucleic acid portion.
In the present invention, a modified Cas9 nuclease may be used so that only the first nucleic acid portion 201 is cleaved. Cas9 nuclease has two nuclease domains, a RuvC domain and an HNH domain. For example, by introducing a point mutation into the RuvC domain, the nucleic acid portion 201' in the chain on the motif side adjacent to the protospacer is cleaved. Instead, a Cas9 nuclease that cleaves only at the first nucleic acid portion 201 can be obtained.

FIG. 1D shows a state where the cleaved nucleic acid for RNA detection dissociates into a single strand.
As shown in FIG. 1C, even if the DNA is cleaved in the first nucleic acid portion 201, the complex is still formed by the hybridization. Here, for example, by heating the reaction solution, the respective nucleic acids are dissociated from each other, and the nucleic acid 2′ having the second nucleic acid portion is made single-stranded as shown in FIG. 1(D). You can In addition, since base pair formation and separation are in a dynamic equilibrium state, even if the reaction solution is not heated, a part of the complex is dissociated to form the nucleic acid 2′ having the second nucleic acid part as a single strand. Good.
The single-stranded nucleic acid 2'becomes a molecule that functions as a nucleic acid amplification primer.

FIG. 2(E) shows a state in which the nucleic acid amplification primer has been purified.
As shown in FIG. 2(E), since the tag 5 is linked to the 5'end of the nucleic acid amplification primer 2', the nucleic acid amplification primer 2'is purified using this tag 5 and detected. Other molecules in the reaction solution such as the target RNA and Cas9 nuclease can be removed.
For example, but not limited to, when using biotin as a tag, magnetic beads having streptavidin bound to the surface are added to the reaction solution, and the nucleic acid amplification primer is bound by the binding of biotin and streptavidin. A purified nucleic acid amplification primer can be obtained by recovering the magnetic beads having been adsorbed by a magnet with a magnet and eluting the nucleic acid amplification primer.

The purification shown in FIG. 2(E) does not necessarily have to be performed, and if a nucleic acid amplification primer is produced, the next reaction can proceed. However, in order to prevent the nucleic acid amplification primer from becoming a double-stranded nucleic acid again and non-specific adsorption, it is preferable to purify the nucleic acid amplification primer and use it for the next reaction.

FIG. 2(F) shows a state in which the nucleic acid amplification primer is hybridized with the template nucleic acid, and FIG. 2(G) shows a state in which the template nucleic acid is amplified by the polymerase.
As shown in FIG. 2(F), the template nucleic acid 6 and the polymerase 7 are further added to and mixed with the reaction solution having the purified nucleic acid amplification primer 2'. The template nucleic acid 6 is designed to include a nucleic acid portion 601 having a base sequence complementary to the 5'side portion 201a of the first nucleic acid portion. The second nucleic acid portion 202 is designed to have a base sequence complementary to the nucleic acid on the 3'side of the nucleic acid portion 601 of the template nucleic acid. Therefore, when the nucleic acid amplification primer 2′ hybridizes with the template nucleic acid 6, the nucleic acid amplification primer 2′ continuously forms base pairs with the template nucleic acid 6 including the base at the 3′ end. Thereby, as shown in FIG. 2(G), the extension reaction by the polymerase can be efficiently performed.

Here, even if the 5′-side portion 201a of the first nucleic acid portion and the nucleic acid portion 601 of the template nucleic acid are not designed to be complementary, nucleic acid amplification that does not form a base pair with the template nucleic acid Since the base at the 3'end of the primer 2'is about 3 bases, the extension reaction by the polymerase can proceed. However, in this case, the efficiency of the extension reaction is considerably reduced by the polymerase.
Therefore, in the present invention, the nucleic acid in the 5′-side portion of the first nucleic acid portion may be complementary to a part of the RNA to be detected and at the same time to a part of the template nucleic acid. It is preferable to design

As described above, it is not always necessary to purify the nucleic acid amplification primer shown in FIG. 2(E). When the nucleic acid amplification primer is not purified, the template nucleic acid shown in FIG. 2(F) and the polymerase may be mixed in the first reaction solution shown in FIG. 1(A). That is, RNA to be detected, RNA detecting nucleic acid, Cas9 nuclease, trans-activated crRNA, template nucleic acid and polymerase are mixed from the beginning to prepare a reaction solution, and the reaction i) to iv) is performed. You can also do

When the RNA to be detected is present in the test sample, as shown in FIGS. 1(A) to 2(G), the reactions i) to iii) described above proceed and a normal amplification reaction of the template nucleic acid is performed. Occurs.
On the other hand, when the target detection target RNA does not exist in the test sample, for example, when RNA having low homology with the target detection target RNA exists in the test sample, the above i) to iv) The reaction does not proceed and the normal amplification reaction of the template nucleic acid does not occur.
Therefore, according to the RNA detection method of the present invention, the normal amplification reaction of the template nucleic acid occurs only when the target RNA to be detected is present.

In the RNA detection method of the present invention, when amplifying a template nucleic acid, another primer that hybridizes to a region different from the region of the template nucleic acid to which the nucleic acid amplification primer hybridizes is designed, and PCR using two primers is performed. The template nucleic acid can be amplified by the reaction.

In addition, the LAMP method can also be used to amplify the template nucleic acid. The LAMP method is a nucleic acid amplification method developed by the present applicant (Japanese Patent No. 3313358, etc.), and is a method that enables an isothermal nucleic acid amplification reaction.
In the LAMP method, at least 4 types of primers designed based on the nucleotide sequences of 6 regions on the template nucleic acid are used as primers, and each primer is an inner primer F (FIP), an inner primer B (BIP ), outer primer F (F3), outer primer B (B3). In addition to this, loop primer F (LF) and loop primer B (LB) can also be used.
When the primer for nucleic acid amplification in the present invention is designed to be the inner primer of the LAMP method, as in FIG. 3 nucleic acid portions 203 are provided. The base sequence of the third nucleic acid portion is designed so as to include the same base sequence as a part of the base sequence of the region 5′ to the region complementary to the second nucleic acid portion in the template nucleic acid.
On the other hand, when the nucleic acid amplification primer of the present invention is designed to be the outer primer of the LAMP method, it is not necessary to provide the third nucleic acid portion in the RNA detection nucleic acid.

The amplified template nucleic acid is, for example, but not limited to, by reacting with an intercalator for staining, or by using a primer labeled with a fluorescent dye or the like for nucleic acid amplification, It is possible to detect using any means.

According to the RNA detection method of the present invention, the amplification reaction of the template nucleic acid occurs only when the RNA to be detected is present, so that the RNA to be detected can be detected using the amplification of the template nucleic acid as an index.
Further, the RNA detection method of the present invention does not directly amplify the RNA to be detected even though it is a method utilizing nucleic acid amplification, and thus even a short chain RNA of 10 to 100 bases which is difficult to directly amplify It has an effect that it can be suitably detected.

1-2. Second Embodiment In the first embodiment of the RNA detection method of the present invention, the RNA to be detected was crRNA, but in the RNA detection method of the present invention, not only crRNA but also any nucleotide sequence RNA can be the detection target. When RNA other than crRNA is to be detected, an adapter nucleic acid containing a base sequence derived from the repeat sequence of crRNA is ligated to the RNA to be detected, whereby the nucleic acid molecule can be recognized by the CRISPR/Cas9 system. ..
FIG. 3 is a drawing schematically showing a second embodiment of the RNA detection method of the present invention in which an adapter nucleic acid is ligated. FIG. 3(A) shows a state in which an adapter nucleic acid, RNA to be detected, and RNA ligase are mixed, and FIG. 3(B) shows a state in which the adapter nucleic acid is linked to the RNA to be detected.

