Probe and use method thereof

Abstract

To provide a probe for detecting a target nucleic acid and a method for using the same.SOLUTION: A probe comprises an oligonucleotide region that defines a capture sequence for specific hybridization with a target RNA. The oligonucleotide region comprises: a first unit derived from a nucleoside represented by the following formula (1) and / or another specific structure; and at least one second unit comprising a photo-crosslinking reactive group for photo-crosslinking pyrimidine bases within the target RNA.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present specification relates to a probe for detecting a target nucleic acid and a method for using the same. [Background technology]

[0002] In the medical field, it is important to identify the presence or absence and type of pathogen of an infectious disease, and to evaluate the presence or absence and localization of gene products in gene-related diseases.

[0003] For example, PCR is recommended for infectious disease testing because of its specificity and detection sensitivity. PCR is theoretically capable of detecting a single virus, and is expected to identify pathogens with high sensitivity.

[0004] In situ hybridization (ISH) is a technique that uses probes to visualize and detect the localization of target nucleic acids in cells, tissue sections, and even whole tissues. For example, ISH in pathological specimens is useful in the diagnosis of infectious diseases and tumors, which can be diagnosed by detecting target nucleic acids derived from target genes. ISH has become a useful diagnostic tool for diseases in which gene mutations and amplifications are observed.

[0005] Known probes for detecting target nucleic acids using these technologies include natural probes using natural nucleotides such as DNA, artificial nucleic acid probes using non-natural backbones, and photocrosslinking artificial nucleic acid probes using photocrosslinking artificial nucleic acids (Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2022-39526 Summary of the Invention [Problem to be solved by the invention]

[0007] However, in the case of PCR, there are cases where false negatives are generated due to low yields in the extraction process from biological samples and inhibition of reverse transcription reaction and PCR reaction by impurities, resulting in reduced sensitivity.

[0008] In addition, in the case of ISH, in order to improve detection sensitivity, it is important to ensure the specificity of hybridization to the target nucleic acid, but there are limitations to the improvement of detection sensitivity and detection accuracy by adjusting the Tm and stringency of the probe.

[0009] Furthermore, each type of probe has its own inherent problems. Natural probes can be easily synthesized, but have low nuclease resistance and the hybridization product has low washing durability because hybridization with the target nucleic acid depends only on hydrogen bonds, making it difficult to improve detection sensitivity and detection accuracy. Although artificial nucleic acid probes have excellent nuclease resistance, they have low washing durability because hybridization depends only on hydrogen bonds, so there is still a limit to improving detection sensitivity. It has been found that photocrosslinking-type artificial nucleic acid probes have excellent specificity and washing durability because they are covalently bonded to the target nucleic acid by photocrosslinking, but have a problem in that detection sensitivity is easily reduced because of low nuclease resistance.

[0010] For this reason, at present, there is still a demand for probes that detect target RNA by hybridization with improved detection sensitivity and detection accuracy.

[0011] The present specification provides probes that can contribute to the sensitivity and accuracy of detecting target RNA, and methods of using the same. [Means for solving the problem]

[0012] The present inventors have focused on the possibility that a probe having both nuclease resistance and washing durability can detect target RNA with high sensitivity and high accuracy. Therefore, the inventors have investigated combinations of photocrosslinkable artificial nucleosides, which have excellent sequence specificity and washing durability, with various artificial nucleosides that are considered to have excellent nuclease resistance. As a result, the inventors have found that a combination of a photocrosslinkable artificial nucleoside unit and an artificial nucleoside unit having a specific acyclic skeleton is effective for constructing a specific and highly sensitive probe, and as a result, the target RNA can be detected with high sensitivity and high accuracy. According to the present specification, the following means are provided.

[0013] [1] comprising an oligonucleotide region defining a capture sequence that specifically hybridizes to a target RNA; The oligonucleotide region is a probe having a first unit derived from a nucleoside represented by the following formula (1) and / or (2) and at least one second unit containing a photocrosslinking reactive group that photocrosslinks pyrimidine bases in the target RNA. [ka] [ka] (In formula (1) and formula (2), each B independently represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group.) [2] The probe according to [1], wherein the second unit is a unit derived from one or two types of nucleosides selected from the group consisting of units represented by the following formulas (3) and (4): [ka] [ka] [3] The probe described in either [1] or [2], wherein the oligonucleotide region has the first unit as all remaining units other than the second unit. [4] The probe according to any one of [1] to [3], comprising a label element for detecting the target RNA at the 3' end and / or the 5' end of the oligonucleotide region. [5] A method for detecting a target RNA, comprising: (a) hybridizing a probe comprising an oligonucleotide region defining a capture sequence that specifically hybridizes to the target RNA with the target RNA, the oligonucleotide region having a first unit represented by the following formula (1) and / or (2) and at least one second unit containing a photocrosslinking reactive group that photocrosslinks pyrimidine bases in the target RNA; (b) forming a crosslinked structure by light irradiation in a hybridization product in which the target RNA and the probe are hybridized; (c) removing the hybridization products other than the hybridization products having the cross-linked structure after the step (b); A method of providing. [Brief description of the drawings]

