Method and assay kit for detecting single base substitution in group of short-chain RNAS having sequence diversity on 3' side

Abstract

According to one embodiment, there is provided a method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity on a 3' side. The method including: bringing into contact, a solution containing the group of short-chain RNAs, a guide RNA containing a sequence that is complementary to the target sequence, a sequence-specific endonuclease that recognizes the guide RNA-target nucleic acid complex, and a reporter substrate; measuring a signal derived from the reporter substrate; and determining the presence or absence of a single base substitution in the target nucleic acid based on the intensity of the signal.

Description

METHOD AND ASSAY KIT FOR DETECTING SINGLE BASE SUBSTITUTION IN GROUP OF SHORT-CHAIN RNAS HAVING SEQUENCE DIVERSITY ON 3' SIDECross-Reference to Related Applications

[0001] This application is based upon and claims the benefit of priority from Japanese Patent Application No. 2024-098124, filed June 18, 2024, the entire contents of which are incorporated herein by reference.Field

[0002] Embodiments described herein relate generally to a method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity at the 3’ side.Background

[0003] Genome editing has enabled various genetic modifications in microorganisms, animals, and plants. Genome editing is a technique that uses DNA cleavage enzymes to induce specific double-stranded DNA breaks in target genes, and then precisely modifies the genes by utilizing the repair process. In recent years, in addition to editing by DNA cleavage, techniques have been developed to introduce functional domains such as DNA-modifying proteins to enable sequence-specific modifications and labeling. General genome editing is a technique that uses DNA cleavage enzymes to induce specific double-stranded DNA breaks in target genes, and then precisely modifies the genes by utilizing the repair process.

[0004] On the other hand, it has been reported that microRNA can be used to detect and identify various types of cancer. For example, it has been proposed that the sequence ratio of single base substitutions in microRNA can be used as an indicator. However, since general sequence analysis requires a lot of time and cost, it is difficult to apply it to the diagnosis of many samples, such as screening tests.

[0005] FIG. 1 is a schematic diagram showing a flow of a method according to the first embodiment.FIG. 2 is a schematic diagram showing a concept of the first embodiment.FIG. 3 is a schematic diagram showing the concept of the first embodiment.FIG. 4 is a graph showing experimental results.

[0006] In general, according to one embodiment, there is provided a method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity at the 3’ side. The method includes the following: bringing into contact, a solution containing the group of short-chain target RNAs with a guide RNA having a sequence that is complementary to the target sequence including a single nucleotide substitution site, a sequence-specific endonuclease that recognizes the guide RNA-target nucleic acid complex, and a reporter substrate that generates a signal upon the trans-cleavage activity of the sequence-specific endonuclease with each other; measuring a signal generated from the reporter nucleic acid; and determining the presence or absence of a single base substitution in the test nucleic acid based on the intensity of the signal. The guide RNA includes a single-stranded structure and an intramolecular hairpin structure on the 5’ side of this single-stranded structure. The single-stranded structure includes a complementary sequence of the target sequence, and the length of the complementary sequence from the 5’ end to the single-base substitution site is 0 to 10 bases.

[0007] Embodiments will now be described with reference to the accompanying drawings. In these embodiments, substantially identical structural elements are denoted by the same symbols, and some of the descriptions may be omitted. Note that the drawings are schematic, and the relationship between the thickness and planar dimensions of each part, the ratio of the thickness of each part, etc., may differ from the actual ones.

[0008] (First Embodiment) The first embodiment is a method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity at the 3’ side. As shown in FIG. 1, the method includes the following three items: - bringing into contact, a solution containing a group of short-chain RNAs with sequence diversity at the 3’ side, a guide RNA containing a complementary sequence to a target sequence with a single base substitution site, sequence-specific endonuclease corresponding to the guide RNA, and a reporter substrate that generates a signal upon the trans-cleavage activity of the sequence-specific endonuclease with each other; (S11); - measuring a signal generated from the reporter substrate (S12); and - determining whether or not there is a single base substitution in the group of short-chain RNAs in the target nucleic acid based on the intensity of the signal (S13).

[0009] The target nucleic acid may as well be a group of short-chain RNAs that have a single base substitution site to be detected, or it may be a group of nucleic acids that includes such a group of short-chain RNAs. For example, the solution containing such a target nucleic acid may be an aqueous solution containing nucleic acids derived from humans or other animals or plants. The short-chain RNAs can be relatively short-chain RNAs having a base length of more than 10 bases to 100 bases, 15 bases or longer to 50 bases, 15 bases or longer to 30 bases, or 15 bases or longer to 25 bases. Such short-chain RNAs can be, for example, microRNAs, PIWI-interacting RNAs, and small interfering RNAs. The solution containing the group of short-chain RNAs of the target nucleic acids may as well be referred to as the test solution.