As shown in FIG. 3(A), when ligating the adapter nucleic acids, the RNA 1 to be detected, the adapter nucleic acid 8 and the RNA ligase 9 are mixed, and a ligation reaction catalyzed by the RNA ligase 9 is performed. As a result, as shown in FIG. 3B, the adapter nucleic acid 8 can be linked to the 3′ side of the RNA 1 to be detected.
Here, the 5'end of the adapter nucleic acid 8 is preferably phosphorylated or adenylated. In addition, it is preferable that the 3'end of the adapter nucleic acid 8 is phosphorylated, so that self-ligation between the adapter nucleic acids 8 can be suppressed.

The base sequence of the adapter nucleic acid 8 may be any base sequence that allows the CRISPR/Cas9 system to function, and is not limited thereto. For example, a nucleic acid portion derived from a repeat sequence of natural crRNA. It is possible to use a base sequence identical to the base sequence of, or a part of the base sequence, or a base sequence highly homologous to these base sequences. Specific base sequences are not limited to these, but the following base sequences can be used, for example.
5'-GUUUUAGAGCUAUGCUGUUUUG-3' (SEQ ID NO: 1)
5'-GUUUUAGAGCUAUGCU-3' (SEQ ID NO: 2)
5'-GUUUUAGAGCUA-3' (SEQ ID NO: 3)

As the RNA ligase 9, T4 RNA Ligase 1 and T4 RNA Ligase 2 can be used, although not limited thereto.
The details of the ligation reaction can be performed, for example, according to the method described in Jerome E. Lee et al., Journal of Visualized Experiments, 2014, Vol.93, e52095.

After connecting the adapter nucleic acid 8 to the RNA 1 to be detected, the reactions i) to iv) can be performed as in the first embodiment. That is, in the reaction shown in FIGS. 1(A) to 2(G), the nucleic acid portion 101 having the base sequence derived from the spacer sequence is replaced with the RNA 1 to be detected, and the nucleic acid portion 102 having the base sequence derived from the repeat sequence is the adapter. The reaction proceeds with the nucleic acid 8 being replaced.

The base sequence of the transactivating crRNA used in the RNA detection method of the present invention may be any base sequence that functions the CRISPR/Cas9 system, but is not limited thereto. For example, the same nucleotide sequence as the nucleotide sequence of natural trans-activated crRNA, an active nucleotide sequence of a part thereof, or an active nucleotide sequence highly homologous to these nucleotide sequences can be used. Specific base sequences are not limited to these, but the following base sequences can be used, for example.
5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO: 4)
5'-UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 5)
5'-AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 6)

The adapter nucleic acid or trans-activated crRNA is preferably RNA so that it functions efficiently in the CRISPR/Cas9 system, but part or all may be DNA or artificial nucleic acid. It may be a nucleic acid in which a part of is modified.
The base sequence of the adapter nucleic acid and the base sequence of the transactivating crRNA are preferably designed to be optimized according to the type of Cas9 nuclease used.

In the RNA detection method of the present invention, prior to the step i), an adapter nucleic acid containing a nucleic acid portion derived from a repeat sequence of crRNA is ligated to RNA to be detected, so that not only crRNA but also any RNA is detected. It will be possible to design the base sequence of the RNA detection nucleic acid or template nucleic acid so that the CRISPR/Cas9 system will function corresponding to the RNA of the base sequence of, and RNA of any base sequence can be detected. Produce an effect.

1-3. Third Embodiment In the RNA detection method of the present invention, before the step i), an adapter nucleic acid can be linked to the RNA to be detected to form a so-called "guide RNA (sgRNA)". A guide RNA is a molecule in which an adapter nucleic acid having a nucleic acid portion derived from a repeat sequence of crRNA and a nucleic acid portion derived from a transactivating crRNA is linked to a detection target RNA, and functions of the crRNA and the function of the transactivating crRNA. It becomes a molecule that has both.
4 and 5 are drawings schematically showing a third embodiment of the RNA detection method of the present invention, including the production of guide RNA.

FIG. 4(A) shows a state in which an adapter nucleic acid, RNA to be detected, and RNA ligase are mixed, and FIG. 4(B) shows a state in which the adapter nucleic acid is linked to the RNA to be detected.
As shown in FIG. 4(A), when the guide RNA is prepared, RNA 1 to be detected, adapter nucleic acid 8 and RNA ligase 9 are mixed, and a ligation reaction by RNA ligase 9 is performed. The adapter nucleic acid 8 has a nucleic acid portion 801 having a base sequence derived from the repeat sequence of crRNA and a nucleic acid portion 802 having a base sequence derived from trans-activated crRNA. By the ligation reaction catalyzed by RNA ligase 9, as shown in FIG. 4(B), the guide nucleic acid 10 can be prepared by ligating the adapter nucleic acid 8 to the 3′ side of the RNA 1 to be detected.
The adapter nucleic acid 8 is preferably RNA so as to function in the CRISPR/Cas9 system, but may be partially modified RNA, or partially RNA or RNA that is artificial nucleic acid. May be.

The base sequence of the adapter nucleic acid 8 used for preparing the guide RNA may be any sequence as long as it is a base sequence that functions the CRISPR/Cas9 system. For example, the active portion of the nucleic acid derived from the repeat sequence of natural crRNA. It is possible to use a nucleotide sequence designed to form a loop structure by linking the nucleotide sequence of 1) with the nucleotide sequence of the active portion of natural trans-activated crRNA. As specific base sequences, for example, the following base sequences can be used.
5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO: 7)
5′-GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC-3′ (SEQ ID NO: 8)
5′-GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC-3′ (SEQ ID NO: 9)
5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 10)
5'-GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 11)
5'-GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 12)
5'-GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 13)

FIG. 5 is a drawing schematically showing the formation of a complex and the degradation of nucleic acid by Cas9 nuclease when using guide RNA. FIG. 5(A) shows a state in which a test sample containing RNA to be detected is mixed with a reagent, FIG. 5(B) shows a state in which a complex is formed, and FIG. 5(C) shows a complex. The first nucleic acid part in the inside shows a state of being decomposed by Cas9 nuclease.
As shown in FIG. 5(A), the guide RNA 10, the RNA detection nucleic acid 2 and the Cas9 nuclease 3 are present in the mixed solution. Unlike the first embodiment, the third embodiment using a guide RNA does not require trans-activated crRNA. This is because the guide RNA 10 contains the nucleic acid portion 802 having a base sequence derived from trans-activated crRNA.

As shown in FIG. 5B, the guide RNA 10, the RNA detection nucleic acid 2 and the Cas9 nuclease 3 form a complex. Here, the guide RNA 10 forms a loop structure. The complex shown in FIG. 5B is a complex having a structure similar to that of the complex shown in FIG.
In the complex shown in FIG. 5B, the first nucleic acid portion 201 of the RNA detecting nucleic acid hybridizes with the detection target RNA 1 contained in the guide RNA 10.

As shown in FIG. 5(C), when the complex is formed, as in the case of the first embodiment, a site (201′) separated from the protospacer-adjacent motif 202b′ by about 3 bases on the 5′ side. DNA cleavage by Cas9 nuclease occurs in (a)) and in the 5'side 201a of the first nucleic acid portion which is a complementary site.
Then, in the third embodiment, the same reaction as in the first embodiment can be performed thereafter. That is, by performing the reactions shown in FIGS. 1(D) to 2(G), amplification of the template nucleic acid occurs, and the RNA to be detected can be detected using this as an index.

1-4. Fourth Embodiment In the first to third embodiments, the double-stranded nucleic acid is used as the RNA detecting nucleic acid, but when NmeCas9 is used as the Cas9 nuclease, it is used as the RNA detecting nucleic acid. Single-stranded nucleic acids can be used.
FIG. 6 is a drawing schematically showing a fourth embodiment of the RNA detection method of the present invention, which uses a single-stranded nucleic acid as the RNA detection nucleic acid.