[0014] [Figure 1] FIG. 2 shows the results of recovery of target RNA in Example 1 (with or without photocrosslinking). [Diagram 2] FIG. 1 shows the recovery results of target RNA (difference in backbone) in Example 1. [Diagram 3] FIG. 1 shows the detection results of target RNA by CISH in Example 2 (with or without photocrosslinking and differences in backbones). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The disclosure of the present specification relates to a probe for detecting a target RNA and a method for using the same, etc. The probe disclosed in the present specification (hereinafter also simply referred to as the present probe) has a first unit, and therefore has excellent nuclease resistance and hybridizes with high affinity to the target RNA, making it possible to efficiently and / or more efficiently obtain a genuine hybridization product without mismatches.

[0016] In addition, since the probe has a second unit, a true crosslinked hybridization product can be formed by photocrosslinking with light irradiation in a true hybridization product with a target RNA. On the other hand, since the probe has a second unit, a crosslinked structure is unlikely to be formed in a hybridization product with a mismatch. As a result, a crosslinked structure is formed specifically only in the true hybridization product, and a true crosslinked hybridization product is generated. The true crosslinked hybridization product has excellent washing durability, which allows it to maintain a hybridized state even under strong washing conditions.

[0017] As described above, the present probe has excellent nuclease resistance and RNA affinity, and by forming a crosslinked structure, a true crosslinked hybridization product can be obtained that has excellent specificity for the target sequence and washing durability. Therefore, the present probe can suppress false negatives, detect true positives with high sensitivity, and detect true negatives with high specificity. In other words, the target RNA can be detected with high sensitivity and accuracy.

[0018] This probe is effective when using test samples that may contain biological contaminants such as nucleic acids other than the target RNA, ribonucleases, etc. In other words, by using this probe in the detection of target RNA in various biological test solutions (PCR method, LAMP method) and in the detection of the localization of target nucleic acid in cells and tissues (ISH method), it is possible to accurately capture the target RNA and remove contaminants, thereby enabling the detection of target RNA with high sensitivity and accuracy.

[0019] Furthermore, a method for detecting a target RNA using the present probe and a target RNA detection kit including the present probe are also useful in the same manner as described above.

[0020] The present probe and its usage will be described in detail below.

[0021] (probe) The probe comprises an oligonucleotide region that defines a capture sequence that specifically hybridizes to a target RNA.

[0022] In the present specification, the term "RNA" includes not only natural nucleic acids, which are RNAs retained by living organisms, but also modified RNAs, which include modifications to these components, so long as they have the ability to hybridize and crosslink with the present probe. The RNA is not particularly limited as long as it is naturally derived RNA that can be retained by living organisms or RNA introduced from the outside, and includes mRNA, tRNA, rRNA, ncRNA such as siRNA and miRNA, ribozymes, and double-stranded RNA.

[0023] The target RNA is an RNA to be detected by hybridizing with the present probe. The type of RNA is not particularly limited, and may be RNA derived from a living organism or a non-living organism.

[0024] The target RNA is not particularly limited, and examples thereof include RNA derived from animals including mammals such as humans, RNA derived from plants, microorganisms and viruses, RNA generated from various DNAs by reverse transcription reaction, RNA added as a label, etc. More specifically, such RNA includes reverse transcribed RNA from RNA or DNA associated with the source of infectious diseases in animals or plants, reverse transcribed RNA from RNA or DNA associated with gene-related diseases including tumors in animals, etc.