[0010] With reference to FIG. 2, a target nucleic acid 11 and a guide RNA 21 will be described. The target nucleic acid 11 includes a sequence diversity portion 32 on the 3’ side and a target sequence 22a on the 5’ side. The target sequence 22a has a single base substitution site 12a in its sequence. That is, in target nucleic acid 11, the sequence on the 3’ side following target sequence 22a can be any sequence. Further, the target nucleic acid 11 may also include an arbitrary sequence on a side of the 5’ side with respect to the target sequence 22a. The target sequence 22a can be selected from a part of microRNA sequence, for example, a sequence in microRNAs database or a result of sequence analysis of microRNAs. The target sequence 22a may be selected in advance as desired.

[0011] The guide RNA 21 includes a single-stranded structure 22b and an intramolecular hairpin structure 23. The single-stranded structure 22b includes a sequence 22c complementary to the target sequence 22a of the target nucleic acid 11. Therefore, it can also be stated that the single-stranded structure 22b includes a target sequence recognition site 22c. The target sequence recognition site 22c has a single base substitution site 12b at a position corresponding to the single base substitution site 12a of the target nucleic acid 11. The length from the 5’ end of the target sequence recognition site 22c to the single base substitution site can be 0 to 10 bases. Here, the target sequence recognition site 22c can be, for example, a complementary sequence of the target sequence 22a. Note here that the sequence of the single-stranded structure 22b other than the single-base substitution site 12b may contain one or two bases that are not complementary to the target sequence 22a. FIG. 2 shows an example in which the length of the target sequence recognition site 22c is equal to the length of the single-stranded structure 22b, but the structure is not limited to this. Note that the single-stranded structure 22b may be longer. The intramolecular hairpin structure 23 is a part of sequence-specific endonuclease recognition site (that is, direct repeat), which recognizes and binds to the corresponding sequence-specific endonuclease. For example, the sequence-specific endonuclease may be an enzyme having collateral activity, for example, a Cas protein, CRISPR-Cas13 or the like.

[0012] The sequence of the target sequence recognition site 22c of the guide RNA 21 is selected according to the sequence of the target nucleic acid 11 to be detected. Thus, it recognizes a specific group of short-chain RNAs having sequence diversity on the 3’ side as the target nucleic acid, and specifically binds to them. In this manner, the sequence-specific endonuclease is activated. Here, the single base substitution site 12b of the target sequence recognition site 22c is selected for the type of base to be detected. In other words, it is a base corresponding to either one of the wild type and the mutant type is selected. That is, even for single base substitution site, if the bases of the guide RNA 21 and the target nucleic acid 11 are complementary to each other, the sequence-specific endonuclease is activated. With this configuration, the target nucleic acid group can be accurately detected regardless of sequence diversity on the 3’side. In the case of microRNAs and the like, regardless of whatever the type of the microRNA, information on the presence of mutations in sequences that have a common target nucleic acid sequence can be useful for detecting or identifying diseases such as cancer.

[0013] With reference to FIG. 3, a specific example of discriminating between the target mutant type A and the non-target wild type C will be explained. For example, when determining the base sequence of a single base substitution site as to miR_1 contained in the target nucleic acid group, the target sequences are a miR_1-standard including a standard sequence 32a (part (a) of FIG. 3) and a miR_1-3’-modification including a modified sequence 32b (part (b) of FIG. 3). For each of these, for example, the target mutant type A and the non-target wild type C are assumed for the single base substitution site 12a. In other words, the microRNA with the standard sequence including the single base substitution is the miR_1-standard, and more specifically, the target nucleic acid 11a including the mutant type A and the non-target nucleic acid 31a including the wild type C. The microRNA including the 3’ side-modified sequence including a single-base substitution is miR_1-3’ modified, and more specifically, the target nucleic acid 11b including the mutant type A and the non-target nucleic acid 31b including the wild type C.