FIG. 6A shows a state in which a test sample containing RNA to be detected and a reagent are mixed. Detection target RNA 1, RNA detection nucleic acid 2, Cas9 nuclease 3, template nucleic acid 6, and polymerase 7 are present in the mixed reaction solution.
As shown in FIG. 6(A), the RNA detecting nucleic acid 2 is a single-stranded nucleic acid and has a full length of DNA. The RNA detecting nucleic acid 2 includes a first nucleic acid portion 201 having a base sequence complementary to at least a part of the detection target RNA and a second nucleic acid portion 202 having a base sequence complementary to a part of the template nucleic acid 6. Designed to include and. Here, the second nucleic acid portion 202 is linked to the 5′ side of the first nucleic acid portion 201. The first nucleic acid portion 201 preferably has 17 bases or more.

The first nucleic acid portion 201 can be divided into a 5'side portion 201a and a 3'side portion 201b based on the characteristics of its base sequence. The 5′-side portion 201a is a portion designed not only to be complementary to the RNA to be detected but also to have a base sequence complementary to the template nucleic acid.
The template nucleic acid 6 is provided with a nucleic acid portion 601 having the same base sequence as a part of RNA to be detected. This makes it possible to design the 5′-side portion 201a of the first nucleic acid portion so as to have a base sequence that is not only complementary to the RNA to be detected but also complementary to the template nucleic acid.
The 5'side portion 201a complementary to both the RNA to be detected and the template nucleic acid is preferably provided with one or more bases, more preferably with 2 to 10 bases. However, the RNA detection method of the present invention can be performed without the 5′-side portion 201a.

In the fourth embodiment, since the RNA detection nucleic acid and the template nucleic acid can be designed corresponding to the RNA having the arbitrary base sequence, the RNA having the arbitrary base sequence can be detected.
In addition, in the fourth embodiment, it is not necessary to provide a protospacer-adjacent motif in the nucleic acid for RNA detection.

In the fourth embodiment, NmeCas9 nuclease, which is a Cas9 nuclease derived from Neisseria meningitidis (N. meningitidis), is used as the Cas9 nuclease (WO2014/190181).
NmeCas9 does not require a transactivating crRNA or a protospacer-adjacent motif, and furthermore, can cleave DNA in an RNA-DNA complex of at least 17 bases or more regardless of its sequence. NmeCas9 is said to have DNase H activity in comparison with RNase H, which cleaves the DNA chain in the RNA-DNA complex and degrades the RNA chain in the RNA-DNA complex (Mol Cell., 2015. , Oct 15, 60(2), pp.242-255).

FIG. 6B shows a state in which the nucleic acid for RNA detection and the RNA to be detected hybridize to form a complex.
As shown in FIG. 6(B), the first nucleic acid portion 201 of the RNA detecting nucleic acid 2 has a base sequence complementary to a part of the RNA 1 to be detected. It can hybridize with RNA1 to be detected to form a complex.

FIG. 6(C) shows a state in which the first nucleic acid portion in the complex is cleaved by Cas9 nuclease.
As shown in FIG. 6(C), when the complex is formed, Cas9 nuclease 3 (NmeCas9 nuclease) causes about 3 bases 3′ from the boundary between the first nucleic acid portion 201 and the second nucleic acid portion 202. Cut the DNA at the position. The cleavage site can be slightly shifted by adding trans-activated crRNA or the like.

FIG. 7D shows a state where the cleaved nucleic acid for RNA detection is dissociated into a single strand.
As shown in FIG. 6C, even if the DNA is cleaved in the first nucleic acid portion 201, the 5′-side portion 201a of the first nucleic acid portion is in a state of being hybridized with the detection target RNA1. However, since base pair formation and separation are in a dynamic equilibrium state, nucleic acid molecules can be dissociated from each other as shown in FIG. 7D, or by heating the reaction solution, It can also promote the dissociation of nucleic acid molecules.
Among the dissociated nucleic acid molecules, the nucleic acid 2′ having the second nucleic acid portion becomes a molecule that functions as a nucleic acid amplification primer.

FIG. 7(E) shows a state in which the nucleic acid amplification primer is hybridized with the template nucleic acid, and FIG. 7(F) shows a state in which the template nucleic acid is amplified by the polymerase.
As shown in FIG. 7(E), the template nucleic acid 6 is provided with a nucleic acid portion 601 having a complementary base sequence to the 5'side portion 201a of the first nucleic acid portion. The second nucleic acid portion 202 is designed to have a base sequence complementary to the nucleic acid on the 3'side of the nucleic acid portion 601 of the template nucleic acid. Therefore, when the nucleic acid amplification primer 2'hybridizes with the template nucleic acid 6, it continuously forms base pairs with the template nucleic acid 6 including the 3'end thereof, as shown in FIG. 7(F). In addition, the amplification reaction by the polymerase 7 becomes possible.

As described above, in the fourth embodiment, it is not necessary to ligate an adapter nucleic acid to an RNA to be detected, a transactivating crRNA or a protospacer-adjacent motif is not necessary, and RNA having an arbitrary nucleotide sequence can be detected. Is possible.

1-5. Detailed Description of RNA Detection Method of the Present Invention In the present invention, the RNA to be detected may be naturally occurring RNA or artificially synthesized RNA. Further, it is not limited to a molecule having a full length of RNA, but may be a partially modified RNA, or a part of DNA or an artificial nucleic acid.
For the purpose of clinical diagnosis, it is preferable to artificially extract natural RNA existing in the living body as the “RNA to be detected”.
Examples of natural RNA existing in the living body include, but are not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (small nuclear RNA). : SnRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA) and the like.

The length of the RNA to be detected is not particularly limited as long as it can be recognized by the CRISPR/Cas9 system, but is usually 5 to 1000 bases.
The RNA detection method of the present invention is a method that utilizes nucleic acid amplification, but since it does not directly amplify the RNA to be detected, it is suitable even for short-chain RNA of 10 to 100 bases that is difficult to directly amplify. Can be detected.
The RNA to be detected is more preferably 15 to 50 bases, and miRNA can also be suitably detected.

As the “template nucleic acid” used in the present invention, DNA, RNA or an artificial nucleic acid which is an analog thereof can be used, but DNA is preferably used because amplification is easy. As the "template nucleic acid", a stable double-stranded nucleic acid can be used, but a single-stranded nucleic acid may be used.
In the present invention, as the artificial nucleic acid, for example, an artificial nucleic acid having a modified nucleotide skeleton composed of sugar and phosphate, or an artificial nucleic acid having a modified base portion can be used. Examples of the artificial nucleic acid having a modified nucleotide skeleton include, but are not limited to, a peptide nucleic acid having a peptide chain as a skeleton (Peptide Nucleic Acid: PNA), and a locked nucleic acid having a sugar with a crosslinked structure (Locked Nucleic Acid). Nucleic Acid: LNA), a glycol nucleic acid having a glycol skeleton linked by a phosphodiester bond (Glycol Nucleic Acid: GNA), and the like. Further, the artificial nucleic acid having a modified base portion is not limited to these, for example, as a base, 2-amino-6-dimethylaminopurine, pyridone-2-one, 2-amino-6-( Examples thereof include artificial nucleic acids using 2-thienyl)purine, pyrrolopyridine, methylisocarbosteryl, azaadenine, azaguanine and the like.