[0025] The target RNA is detected as various biological samples or extracts from biological samples. Biological samples are not particularly limited, but include so-called medical and sanitary specimen samples, such as various biological liquid samples such as saliva, blood, and urine, which are artificially collected or naturally isolated from humans and animals such as livestock, cell samples, and tissue samples, as well as processed samples in which nucleic acids have been extracted, amplified, reverse transcribed, etc. from these samples. Biological samples also include biological samples of various parts or forms artificially collected or naturally isolated from plants or other organisms, or processed samples. Biological samples also include samples for non-medical purposes that may contain RNA.

[0026] (Oligonucleotide Region) The probe has an oligonucleotide region at least in part. The oligonucleotide region defines a capture sequence that specifically hybridizes to the target RNA. The capture sequence is a sequence that specifically hybridizes to at least a part of the target sequence of the target RNA. The target sequence is not particularly limited, but may be a unique sequence that characterizes the target RNA to be detected by the probe. The target sequence may also be a captured sequence for capturing a target RNA that is not the unique sequence but contains the unique sequence, in order to capture the target RNA that contains the unique sequence. For example, the target sequence is at least a part of the sequence of RNA and other sequence features associated with a specific gene, RNA mutations and other sequence features associated with a gene that characterizes an individual, RNA, mutations in the RNA, other sequence features associated with a gene that characterizes a disease or pathogen in an animal or plant, and the like.

[0027] The capture sequence defined by the oligonucleotide region has a degree of complementarity that allows it to specifically hybridize to the target sequence. The identity of the capture sequence to the complementary sequence of the target sequence is not particularly limited, but is, for example, 80% or more, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 96% or more, for example, 98% or more, or for example, 99% or more. The identity of the capture sequence excluding the photocrosslinking reactive group to the complementary sequence is, for example, 90% or more, for example, 95% or more, for example, 98% or more, for example, 99% or more, or for example, 100%.

[0028] In this specification, the identity or similarity of a base sequence or an amino acid sequence is a relationship between two or more proteins or two or more polynucleotides, as determined by comparing the sequences, as known in the art. In the art, "identity" means the degree of sequence invariance between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or, in some cases, by alignment between a stretch of such sequences. Also, similarity means the degree of correlation between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or, in some cases, by alignment between a stretch of partial sequences. More specifically, it is determined by sequence identity and conservation (substitutions that maintain the physicochemical properties of specific amino acids in a sequence or in a sequence). Note that similarity is referred to as Similarity in the results of sequence homology search by BLAST, which will be described later. Methods for determining identity and similarity are preferably designed to align the sequences to the maximum extent. Methods for determining identity and similarity are provided as publicly available programs. For example, the alignment can be determined using the BLAST (Basic Local Alignment Search Tool) program by AltschTl et al. (e.g., AltschTl SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., NTcleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use the default values.

[0029] Those skilled in the art can appropriately set the length and sequence of the capture sequence, taking into consideration the temperature during the formation of the hybridization product. The length of the capture sequence is not particularly limited, but if the capture sequence is too long, the Tm value becomes too high, making washing difficult, and if it is too short, the specificity for the target RNA decreases. For these reasons, the capture sequence is, for example, 10mer or more and 50mer or less, or, for example, 15mer or more and 50mer or less, or, for example, 20mer or more and 35mer or less.

[0030] (First unit) The probe comprises, in the oligonucleotide region, a first unit derived from one or more nucleosides selected from the group consisting of, for example, formula (1) and formula (2). The first unit contributes to the affinity of the probe for RNA (hybridization efficiency) and nuclease resistance. These nucleosides are linked by phosphodiester bonds.

[0031] [ka]

[0032] [ka]

[0033] In formula (1) and formula (2), B can be independently selected from a purine-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purine-9-yl group, and a substituted 2-oxo-pyrimidin-1-yl group. Such bases include known natural bases as well as artificial bases.

[0034] That is, B may be a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group, as well as 2,6-dichloropurin-9-yl or 2-oxo-pyrimidin-1-yl, and may further be 2-oxo-4-methoxy-pyrimidin-1-yl, 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl, or 2,6-dimethoxypurin-9-yl.

[0035] Further examples include 2-oxo-4-amino-pyrimidin-1-yl with a protected amino group, 2-amino-6-bromopurin-9-yl with a protected amino group, 2-amino-6-hydroxypurin-9-yl with a protected amino group, 2-amino-6-hydroxypurin-9-yl with a protected amino group and / or hydroxyl group, 2-amino-6-chloropurin-9-yl with a protected amino group, 6-aminopurin-9-yl with a protected amino group, and 4-amino-5-methyl-2-oxo-pyrimidin-1-yl with a protected amino group. The protecting groups for the hydroxyl group and amino group include various known protecting groups.