[0014] The guide RNA 21 comprises a complementary base U of the target mutant type A at the single-base substitution site. Alternatively, if the wild type C is targeted, the base at the single-base substitution site should only be the complementary base G of the wild type C. In the method, when the guide RNA 21 encounters the corresponding sequence-specific endonuclease 35, they bind to each other to form a complex 37 (also referred to as a "guide RNA - sequence-specific endonuclease"). When the target sequence recognition site 22c of the complex 37 encounters the target sequence 11a including its complementary strand (that is, the target nucleic acid 11), a double strand is formed there. Further, the sequence-specific endonuclease 35 is activated, and the sequence-specific endonuclease activity (that is, nucleic acid cleavage activity) 38 is expressed, and the target nucleic acid 11a is cleaved at the cleavage recognition site (not shown in the figure).

[0015] For example, in the formation of a base sequence-specific double strand for such a target sequence, when the guide RNA 21 is a crRNA, for example, the crRNA may bind to the target sequence as a discriminating molecule that can identify a difference in one base, and thus the sequence-specific endonuclease (for example, Cas protein) may be activated to cleave the target sequence.

[0016] On the other hand, in this reaction system, there is a reporter signal that can be detected by the activation of the aforementioned sequence-specific endonuclease. An example of this is a reporter nucleic acid 41a, which will now be described. The reporter nucleic acid 41a comprises a signal substance 42, a masking substance 45, and an oligonucleotide portion. The oligonucleotide portion comprises a cleavage recognition site by the sequence-specific endonuclease. The length of the oligonucleotide portion can be, for example, 50 bases or less. The reporter nucleic acid 41a is cleaved at the cleavage recognition site of the sequence-specific endonuclease by the nucleic acid cleavage activity 38 of the sequence-specific endonuclease 35 that is activated by the formation of the double strand. As a result, the signal emitting from the signal substance 42 that was inhibited by the masking substance 45 becomes detectable. The signal substance 42 may be, for example, a fluorescent substance, a chemiluminescent substance, or a chromogenic substance. The signals can be detected by the cleavage of the reporter nucleic acid 41a, caused by the act of the cleavage activity 38 of activated sequence-specific endonuclease 35. With on such a configuration and function, it can be said that the signal substance 42 is a substance that specifically transmits specific information.

[0017] The signal 43 from the signal substance 42 is detected by an appropriate detection mechanism or detection device according to the type of the signal. For example, the method may be carried out in a real-time PCR system or microplate reader that comprises such a detection mechanism. The bringing into contact (S11) can be carried out by allowing the reaction to proceed for 1 to 120 minutes under conditions of 25°C to 55°C. Note that the reaction temperature should preferably be 37°C. The detection of the signal 43 may be carried out along with time in parallel with the reaction caused by the bringing into contact described above, according to the type of signal substance, or it may be measured once, once or twice, or more than twice, for example, once to 120 times, at different points in time after a certain amount of time has elapsed.

[0018] In the method, the determining the presence or absence of a single base substitution in the target nucleic acid may further include determining the presence or absence of a single base substitution in the target nucleic acid based on whether or not there is a difference between the signal of the standard nucleic acid and the signal of the nucleic acid derived from the target substance. The signal of the standard nucleic acid may be measured in advance, or it may be measured before or after or in parallel with the measurement of the signal from the test substance, that is, the target nucleic acid. The presence or absence of a single base substitution may be determined, for example, from the presence or absence of a detection signal, the presence or absence of fluorescence intensity, the magnitude of the detection signal, or the difference in the magnitude of the fluorescence intensity. For example, even when a standard nucleic acid is not used, it is possible to make a determination similarly based on the presence or absence of a detection signal or the relative size of the detection signal. Standard nucleic acids can be designed based on the target sequence, for example, and are a sequence of microRNA in a database that includes the target sequence or a sequence obtained from sequence analysis such as next-generation sequencing. In addition, if desired, multiple types of guide RNAs 21 that have different types of bases assigned to the single base substitution sites may as well be used in combination. In this case, a comprehensive judgment may be made after making a judgment for each type of guide RNA 21. Furthermore, the labels can be designed as well so that the signals to be detected can be distinguished for each type of RNA 21.

[0019] In the case of conventional sequence analysis, it takes a lot of time and cost to detect single base substitutions, and therefore it is difficult to apply it to the diagnosis of many samples. However, according to the method according to the embodiment, it is possible to perform detection in a short time and at low cost. Further, the presence or absence of single base substitutions in the target nucleic acid group can be accurately detected regardless of sequence diversity on the 3’ side of microRNAs. y considering the sequence diversity of the 3’ side of the single base substitution site of microRNAs, it can be used for the detection or identification of diseases such as cancer while including information on the diversity of the 3’ side of microRNAs.