The “template nucleic acid” used in the present invention does not necessarily have to be separately designed corresponding to the sequence of RNA to be detected, and one template nucleic acid can be commonly used in the detection of various kinds of RNA to be detected. Is. Here, the “template nucleic acid” preferably has low homology to any of various types of RNA to be detected, whereby the template nucleic acid hybridizes with the first nucleic acid portion of the RNA detection nucleic acid. Can be prevented.
More preferably, a template nucleic acid having a non-natural artificial base sequence is used as the “template nucleic acid”. Here, the "non-natural artificial base sequence" refers to a base sequence artificially created, not a base sequence of a nucleic acid of biological origin, for any of the base sequences of nucleic acids of biological origin registered in a gene database. However, those having low homology are preferable. By using a non-natural artificial base sequence, the template nucleic acid will hybridize with the first nucleic acid portion of the nucleic acid for RNA detection, and the template nucleic acid will hybridize with the biological nucleic acid present in the test sample. It is possible to prevent this from happening and further reduce the risk of contamination.

The “nucleic acid for RNA detection” used in the present invention includes a first nucleic acid portion having a base sequence complementary to at least a part of RNA to be detected and a second nucleic acid portion having a base sequence complementary to a part of a template nucleic acid. And the nucleic acid portion thereof.
Here, the first nucleic acid portion may have a base sequence complementary to all the base sequences of the detection target RNA, or has a base sequence complementary to only a part of the detection target RNA. It may be one.

In the present invention, “having a complementary base sequence” means, for example, a base pair of A (adenine) and T (thymine), a base pair of G (guanine) and C (cytosine), and A (adenine). It has a base sequence that can form a base pair that can bond with each other by hydrogen bond, such as a base pair with U (uracil) and a base pair with 2-amino-6-dimethylaminopurine and pyridone-2-one. However, it may have a base that is a mismatch that does not form a base pair that can be bonded by a hydrogen bond. However, in the first nucleic acid portion and the second nucleic acid portion, the mismatch is preferably 20% or less, and more preferably 5% or less.

The nucleic acid for RNA detection used in the present invention may be a double-stranded nucleic acid, or may be a single-stranded nucleic acid depending on the type of Cas9 nuclease as in the fourth embodiment.
The first nucleic acid part of the nucleic acid for RNA detection is preferably DNA so that it functions in the CRISPR/Cas9 system, but may be partially modified DNA, or partly RNA or It may be DNA which is an artificial nucleic acid. When using Cas9 nuclease capable of cleaving RNA, the first nucleic acid portion can be RNA. As a Cas9 nuclease capable of cleaving RNA, for example, C2c2 (Cas13a) can be used.
The portion of the nucleic acid for RNA detection other than the first nucleic acid portion is preferably DNA, but it may be RNA, or modified DNA or RNA, or artificial nucleic acid may be used. Moreover, the nucleic acid for RNA detection may be a nucleic acid bound to another substance such as a tag or a label.

The first nucleic acid portion is not particularly limited in its base length or sequence as long as it has a base sequence complementary to at least a part of the RNA to be detected, but is usually 5 to 5000 bases, and more preferably It is 10 to 100 bases, and more preferably 15 to 50 bases.
The second nucleic acid portion is not particularly limited in its base length and sequence as long as it has a base sequence complementary to a part of the template nucleic acid, but has a base length and a Tm value suitable for hybridization. It is preferable to design the sequence. The base length of the second nucleic acid portion is usually 5 to 1000 bases, more preferably 10 to 100 bases, still more preferably 15 to 30 bases.
The second nucleic acid portion may have a portion complementary to the template nucleic acid and at the same time complementary to the RNA to be detected.

The RNA detecting nucleic acid used in the present invention has a second nucleic acid portion linked to the 5'side of the first nucleic acid portion. Here, an intervening sequence of about 1 to 3 bases which is neither complementary to the nucleic acid to be detected nor the template nucleic acid can be provided between the first nucleic acid part and the second nucleic acid part.

The RNA detecting nucleic acid used in the present invention may have a nucleic acid portion other than the first nucleic acid portion and the second nucleic acid portion. For example, a 5'side of the second nucleic acid portion can be linked to a third nucleic acid portion that is not complementary to the base sequence of the template nucleic acid. Further, the 3'side of the first nucleic acid portion can be linked to a fourth nucleic acid portion which is not complementary to the base sequence of the RNA to be detected.
The third nucleic acid portion can also have a specific function. For example, but not limited to these, the third nucleic acid may include the same base sequence as a part of the base sequence of the region 5′ to the region complementary to the second nucleic acid portion in the template nucleic acid. By designing the nucleic acid portion of, the primer for nucleic acid amplification generated from the nucleic acid for RNA detection can be used as an inner primer for the LAMP method.
In addition, the fourth nucleic acid portion can also have a specific function. For example, but not limited to these, the fourth nucleic acid portion has a non-natural artificial base sequence and is designed so as not to hybridize with the template nucleic acid, so that the first nucleic acid portion is contained in the test sample. In the case where the target nucleic acid hybridizes with a nucleic acid other than the detection target RNA, or the detection target RNA has a high homology with the template nucleic acid and the first nucleic acid portion hybridizes with the template nucleic acid, Since the nucleic acid portion of does not hybridize with the nucleic acid or the template nucleic acid in the test sample, it is possible to prevent an unintended amplification reaction.

In the nucleic acid for RNA detection, the first nucleic acid portion needs to be individually designed according to the sequence of the detection target RNA, but the second to fourth nucleic acid portions may not necessarily be designed depending on the sequence of the detection target RNA. A common sequence can be used in the detection of various types of detection target RNA without designing. Therefore, in the RNA detection method of the present invention, when designing a nucleic acid for RNA detection, at least only the first nucleic acid portion may be designed according to the sequence of the RNA to be detected, and it can be designed easily.

The nucleic acid for RNA detection and the template nucleic acid used in the present invention are not limited to these, but can be chemically synthesized by using, for example, a solid phase synthesis method. When a double-stranded nucleic acid is used as a nucleic acid for RNA detection, it can also be obtained by PCR amplification using a primer to which a tag such as biotin is added.

The RNA detecting nucleic acid used in the present invention preferably has a modification at the 3′ end that terminates the amplification reaction. By providing such a modification, the nucleic acid for RNA detection, in which the first nucleic acid portion is not removed, hybridizes with a template nucleic acid or a nucleic acid other than the RNA to be detected in the test sample to generate a nucleic acid amplification reaction. It is possible to prevent it.
Modifications that terminate the amplification reaction include, but are not limited to, for example, modification in which the 3'position OH group of ribose at the terminal of the polynucleotide is replaced with another substance such as a phosphate group. Thereby, the extension reaction by the polymerase can be stopped.
Alternatively, as described above, the fourth nucleic acid portion is ligated to the 3'end of the first nucleic acid portion and designed so that the fourth nucleic acid portion has a non-natural artificial base sequence and does not hybridize with the template nucleic acid. Therefore, even if the first nucleic acid portion has hybridized with a nucleic acid other than the detection target RNA in the test sample, or even if the first nucleic acid portion has hybridized with the template nucleic acid, Since the nucleic acid portion of 4 does not hybridize with the nucleic acid or the template nucleic acid in the test sample, it is possible to prevent an unintended nucleic acid amplification reaction from occurring.

As the "Cas9 nuclease" used in the present invention, a natural Cas9 nuclease can be used, or a Cas9 nuclease obtained by artificially modifying it can also be used.
Cas9 nucleases found in nature are roughly classified into Cas9 nucleases belonging to class 1 in which a plurality of effector factors including nucleases are involved in cleavage, and Cas9 nucleases belonging to class 2 requiring only nucleases.
In the present invention, any class of Cas9 nucleases can be used, but it is preferable to use Class 2 Cas9 nucleases with few effector factors to be used.