[0036] Further examples include 6-aminopurin-9-yl (adenine), 2-amino-6-hydroxypurin-9-yl (guanidine), 2-oxo-4-amino-pyrimidin-1-yl (cytosine), 2-oxo-4-hydroxy-pyrimidin-1-yl (uracil), or 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (thymine).

[0037] Further examples include 4-amino-5-methyl-2-oxo-pyrimidin-1-yl (methylcytosine), 2,6-diaminopurin-9-yl, 6-amino-2-fluoropurin-9-yl, 6-mercaptopurin-9-yl, 4-amino-2-oxo-5-chloro-pyrimidin-1-yl, or 2-oxo-4-mercapto-pyrimidin-1-yl.

[0038] Further examples include 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, and 2-amino-6-bromopurin-9-yl.

[0039] The substituent in each of the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group is any one of a hydroxyl group, a protected hydroxyl group, a lower alkoxy group, a mercapto group, a protected mercapto group, a lower alkylthio group, an amino group, a protected amino group, an amino group substituted with a lower alkyl group, a lower alkyl group, a lower alkoxymethyl group, or a halogen atom, or any combination of two or more thereof. The lower alkyl group is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, the lower alkoxy group is a lower alkyl ether group, and the lower alkylthio group is a lower alkyl thio group. The protecting group for a hydroxyl group is not particularly limited, but representative examples include, for example, an aliphatic acyl group, an aromatic acyl group, a lower alkoxymethyl group, an oxycarbonyl group which may have an appropriate substituent, a tetrahydropyranyl group which may have an appropriate substituent, a tetrathiopyranyl group which may have an appropriate substituent, a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups (however, the substituent in the substituted aryl group mentioned above means a lower alkyl, a lower alkoxy, a halogen atom, or a cyano group), or a silyl group. The protecting group for an amino group is not particularly limited, but examples include a hydroxyl group, In addition to those exemplified as the protecting group, examples thereof include a benzyl group, a methylbenzyl group, a chlorobenzyl group, a dichlorobenzyl group, a fluorobenzyl group, a trifluoromethylbenzyl group, a nitrobenzyl group, a methoxyphenyl group, a methoxymethyl (MOM) group, an N-methylaminobenzyl group, an N,N-dimethylaminobenzyl group, a phenacyl group, an acetyl group, a trifluoroacetyl group, a pivaloyl group, a benzoyl group, a phthalimido group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, a benzyloxycarbonyl group, a t-butoxycarbonyl (Boc) group, a 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc) group, a 9-fluorenylmethoxycarbonyl group, a benzyloxymethyl (BOM) group, and a 2-(trimethylsilyl)ethoxymethyl (SEM) group.More preferred are benzyl, methoxyphenyl, acetyl, trifluoroacetyl (TFA), pivaloyl, benzoyl, t-butoxycarbonyl (Boc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM), and 2-(trimethylsilyl)ethoxymethyl (SEM), and particularly preferred are benzyl, methoxyphenyl, acetyl, benzoyl, and benzyloxymethyl.

[0040] Preferred examples of the substituted purin-9-yl group include, for example, 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, and 6-mercaptopurin-9-yl. If the above-mentioned substituents contain an amino group or a hydroxyl group, preferred examples include substituents in which the amino group and / or the hydroxyl group are protected.

[0041] Substituted 2-oxo-pyrimidin-1-yls include, for example, 2-oxo-4-amino-pyrimidin-1-yl, 1H-(1,2,4-triazol-1-yl)-pyrimidin-1-yl, 4-1H-1,4-amino-2-oxo-5-chloro-pyrimidin-1-yl, 2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidin-1-yl, 2-oxo-4-hydroxy-pyrimidin-1-yl, 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl, or 4-amino-5-methyl-2-oxo-pyrimidin-1-yl. Further preferred examples include 2-oxo-4-methoxy-pyrimidin-1-yl and 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl.

[0042] B in formula (1) and formula (2) is selected from, for example, adenine, guanine, cytosine, and thymine.

[0043] The first unit represented by formula (1) is a unit derived from L-threoninol, also called L-αTNA, and is disclosed in International Publication No. 2010 / 001902 and JP 2012-170373 A. The first unit represented by formula (2) is a unit derived from serinol, also called SNA, and is disclosed in the same publication as the unit represented by formula (1). Those skilled in the art can appropriately synthesize oligonucleotides containing these units.