[0020] (Second Embodiment) The second embodiment is an assay kit for detecting single base substitutions in a group of short-chain RNAs having sequence diversity on the 3’ side. The kit comprises a guide RNA that includes a complementary sequence for target nucleic acid having a single base substitution site, a sequence-specific endonuclease corresponding to the guide RNA, and a reporter substrate that becomes detectable by activation of sequence-specific endonuclease.

[0021] As shown in FIGS. 2 and 3, the guide RNA 21 includes a single-stranded structure 22b, and includes an intramolecular hairpin structure 23 at its 5’ side. The single-stranded structure includes a complementary sequence 22c of the target sequence at its 5’ side, and the length from the 5’ end side of the complementary sequence 22c to the single base substitution site is 0 to 10 bases.

[0022] The sequence-specific endonucleases may be an enzyme with collateral activity, for example, a Cas protein, such as CRISPR-Cas13 or the like. The label that becomes detectable by the activation of such a sequence-specific endonuclease may be, for example, a reporter nucleic acid 41a, which will now be described below.

[0023] As shown in FIG. 3, the reporter nucleic acid 41a comprises a signal substance 42, a masking substance 45, and an oligonucleotide portion. The oligonucleotide portion comprises a cleavage recognition site for the sequence-specific endonuclease. The length of the oligonucleotide portion can be, for example, 50 bases or less. The reporter nucleic acid 41a is cleaved at the cleavage recognition site of the sequence-specific endonuclease by the nucleic acid cleavage activity 38 of the sequence-specific endonuclease 35 which is activated by the formation of a double strand. As the result, the signal from the signal substance 42, which is inhibited by the masking substance 45, becomes detectable. For example, the signal substance 42 can be a fluorescent substance, chemiluminescent substance, chromogenic substance or the like. For example, the following are examples of quenchers as a fluorescent substance and a masking substance 45: Fluorescent substances: FAM, HEX, VIC, ROX, Cy5, Texas Red, JOE Masking substances: Iowa Black (registered trademark), BHQ, TAMRA, ZEN For example, a combination of at least one fluorescent substance and a masking substance selected from these groups can be used. For example, combinations of fluorescent substances and quenchers include FAM and Iowa Black (registered trademark) FQ, FAM and BHQ-1, ROX and Iowa Black (registered trademark) Q, ROX and BHQ-2, Cyanine 5 and IOWA Black (registered trademark) RQ, Cyanine 5 and BHQ-2, but the combinations are not limited to these.

[0024] The assay kit may comprise each of the component ingredients in a state of being dried by freeze-drying or other means and stored in a container. Alternatively, each of the ingredients may be maintained in an appropriate solution and stored in an appropriate container, and the ingredients may be provided together as an assay kit. With the assay kit thus provided, it is possible to detect single base substitutions in a short time and at low cost. Further, the presence or absence of single base substitutions in the target nucleic acid group can be detected while maintaining the diversity of the target nucleic acid group, without being sorted out by the diversity of the 3’ side. As to the importance of consideration of the sequence diversity of the 3’ side of the single base substitution site of the target nucleic acid, it can be used for the detection or identification of diseases such as cancer while including information on the diversity of the 3’ side of the target nucleic acid, for example, by using microRNA and the like as the test substance.

[0025] Examples The followings are examples in which a method according to the embodiment is carried out.

[0026] Experiment 1: Detection of single base substitutions in two groups of microRNAs having sequence diversity at the 3’ side. Four types of microRNAs shown in FIG. 3 were prepared. For the microRNAs, miR_1 was used. The target sequences were miR_1-standard and miR_1-3’-modification. The miR_1-standard contains the standard sequence 32a (part (a) of Figure 3) as the sequence diversity portion, and the standard sequence 32a is UUUUGUG (sequence number a). The miR_1-3’-modification contains the modified sequence 32b (part (b) of Figure 3) as the sequence diversity portion, and the modified sequence 32b is UGUUGU (sequence number b). Furthermore, the single base substitution site 12a was targeted as the mutant type A and non-targeted as the wild type C. That is, the microRNA having the standard sequence including the single base substitution is the miR_1-standard, and more specifically, the target nucleic acid 11a including the mutant type A (sequence number 1) and the non-target nucleic acid 31a including the wild type C (sequence number 2). The microRNA including the 3’ side-modified sequence including a single-base substitution is miR_1-3’ modified, and more specifically, the target nucleic acid 11b including the mutant type A (sequence number 3) and the non-target nucleic acid 31b including the wild type C (sequence number 4). These nucleic acids were synthesized, and the obtained nucleic acids were maintained each in buffer solutions.