Class 2 Cas9 nucleases are further classified into type II, type V and type VI.
Examples of the type II Cas9 nuclease include SpyCas9 nuclease derived from Streptococcus pyogenes (Streptococcus pyogenes), SaCas9 nuclease derived from Staphylococcus aureus (Staphylococcus aureus), and NmeCas9 nuclease derived from Neisseria meningitidis (N. meningitidis). Can be used.

SpyCas9 nuclease is a Cas9 nuclease that is often used in genome editing technology, and many variants thereof have been developed. SaCas9 nuclease is a Cas9 nuclease characterized by having a smaller molecular size than SpyCas9 nuclease. SpyCas9 nuclease and SaCas9 nuclease can be suitably used in the first to third embodiments of the RNA detection method of the present invention.
As described above, NmeCas9 nuclease is a Cas9 nuclease that can be used in the fourth embodiment of the RNA detection method of the present invention, and when this is used, a single-stranded nucleic acid is used as a nucleic acid for RNA detection. be able to. And when NmeCas9 nuclease is used, it is not necessary to ligate an adapter nucleic acid to the RNA to be detected, and it is possible to detect RNA of any base sequence without the need for trans-activated crRNA or protospacer adjacent motif. Is.
When NmeCas9 nuclease is used, a double-stranded nucleic acid can be used as a nucleic acid for RNA detection, but in this case, an adapter nucleic acid or trans-activated crRNA is required.

As the type V Cas9 nuclease, for example, Cpf1 (AsCpf1) nuclease derived from Acidaminococcus sp. BV3L6, Cpf1 (LbCpf1) nuclease derived from Lachnospiraceae bacterium ND2006, and the like can be used.
As the type VI Cas9 nuclease, for example, C2c2 (Cas13a) nuclease can be used.

When Cpf1 (AsCpf1) nuclease or Cpf1 (LbCpf1) nuclease is used, transactivating crRNA is not required, and the nuclease cleaved end becomes the protruding end.
When C2c2 (Cas13a) nuclease is used, the RNA detecting nucleic acid can be RNA.

The Proto-spacer Adjacent Motif (PAM) must be designed according to the type of Cas9 nuclease. For example, when SpyCas9 is used, it is 5′-NGG-3′, when SaCas9 is used, 5′-NNGRR(T)-3′, and when NmeCas9 is used, 5′-NNNNGATT-3. ’ The sequence of the Cpf1 (AsCpf1) and Cpf1 (LbCpf1) protospacer-adjacent motifs is 5'-TTTN-3'.

Whether the nucleic acid for RNA detection is single-stranded or double-stranded, whether the nucleic acid for RNA detection is DNA or RNA, and whether transactivating crRNA is required, depending on the type of Cas9 nuclease The conditions are different. Further, the sequence of the motif adjacent to the protospacer recognized by Cas9 nuclease also differs depending on the type of Cas9 nuclease. Therefore, in the RNA detection method of the present invention, it is necessary to design the RNA detection nucleic acid according to the type of Cas9 nuclease.

As the “polymerase” used in the present invention, a polymerase that synthesizes DNA using DNA as a template (DNA-dependent DNA polymerase) or a polymerase that synthesizes DNA using RNA as a template (RNA-dependent DNA polymerase) can be used. It is preferable to use a DNA-dependent DNA polymerase because of the ease of nucleic acid amplification.

In the step i) of the RNA detection method of the present invention, a test sample containing RNA to be detected is brought into contact with a nucleic acid for RNA detection to hybridize the first nucleic acid portion to the RNA to be detected to form a complex. Is a step of forming.
Here, “hybridize” means that the base of the RNA to be detected and the base of the first nucleic acid portion are bonded by hydrogen bond.

The conditions for contacting the test sample containing the RNA to be detected and the nucleic acid for RNA detection are not particularly limited as long as they can hybridize and form a complex, but those with high homology It is preferable that the reaction conditions are such that only those can hybridize.
As such reaction conditions, conditions such as temperature, salt concentration, pH, and organic solvent to be added can be set in consideration of the base sequence of the RNA to be detected and the base sequence of the first nucleic acid portion.

The step ii) of the RNA detection method of the present invention is a step of degrading a part of the first nucleic acid part in the complex with Cas9 nuclease.
Here, "decomposition" means any decomposition as long as the decomposition of at least a part of the covalent bond of the nucleotide skeleton of the first nucleic acid part results in the cleavage of the nucleotide skeleton. Good. Further, the “part of the first nucleic acid portion” may be a covalent bond at the boundary portion between the first nucleic acid portion and the second nucleic acid portion.

The reaction conditions for decomposing with Cas9 nuclease are not particularly limited as long as they are the reaction conditions for decomposing the first nucleic acid part, but the optimum temperature, the optimum salt concentration, and the optimum salt concentration depending on the type of Cas9 nuclease used It is preferable to carry out the reaction at a suitable pH using a solvent containing necessary metal ions and the like.

The step iii) of the RNA detection method of the present invention is a step of converting a nucleic acid having a second nucleic acid portion among RNA detection nucleic acids into a single-stranded nucleic acid to be used as a nucleic acid amplification primer.
To make a single strand, the reaction solution may be heated to promote the dissociation of the nucleic acids, or the nucleic acids may be naturally dissociated to form a single strand without heating. Good.
In addition, the nucleic acid amplification primer does not necessarily have to be a single strand in its entire length, and if most of the 3'-end side is a single strand, it is in a state of being partially double-stranded. Can also function as a primer for nucleic acid amplification.

The step iv) of the RNA detection method of the present invention is a step of hybridizing a nucleic acid amplification primer to a template nucleic acid and performing a reaction of amplifying the template nucleic acid with a polymerase.
Here, the reaction for amplifying the template nucleic acid is not particularly limited as long as it is a nucleic acid amplification method that requires a primer, but is not limited thereto, and for example, PCR method, LAMP method, SDA method, etc. are used. Can be done by

In the PCR (Polymerase Chain Reaction) method, two primers that are complementary to the template nucleic acid and hybridize with the sense strand and antisense strand of the template nucleic acid are designed and used. Then, 1) heat denaturing the double-stranded nucleic acid to denature it into a single-stranded nucleic acid, 2) performing annealing to hybridize the primer to the template nucleic acid, and 3) starting the template nucleic acid with a polymerase starting from the primer. By performing an extension reaction for synthesizing a nucleic acid as a template and repeating steps 1) to 3), the nucleic acid in the region sandwiched by the primers can be amplified.

When the PCR method is used in the RNA detection method of the present invention, the nucleic acid amplification primer of the present invention is used as one primer, and the other primer is designed and used based on the base sequence of the template nucleic acid.
In the annealing of the PCR method, Tm (melting temperature) is calculated based on the base sequence of the second polynucleotide portion of the nucleic acid amplification primer of the present invention or the base sequence of the other primer, and the annealing temperature is Tm. It is preferable to set the temperature to a lower temperature.
The extension reaction is usually performed at about 67 to 77°C, and the thermal denaturation is usually performed at about 90 to 98°C.
Since the annealing, the extension reaction, and the thermal modification are performed at different temperatures, a thermal cycler is usually used as a device for rapidly transferring these temperatures.
A thermostable DNA polymerase is usually used in the PCR method, and in that case, a buffer solution containing four types of substrates (dATP, dGTP, dCTP, dTTP) and divalent magnesium ions is provided to maintain an optimum pH. Use reaction buffer.

The LAMP method is a nucleic acid amplification method developed by the present applicant (Japanese Patent No. 3313358 and the like), and is a method that enables a nucleic acid amplification reaction at an isothermal temperature using a strand displacement type polymerase.
In the LAMP method, an inner primer pair (FIP and BIP) and an outer primer pair (F3 and B3) can be used, and in addition, a loop primer pair (LF and LB) can be used.
The outline of the nucleic acid amplification reaction of the LAMP method is schematically shown in FIGS.