[0044] The phosphate group in the first unit represented by formula (1) and formula (2) may be appropriately modified, for example, by thiolation.

[0045] The first unit is not particularly limited and may be any of the units of formula (1) and formula (2), but in consideration of the detection sensitivity of the target RNA, it may be preferable to use the unit represented by formula (1). The oligonucleotide region may have other nucleoside units other than the first unit and the second unit. Such other units are, for example, natural ribonucleotides.

[0046] The number of first units in the oligonucleotide region is not particularly limited, but taking into consideration affinity for the target RNA and nuclease resistance, it is, for example, 70% or more of the number of nucleoside units constituting the entire capture sequence, such as 70% or more, such as 75% or more, such as 80% or more, such as 90% or more, or such as 95% or more, and all other than the second units described below are first units.

[0047] Although there is no particular limitation, in consideration of exonucleases that degrade RNA, it is preferable that the probe has one or more first units at one or both of the two ends.

[0048] (Second Unit) The oligonucleotide region has at least one second unit in the capture sequence, which contains a photocrosslinking reactive group that photocrosslinks a predetermined base in the target sequence. The photocrosslinking reactive group is a group that can form a crosslinked structure by forming a covalent bond with a predetermined base of a hybridized complementary strand upon irradiation with a predetermined light. The second unit contributes to the target sequence specificity and washing durability of the present probe.

[0049] The photocrosslinking reactive group possessed by the second unit is not particularly limited, and examples thereof include 3-cyanovinylcarbazole group and its derivatives (WO2009 / 066447 (EP2216338, TS 2010-274000A), Yoshinaga Yoshimoto et al., Organic Letters 10:3227-3230(2008)), p-carbamoylvinylphenol group (Takehiro Ami et al., Organic & BiomolecTlar Chemistry 5:2583-2586(2007)), 4,5',8-trimethylpsoralen group (Akio Kobori et al., Chemistry Letters 38:272-273(2009)), and N 3 -methyl-5-cyanovinyluracil group (Kenzo FTjimoto et al., Chemical CommTnications: 3177-3179 (2005)), pyranocarbazole (JP Patent Publication 2019-26569 and WO2019 / 022158). All of these form a crosslinked structure by covalent bond between the pyrimidine ring of the pyrimidine base at the base position (+1 position: a position shifted by one base to the 3' side from the paired base) adjacent to the 3' side of the base (paired base) in the target sequence that pairs with the photocrosslinking reactive group by light irradiation. Among them, 3-cyanovinylcarbazole may be preferable from the viewpoint of reaction rate, and pyranocarbazole may be preferable from the viewpoint of being able to form a crosslinked structure by visible light irradiation. The second unit generally has one photocrosslinking reactive group.

[0050] When a 3-cyanovinylcarbazole group or a derivative thereof is used as the photocrosslinking reactive group, a covalent crosslinked structure is formed between the molecule and the pyrimidine ring of the pyrimidine base at the base position adjacent to the 3' side of the base in the target sequence that pairs with the molecule (+1 position: a position shifted by one base toward the 3' side from the paired base).

[0051] The second unit may have a backbone structure carrying a photocrosslinking reactive group, such as a deoxyribose-derived backbone, a ribose-derived backbone, a D-threoninol-derived backbone, or the serinol backbone of the first unit represented by formula (2). These nucleosides are linked by phosphodiester bonds.

[0052] When the oligonucleotide region has a unit represented by formula (1) as the first unit, the backbone structure of the second unit may preferably be a D-threoninol-derived backbone.

[0053] Nucleosides derived from the second unit modified with 3-cyanovinylcarbazole group or its derivative include, for example, CNV-K (deoxyribose modified) represented by the following formula (3) and CNV-D (D-threoninol modified) represented by the same formula (4) (both manufactured by NICCA Chemical Co., Ltd.). These nucleosides have a faster reaction rate and superior crosslinking efficiency compared to other photocrosslinking reactive groups. The phosphate group in the first unit represented by formula (3) and formula (4) may be appropriately modified, for example, by thiolation.

[0054] [ka]

[0055] [ka]

[0056] The pyrimidine base in the target sequence that forms a crosslinked structure with the photocrosslinking reactive group is cytosine (C) or uracil (TT). These bases may be modified as long as they can be photocrosslinked with the photocrosslinking reactive group. Examples of such modified bases include, but are not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, etc.