[0027] Similarly, a guide RNA 21 (sequence number 5) was synthesized. The guide RNA 21 comprises a complementary base of the target mutant type A in the single base substitution site. Apart from this, a FAM as a fluorescent substance was attached to the 5’ end side of the oligonucleotide represented by sequence number c and IOWA Black (registered trademark) FQ as a quencher was attached to the 3’ end side, and thus a detection nucleic acid was obtained.

[0028] The full-length nucleotide sequences of these are shown in Table 1. Further, in Table 2, these are listed in correspondence with the guide RNA for detecting mutations (abbreviated as "gRNA"), the target sequence, and the non-target sequence. Table 3 summarizes the features of designs of miR_1. From Tables 2 and 3, it can be seen that the miR_1-standard and miR_1-3’ modified gRNAs have the following features. That is, there are 4 bases of complementary nucleotides in the sequence downstream of the mutation in each case, and the number of bases of diversity allowing sequences on the 3’ side is 6 in each case. Further, the insertion position of the single-base substitution identification site to gRNA is the single-base substitution position of the microRNA sequence, that is, the fifth from the 5’ end of the complementary sequence of the target sequence, and the insertion position of the mismatch site included in the complementary sequence of the target sequence is the ninth from the 5’ end of the complementary sequence of the target sequence. In addition, the reporter nucleic acid used was a poly U reporter nucleic acid, and the fluorescent substance used was FAM, and the quencher substance used was Iowa Black (registered trademark) FQ, and these are collectively shown in Table 4. The complementary base pair is a combination of A-U and G-C.

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] As sequence-specific endonuclease, CRISPR-Cas13a (manufactured by Genscript, product number Z03472), which is a Cas protein, was prepared. The recognition sequence, cleavage sequence, and guide RNA structure corresponding to the CRISPR-Cas13a can be selected as needed.

[0035] The microRNA having the standard sequence including a single base substitution is miR_1-standard, and more specifically, the target nucleic acid 11a (sequence number 1, "target (mutant type A)" in part (a) of FIG. 3) including a mutant type A and the non-target nucleic acid 31a (sequence number 2, "non-target (wild type C)" in part (a) of FIG. 3) including a wild type C. The microRNA having the 3’ side-modified sequence including a single base substitution is a miR_1-3’-modification, and more specifically, the target nucleic acid 11b including the mutant type A (sequence number 3, "Target (mutant type A)" in part (b) of FIG. 3) and a non-target nucleic acid 31b including the wild type C (sequence number 4, "non-target (wild type C)" in part (b) of FIG. 3). These microRNAs were suspended in buffer solutions respectively at a concentration of 1 × 1010 copies / μL. The suspended solutions were dispensed into respective microplates at a volume of 2 μL. Then, to each, 18 μL of a reaction solution containing the guide RNA 21 (sequence number 5) at a concentration of 100 nM in the final reaction solution and CRISPR-Cas13a at a concentration of 100 nM in the final reaction solution was added, and the mixtures were incubated at 37°C for 5 minutes. The resultants were measured in terms of the fluorescence intensity using a plate reader at an excitation wavelength of 470 nm and a fluorescence wavelength of 520 nm, and the relative fluorescence intensity was calculated using a real-time PCR system for each. The results are shown in FIG. 4 and Table 5.

[0036]

[0037] From the results of FIG. 4 and Table 5, it is clear that the presence or absence of a single base substitution can be detected regardless of the difference in the sequence on the 3’ end side, that is, regardless of the sequence diversity present on the 3’ end side.