As shown in (1) of FIG. 8, when the arbitrary sequence F1c existing on one side chain of the template nucleic acid and the arbitrary sequence F2c existing on the 3'side thereof are selected, the inner primer (FIP) is complementary to F2c. A sequence F2 and a sequence F1c identical to F1c are arranged in this order from 3′ to 5′.
As shown in (6) of FIG. 8, the inner primer (BIP) is selected as B2c when an arbitrary sequence B1c existing on the other strand of the template nucleic acid and an arbitrary sequence B2c existing on the 3'side thereof are selected. It is a primer having complementary sequence B2 and sequence B1c identical to B1c in this order from 3′ to 5′.

Since the template nucleic acid is in a dynamic equilibrium state at around 65°C even when it is a double-stranded nucleic acid, one of the primers anneals to the complementary portion of the template nucleic acid and a strand displacement type polymerase is used. Then, the strand on one side is peeled off by the extension reaction starting from the primer, and a single-stranded state is obtained. Therefore, in the LAMP method, it is not necessary to previously denature the double-stranded nucleic acid into a single-stranded nucleic acid, unlike the PCR method. Then, as shown in (1) of FIG. 8, the inner primer can be annealed to the template nucleic acid that has become a single strand to start the extension reaction. Then, as shown in (2) of FIG. 8, a nucleic acid complementary to the template nucleic acid is synthesized.
In the RNA detection method of the present invention, when the nucleic acid amplification primer is the inner primer of the LAMP method, the second nucleic acid portion is designed to be F2 or B2, and F1c or the third nucleic acid portion is used. Design to have B1c.

As shown in (3) and (6) of FIG. 8, the outer primer means an arbitrary sequence F3c or B3c existing outside (3′ side) of F2c or B2c complementary to the inner primer in the template nucleic acid. A primer having a complementary F3 or B3 sequence.
As shown in (3) of FIG. 8, the outer primer (F3) anneals to the outside of the inner primer (FIP). Next, using the outer primer (F3) as a starting point, a strand displacement-type polymerase synthesizes a nucleic acid that is complementary to the template nucleic acid, and the inner primer (FIP) is used as a starting point to remove the synthesized nucleic acid and synthesize the nucleic acid. To go. As a result, as shown in (4) of FIG. 8, a double-stranded nucleic acid synthesized from the outer primer (F3) as a starting point is produced.

The nucleic acid synthesized starting from the inner primer (FIP) is peeled off to become a single strand by the nucleic acid synthesis starting from the outer primer (F3), but as shown in (5) of FIG. It has complementary regions F1c and F1 on the terminal side. Therefore, as shown in (6) of FIG. 8, self-annealing is caused to form a loop.
As shown in (6) of FIG. 8, the inner primer (BIP) is annealed to the loop-forming nucleic acid, and a complementary nucleic acid is synthesized with this as an origin. In this process, the loop is peeled off and is in a stretched state. Furthermore, the outer primer (B3) anneals to the outside of the inner primer (BIP), and a complementary nucleic acid is synthesized by the strand displacement polymerase starting from this and synthesized first from the inner primer (BIP). The nucleic acid grows while peeling it off. As a result, a double-stranded nucleic acid synthesized from the outer primer (B3) as a starting point is produced as shown in (7) of FIG.

In the RNA detection method of the present invention, the nucleic acid amplification primer can be used as an outer primer of the LAMP method, and in that case, the second nucleic acid portion is designed to be F3 or B3.

As shown in (6) of FIG. 8, the nucleic acid synthesized from the inner primer (BIP) as a starting point is peeled off by the synthesis from the outer primer (B3) to be a single-stranded nucleic acid. This nucleic acid has complementary regions F1c and F1 on the 3'terminal side and complementary regions B1c and B1 on the 5'terminal side, as shown in (8) of FIG. It forms a loop and becomes a dumbbell-shaped nucleic acid.

The dumbbell-shaped nucleic acid has a structure serving as a starting point for causing exponential nucleic acid amplification.
First, as shown in (8) of FIG. 9, nucleic acid synthesis was carried out using F1 at the 3′ end of dumbbell-shaped nucleic acid as a starting point and self-templated, whereby the loop at the 5′ end was peeled off. extend. Furthermore, since the F2c of the loop on the 3'-end side is a single strand, the inner primer (FIP) or loop primer can anneal, and nucleic acid synthesis starting from F2 of FIP is performed. The nucleic acid grows while peeling off the synthesized nucleic acid starting from.

Next, as shown in (9) of FIG. 9, the nucleic acid synthesized from F1 as a single strand was peeled off by the nucleic acid synthesized from FIP to form a single strand, Since it has a region complementary to, it self-anneals to form a loop. Starting from B1 at the 3'end of this loop, self-templated nucleic acid synthesis begins. Then, the nucleic acid synthesized from FIP as a starting point is stretched while being peeled off, resulting in the structure shown in (10) of FIG. 9.
By the above process, the nucleic acid synthesized from FIP as a starting point is peeled off to form a single strand, but as shown in (11) of FIG. A loop forms a dumbbell-shaped nucleic acid.

The structure of the dumbbell-shaped nucleic acid in (11) of FIG. 9 is a structure completely complementary to the structure of (8). Therefore, as in the dumbbell-shaped nucleic acid of (8), self-templated nucleic acid synthesis is performed starting from B1 at the 3'end, and further, an inner primer (BIP) or B2c in the loop at the 3'end side or The loop primer anneals, nucleic acid synthesis is performed starting from B2 of BIP, and the nucleic acid is elongated while peeling off the synthesized nucleic acid starting from B1. As a result, the nucleic acid of (8) is produced again through the same steps as (8), (9) and (11).
By repeating such a cycle of nucleic acid synthesis, nucleic acids such as (10) are synthesized one after another.

In the nucleic acid of (10), BIP or roof primer is annealed to single-stranded B2c to synthesize a nucleic acid while peeling off the double-stranded portion, a nucleic acid having a loop is generated, and the loop is further initiated. By repeating the process of synthesizing the nucleic acid as described above, amplification products having various structures on the same strand, which have mutually repeating sequences, are produced in various sizes.
In the LAMP method, nucleic acids are amplified exponentially by the above amplification reaction.

Strand-displacing DNA polymerase is usually used in the LAMP method. In that case, a buffer solution containing four types of substrates (dATP, dGTP, dCTP, dTTP) and divalent magnesium ions is provided to maintain an optimum pH. Use reaction buffer.
The reaction temperature of the LAMP method is usually 50 to 70°C, preferably 55 to 70°C. The reaction time is usually 1 minute to 10 hours, preferably 5 minutes to 4 hours.

The SDA (Standard Displacement Amplification) method is a method of amplifying a nucleic acid using a restriction enzyme and a strand displacement polymerase. In the SDA method, by inserting a restriction enzyme recognition sequence into the primer in advance, the nick generated by the restriction enzyme gives the starting point of complementary strand synthesis, and the nucleic acid that was previously synthesized is peeled from it, and the nucleic acid is extended. Since the reaction is performed, the nucleic acid can be isothermally amplified without changing the temperature unlike the PCR method.

In the RNA detection method of the present invention, the presence of the nucleic acid to be detected can be detected using the amplification of the template nucleic acid as an index.
The method for confirming the amplification of the template nucleic acid is not limited to these, but for example, it can be optically detected by reacting the amplified nucleic acid with an intercalator and staining, and the primer can be detected. It can be optically detected by pre-labeling with a fluorescent dye and incorporating the fluorescent dye into the amplified nucleic acid. When confirming the amplified nucleic acid, the amplified nucleic acid having a large molecular weight may be separated by electrophoresis, or the amplified nucleic acid may be detected by using FRET (fluorescence resonance energy transfer) with a fluorescent dye. Good.
Examples of the intercalator include, but are not limited to, an intercalator that specifically binds to a double-stranded nucleic acid, such as ethidium bromide, SYBR Green (registered trademark), and Pico Green (registered trademark). be able to.

2. Nucleic acid for detecting RNA The nucleic acid for detecting RNA according to the second invention of the present invention is a reagent used for detecting the target RNA, and the above-mentioned 1. The nucleic acid for RNA detection described in 1. can be used.
The nucleic acid for RNA detection may be a product in a state of being dissolved in a buffer solution, or may be a product in a dried state.
The buffer solution may be any solution as long as it has a composition capable of stably dissolving the nucleic acid for RNA detection. For example, but not limited to, a buffer in which Tris-hydrochloric acid is dissolved, a TE buffer in which Tris-hydrochloric acid and EDTA are dissolved, or the like can be used.

3. RNA detection kit The RNA detection kit of the third invention of the present invention comprises at least:
A) a template nucleic acid that is amplified and serves as an index for detection,
B) A nucleic acid for RNA detection comprising a first nucleic acid part having a base sequence complementary to at least a part of RNA to be detected and a second nucleic acid part having a base sequence complementary to a part of a template nucleic acid ,
C) Cas9 nuclease,
Is contained as a reagent.
The RNA detection kit of the present invention can be used in combination with a commercially available polymerase, but it is preferable that the RNA detection kit itself of the present invention further contains D) polymerase as a reagent.
For these reagents A) to D), see 1. And 2. The same reagents as those described above can be used.

Further, the RNA detection kit of the present invention,
i) contacting a test sample containing RNA to be detected with a nucleic acid for RNA detection to hybridize the first nucleic acid portion to the RNA to be detected to form a complex,
ii) digesting a part of the first nucleic acid part in the complex with Cas9 nuclease,
iii) A nucleic acid having a second nucleic acid portion in the RNA detecting nucleic acid is made into a single-stranded nucleic acid to be used as a nucleic acid amplification primer,
iv) Instructions describing that a primer for nucleic acid amplification is hybridized with a template nucleic acid and a reaction of amplifying the template nucleic acid with a polymerase is performed can be included.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

(Detection of short-chain RNA)
Short RNA was detected using the RNA detection method of the present invention.
The detection of short-chain RNA was performed by the same method as the method described in the second embodiment of the present invention. The experimental process is shown in FIG. 10, the base sequence of the nucleic acid reagent used for detection is shown in FIG. 11(A), the other reagents used are shown in FIG. 11(B), and the base sequence of the template nucleic acid used for detection is shown. Is shown in FIG.
As the RNA to be detected, an artificially synthesized miRNA model sequence (shortRNA-crRNA-A: SEQ ID NO: 21) was used, and a 3'-side crRNA adapter (crRNA-B-PHO: SEQ ID NO: 22) was ligated by ligation, A full-length crRNA (crRNA: SEQ ID NO: 23) was obtained. As shown in FIG. 12, for ligation, a method of not adenylating the 5′ end of the 3′-side crRNA adapter (FIG. 12(A)) and a method of adenylating the 5′ end (FIG. 12(B)) were used. Two methods were used. That is, ligation with the miRNA model sequence was performed using an adapter in which the 5'end was not adenylated (reaction B), and ligation with the miRNA model sequence was performed using an adapter in which the 5'end was adenylated ( Reaction D). As a control, the reaction was performed by each ligation method without adding the miRNA model sequence (reactions A and C).

As shown in FIG. 13(A), in the ligation reaction solution (reactions A to D), trans-activated crRNA (tracrRNA: SEQ ID NO: 24), Cas9 nuclease (Cas9 Nuclease NLS Protein (APB)), and RNA were added. Double-stranded DNA for detection (SpyCas9-pasmid-4/PCR: SEQ ID NO: 20) was added, and the double-stranded DNA was cleaved with Cas9 nuclease ((1) to (4)). As a control, a reaction using distilled water (DW) instead of the ligation reaction solution ((5)) and a reaction using full-length crRNA (crRNA: SEQ ID NO: 23) ((6)) were performed. The reaction was carried out at 37°C for 3 hours.
As Cas9 nuclease, SpyCas9 derived from Streptococcus pyogenes (Streptococcus pyogenes) was used.
The double-stranded DNA for RNA detection has a base sequence complementary to the miRNA model sequence as the first nucleic acid part, and has a base sequence to be the inner primer (BIP) of the LAMP method as the second nucleic acid part. A double-stranded DNA was designed. In addition, the double-stranded DNA was designed so that the antisense strand of the double-stranded DNA for RNA detection contained a base sequence of -AGG- as a sequence serving as a proto-spacer adjacent motif (PAM). Furthermore, biotin for use in purification was linked to the 5'end of the sense strand of the double-stranded DNA for RNA detection.
Capillary electrophoresis was performed to verify double-stranded DNA cleavage by Cas9 nuclease. Bioanalyzer DNA 1000 kit (Agilent) was used for capillary electrophoresis. The result of the capillary electrophoresis is shown in FIG.
As shown in FIG. 13(B), when distilled water (DW), Cas9 nuclease, and transactivated crRNA (tracrRNA) were added to the double-stranded DNA for RNA detection to carry out the reaction, about 100 bp was obtained. Bands were observed ((5)). On the other hand, when double stranded DNA for RNA detection is added with Cas9 nuclease, full-length crRNA (crRNA) and trans-activated crRNA (tracrRNA) and reacted, a band of about 50 bp is observed, It was confirmed that the double-stranded DNA was cut ((6)). Double-stranded DNA was not cleaved when the ligation reaction solution of reactions A and C without addition of miRNA model sequence was used ((1) and (3)), but reaction B with addition of miRNA model sequence and When the ligation reaction solution of D was used, cleavage of double-stranded DNA was observed ((2) and (4)).

The digestion product of Cas9 nuclease was purified using streptavidin beads (Dynabeads M-280 Streptavidin (Thermo)). As a result, the biotin-ligated DNA could be purified.
The DNA obtained by the purification is a DNA having the second nucleic acid portion and serves as an inner primer (BIP) for the LAMP method. The template nucleic acid was amplified by the LAMP method using this primer (Biotin-BIP, gRNA). As the template nucleic acid, the artificial sequence-1 for spyCas9 shown in FIG. 11(C) (SEQ ID NO: 25) was used.
Other primers used in the LAMP method include ART7-FIP (SEQ ID NO: 16) as the inner primer (FIP), ART7-F3 (SEQ ID NO: 14) as the outer primer (F3), and ART7-B3 (ART3 as the outer primer (B3). SEQ ID NO: 15), ART7-LF (SEQ ID NO: 18) was used as a loop primer (LF), and ART7-LB (SEQ ID NO: 19) was used as a roof primer (LB). As the LAMP amplification reaction reagents including these primers, those described in FIG. 11(B) were used.
As a control, LAMP amplification reaction (Control BIP) using synthetic inner primer (ART-BIP-KM29) instead of the primer obtained by purification of Cas9 nuclease digestion product, and purification of Cas9 nuclease digestion product LAMP amplification reaction (DW) using distilled water (DW) instead of the obtained primer, and digestion using distilled water (DW) instead of crRNA when cleaving double-stranded DNA by Cas9 nuclease An amplification reaction (Biotin-BIP, DW) of the LAMP method using the DNA obtained by purifying the product was performed.
The result of real-time measurement of the amount of the amplification reaction product of the LAMP method with a fluorescent dye is shown in the graph of FIG.
As shown in FIG. 14, when a double-stranded DNA was cleaved by Cas9 by adding crRNA derived from the detection target RNA (Biotin-BIP, gDNA), a synthetic inner primer was used (Control BIP) Nucleic acid amplification by the LAMP method equivalent to that was observed. On the other hand, when double-stranded DNA was cleaved by Cas9 without adding crRNA derived from the RNA to be detected (Biotin-BIP, DW), amplification by the LAMP method took a long time and a normal amplification reaction was observed. There wasn't.
From the above, it has been clarified that the RNA to be detected can be detected by using the RNA detection method of the present invention with the amplification of the template nucleic acid as an index.