[0038] The followings are descriptions of examples of further embodiments. (1) A method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity on the 3’ side, the method comprising: bringing into contact, a solution containing a group of short-chain RNAs having sequence diversity on the 3’ side as a target nucleic acid, a guide RNA containing a complementary sequence of a target sequence comprising a single base substitution site, a sequence-specific endonuclease corresponding to the guide RNA, and a reporter substrate that becomes detectable by activation of the sequence-specific endonuclease; measuring a signal derived from the reporter substrate; and determining presence or absence of a single-base substitution in the group of short-chain RNAs in the target nucleic acid based on an intensity of the signal, wherein the guide RNA includes a single-stranded structure and an intramolecular hairpin structure on the 5’ side of the single-stranded structure, the single-stranded structure includes a complementary sequence of the target sequence on the 5’ side, and a length from the 5’ end of the complementary sequence to the single base substitution site is 0 to 10 bases. (2) The method of clause (1), wherein the intramolecular hairpin structure is a sequence-specific endonuclease recognition site. (3) The method of clause (1) or (2), wherein the short-chain RNAs are microRNAs. (4) The method of clause of any one of clauses (1) to (3) wherein the sequence-specific endonucleases have collateral activity. (5) The method of any one of clauses (1) to (4), wherein the reporter substrate is a detection nucleic acid comprising a signal substance that emits a detection signal and a masking substance that inhibits the detection signal, and an oligonucleotide to which the signal substance and the masking substance are attached. (6) The method of clause (5), wherein a length of the oligonucleotide is 50 bases or less. (7) The method of clause (5), wherein the ligand is a fluorescent substance, and the masking substance is a quencher. (8) The method of any one of clauses (1) to (7), wherein measurement of the signal from the detection nucleic acid is performed by a means selected from the group consisting of a real-time PCR system, a microplate reader, a digital PCR system, a fluorescence microscope, and a fluorescence detection device. (9) The method of any one of clauses (1) to (8), wherein the determining of the presence or absence of a single base substitution in the target nucleic acid is performed by determining the presence or absence of a difference between the detection signal obtained by performing similarly for a standard nucleic acid and the signal obtained from the target nucleic acid. (10) An assay kit for use in any one of the methods recited in clause (1) to (9), comprising a guide RNA including a complementary sequence to a target sequence including a single base substitution site, a sequence-specific endonuclease corresponding to the guide RNA, and a detection nucleic acid including a signal substance that becomes detectable by activation of sequence-specific endonuclease and a base sequence recognized by the sequence-specific endonuclease, wherein the guide RNA includes a single-stranded structure, and the 5’ side thereof includes an intramolecular hairpin structure, and the single-stranded structure includes a complementary sequence of the target sequence on the 5’ side thereof, and a length from the 5’ end of the complementary sequence to the single base substitution site is 0 to 10 bases.

[0039] While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.

Claims

1. A method for detecting a single base substitution in a group of short-chain RNAs having sequence diversity on the 3’ side, the method comprising: bringing into contact, a solution containing the group of short-chain target RNAs, a guide RNA containing a sequence that is complementary to the target sequence including a single nucleotide substitution site, a sequence-specific endonuclease that recognizes the guide RNA-target nucleic acid complex, and a reporter substrate that generates a signal upon the trans-cleavage activity of sequence-specific endonuclease; measuring a signal derived from the reporter substrate; and determining presence or absence of a single-base substitution in the group of short-chain RNAs in the target nucleic acid based on an intensity of the signal, wherein the guide RNA includes a single-stranded structure and an intramolecular hairpin structure on the 5’ side of the single-stranded structure, the single-stranded structure includes a complementary sequence to the target sequence on the 5’ side, and a length from the 5’ end of the complementary sequence to the single base substitution site is 0 to 10 bases.

2. The method of claim 1, wherein the intramolecular hairpin structure is a sequence-specific endonuclease recognition site.

3. The method of claim 1, wherein the short-chain RNAs are microRNAs.

4. The method of claim 1, wherein the sequence-specific endonucleases have collateral activity.

5. The method of claim 1, wherein the reporter substrate consists of nucleic acid, a signal substrate that generates a signal, and a quencher substrate that masks a signal, and both substrates are attached to oligonucleotide.

6. The method of claim 5, wherein a length of the oligonucleotide is 50 bases or less.

7. The method of claim 5, wherein the signal substance is a fluorescence and the masking substance is a quencher.

8. The method of claim 1, wherein measurement of the signal generated from the reporter substrate is performed by a means selected from the group consisting of a real-time PCR system, a microplate reader, a digital PCR system, a fluorescence microscope, and a fluorescence detection device.

9. The method of claim 1, wherein the determining of the presence or absence of a single base substitution in the target nucleic acid is performed by determining the presence or absence of a difference between the detection signal obtained by performing similarly for a standard nucleic acid and the signal obtained from the target nucleic acid.

10. An assay kit for use in a method recited in claim 1, comprising a guide RNA including a complementary sequence to a target sequence including a single base substitution site, sequence-specific endonucleases corresponding to the guide RNA, and a reporter that becomes detectable by activation of the sequence-specific endonuclease, wherein the guide RNA includes a single-stranded structure, and an intramolecular hairpin structure at the 5’ side of the single stranded structure, and the single-stranded structure includes a complementary sequence of the target sequence, and a length from the 5’ end of the complementary sequence to the single base substitution site is 0 to 10 bases.

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