INDUSTRIAL APPLICABILITY The RNA detection method, RNA detection primer, and RNA detection kit of the present invention are useful in the industrial fields of clinical tests, clinical test reagents, and research reagents.

1 RNA to be detected
101 nucleic acid part having a base sequence derived from a spacer sequence 102 nucleic acid part having a base sequence derived from a repeat sequence 2 nucleic acid for RNA detection 201 first nucleic acid part 201a 5'side part 201b of the first nucleic acid part 201b first nucleic acid part 3'side part 201' of the nucleic acid part 201a' complementary to the first nucleic acid part 201a' complementary nucleic acid part 201b' to the 5'side part of the first nucleic acid part complementary to the 3'side part of the first nucleic acid part Nucleic acid portion 202 second nucleic acid portion 202a second nucleic acid portion 5'side portion 202b second nucleic acid portion 3'side portion 202' nucleic acid portion 202a' complementary to the second nucleic acid portion 202a' second portion Nucleic acid portion 202b' complementary to the 5'side portion of the nucleic acid portion 203 Nucleic acid portion 203 complementary to the 3'side portion of the second nucleic acid portion Third nucleic acid portion 203' Nucleic acid portion complementary to the third nucleic acid portion 204 fourth nucleic acid portion 204' nucleic acid portion complementary to the fourth nucleic acid portion 3 Cas9 nuclease 4 trans-activated crRNA
5 Tag 6 Template nucleic acid 601 Nucleic acid portion having a nucleotide sequence complementary to the nucleic acid portion 201a 7 Polymerase 8 Adapter nucleic acid 801 Nucleic acid portion having a nucleotide sequence derived from the repeat sequence of crRNA 802 Nucleic acid having a nucleotide sequence derived from trans-activated crRNA part
9 RNA ligase 10 Guide RNA

The present invention relates to an RNA detection method for detecting RNA to be detected by utilizing the principle of nucleic acid amplification such as LAMP method or PCR method. The present invention particularly relates to an RNA detection method that is suitable for detecting short-chain RNA having 10 to 100 bases and can be used for detecting miRNA and the like.
The present invention also relates to a nucleic acid for detecting RNA and a kit for detecting RNA.

INDUSTRIAL APPLICABILITY The RNA detection method, RNA detection nucleic acid , and RNA detection kit of the present invention are useful in the industrial field of clinical tests, clinical test reagents, and research reagents.

Claims (29)

In the RNA detection method for detecting RNA to be detected,
A) A template nucleic acid that is amplified and serves as an index for detection,
B) For RNA detection including a first nucleic acid portion having a base sequence complementary to at least a part of the detection target RNA and a second nucleic acid portion having a base sequence complementary to a part of the template nucleic acid Nucleic acid,
At least C) Cas9 nuclease and D) polymerase,
i) contacting a test sample containing the RNA to be detected with the nucleic acid for RNA detection to hybridize the first nucleic acid portion to the RNA to be detected to form a complex,
ii) decomposing a part of the first nucleic acid portion in the complex with the Cas9 nuclease,
iii) A single-stranded nucleic acid having a second nucleic acid portion in the RNA detecting nucleic acid is used as a nucleic acid amplification primer,
iv) An RNA detection method comprising hybridizing the nucleic acid amplification primer to the template nucleic acid and performing a reaction of amplifying the template nucleic acid with the polymerase. The RNA detection method according to claim 1, wherein a reaction of ligating an adapter nucleic acid containing a base sequence derived from a repeat sequence of crRNA to the detection target RNA is performed before the step i). The RNA detection method according to claim 1 or 2, wherein in the step i), the test sample containing the RNA to be detected is further contacted with trans-activated crRNA. The RNA detection method according to claim 2, wherein the adapter nucleic acid contains a base sequence derived from trans-activated crRNA. The RNA detection method according to any one of claims 1 to 4, wherein the Cas9 nuclease is a Class 2 Cas9 nuclease. The RNA detection method according to claim 5, wherein the Cas9 nuclease is SpyCas9 nuclease. The RNA detection method according to claim 5, wherein the Cas9 nuclease is NmeCas9 nuclease. The RNA detection method according to claim 1, wherein the RNA detection nucleic acid is a double-stranded nucleic acid. The RNA detection method according to claim 7, wherein the RNA detection nucleic acid is a single-stranded nucleic acid. The RNA detection method according to claim 1, wherein the RNA to be detected is a short-chain RNA having 10 to 100 bases. The nucleic acid in the 5'side portion of the first nucleic acid portion is complementary to a part of the detection target RNA and at the same time, is also complementary to a part of the template nucleic acid. The method for detecting RNA according to any one of 10. The RNA detection method according to claim 1, wherein nucleic acid amplification by the LAMP method is performed in the step iv). In the nucleic acid for RNA detection used to detect the RNA to be detected,
A first nucleic acid portion having a base sequence complementary to at least a part of the RNA to be detected,
A nucleic acid for RNA detection, which comprises a second nucleic acid portion having a base sequence complementary to a part of a template nucleic acid that is amplified and serves as an indicator for detection. The nucleic acid for detecting RNA according to claim 13, which is a double-stranded nucleic acid. The nucleic acid for detecting RNA according to claim 13, which is a single-stranded nucleic acid. The RNA detecting nucleic acid according to any one of claims 13 to 15, wherein the first nucleic acid portion has 10 to 100 bases. 14. The nucleic acid in the 5'side portion of the first nucleic acid portion is complementary to at least a part of the RNA to be detected, and at the same time, is also complementary to a part of the template nucleic acid. 17. The nucleic acid for detecting RNA according to any one of 1 to 16. In the RNA detection kit used to detect the RNA to be detected,
A) A template nucleic acid that is amplified and serves as an index for detection,
B) For RNA detection including a first nucleic acid portion having a base sequence complementary to at least a part of the detection target RNA and a second nucleic acid portion having a base sequence complementary to a part of the template nucleic acid An RNA detection kit containing a nucleic acid and C) Cas9 nuclease. The kit for RNA detection according to claim 18, further comprising an adapter nucleic acid containing a nucleotide sequence derived from the repeat sequence of crRNA. The kit for detecting RNA according to claim 18 or 19, further comprising trans-activated crRNA. The kit for RNA detection according to claim 19, wherein the adapter nucleic acid contains a base sequence derived from trans-activated crRNA. The kit for RNA detection according to any one of claims 18 to 21, which further comprises D) a polymerase. The RNA detection kit according to any one of claims 18 to 22, wherein the Cas9 nuclease is a Class 9 Cas9 nuclease. 24. The RNA detection kit according to claim 23, wherein the Cas9 nuclease is SpyCas9 nuclease. 24. The RNA detection kit according to claim 23, wherein the Cas9 nuclease is NmeCas9 nuclease. The RNA detection kit according to any one of claims 18 to 25, wherein the RNA detection nucleic acid is a double-stranded nucleic acid. The RNA detection kit according to claim 25, wherein the RNA detection nucleic acid is a single-stranded nucleic acid. The RNA detection kit according to any one of claims 18 to 27, wherein the RNA to be detected is a short-chain RNA having 10 to 100 bases. The nucleic acid of the 5'side portion in the first nucleic acid portion is complementary to a part of the detection target RNA and at the same time, is also complementary to a part of the template nucleic acid. The kit for detecting RNA according to any one of 28.