[0057] The second unit having a photocrosslinking reactive group is provided in the oligonucleotide region (capture sequence) so as to replace any nucleoside unit that pairs with a base at, for example, the base position (-1 position) adjacent to the 5' side of a pyrimidine base in the target sequence. In other words, the photocrosslinking reactive group is provided so as to replace any nucleoside unit that has a base at the base position (+1 position) adjacent to the 3' side of a purine base (adenine (A) or guanine (G)) in the capture sequence defined by the oligonucleotide region.

[0058] The oligonucleotide region has any number of second units, 1 or more, in the capture sequence. The number of second units in the oligonucleotide region is not particularly limited. Although it varies depending on the length of the capture sequence, when the capture sequence is about 10 to 50 mer, for example, 1 to 5, for example, 1 to 3, for example, 1 to 2, for example, 1. Two or more consecutive second units can be included. When the oligonucleotide region has two or more consecutive second units, an additional second unit can be included. It may be preferable to include an additional covalent bond group at a distance of at least 10 bases from the consecutive covalent bond group.

[0059] The number of second units in the oligonucleotide region can be, for example, 20% or less of the number of nucleoside units constituting the entire capture sequence. If it exceeds 20%, the specificity of hybridization of the capture sequence to the target sequence may decrease. It can also be, for example, 15% or less, for example, 10% or less, or for example, 5% or less.

[0060] The position of the second unit in the oligonucleotide region is not particularly limited, but may be, for example, the 5'-end and / or 3'-end of the capture sequence, and may be a position that suppresses hybridization of the probe to the target sequence as little as possible. Typically, it is the 5'-end region or the 3'-end region. Here, the 5'-end region is, for example, within 20%, for example, within 10%, or for example, within 5% of the total number of bases of the capture sequence from the terminal of the 5'-end, and the 3'-end region is, for example, within 20%, for example, within 10%, or for example, within 5% of the total number of bases of the capture sequence from the terminal of the 3'-end. In addition, the photocrosslinking reactive group may be present, for example, at the 5'-end of the oligonucleotide region, at the end and at the 3'-end, and at least one or more may be present at the 5'-end of the 3'-end.

[0061] In addition, the position of the second unit may be, for example, in the center of the oligonucleotide region, that is, within a range of more than 25% of the base length of the oligonucleotide region from the 5'-end and 3'-end of the oligonucleotide region, if the oligonucleotide region is 10-mer or about 50-mer. If the oligonucleotide region is a certain length or longer, even if it is in the center of the oligonucleotide region, hybridization and photocrosslinking are possible without inhibiting specific hybridization with the target sequence.

[0062] A person skilled in the art would recognize that the present probe having a first unit and a second unit is described in the above-mentioned various documents as well as in JP 2016-145229 A, JP 2017-210448 A, JP 2019-26569 A, etc., and can easily be synthesized based on these publications and the common technical knowledge and well-known techniques regarding synthesis techniques for DNA, etc. at the time of filing this application.

[0063] In addition to the oligonucleotide region that defines the capture sequence, the present probe can be provided with a labeling element for easily detecting the true cross-linked hybridization product that has been truly hybridized to form a cross-linked structure. As such a labeling element, one or more of various known compounds can be combined. For example, fluorescent substances, coloring substances, etc. that can be detected alone, as well as enzymes that develop color upon the supply of a substrate, can be provided as labeling elements at the 3' end and / or 5' end of the oligonucleotide region. In addition, with the intention of detecting using antigen-antibody interactions, biotin-avidin interactions, hydrogen bonds between base pairs, etc., an element on one side of the interaction (antigen or antibody, biotin, etc., DNA chain or RNA chain, etc.) can be provided as a first labeling element at the 3' end and / or 5' end of the oligonucleotide region. In this case, to detect the true cross-linked hybridization product, a complex obtained by interacting with the second labeling element, which is the element on the other side of the interaction, can be detected, etc. Such labeling and detection methods are well known to those skilled in the art.

[0064] In addition to the nucleotide region, the probe may also include a recovery element for easily separating and recovering the true cross-linked hybridization product. Such a recovery element may be, for example, one of the components of an antigen-antibody reaction. Such recovery elements and recovery methods are well known to those skilled in the art.

[0065] As described above, the present probe has an oligonucleotide region that defines the capture sequence of the first unit and the second unit, and therefore can detect the target RNA with high sensitivity and accuracy. Even if the target RNA is in a test sample that contains biological impurities, it can be detected with high sensitivity and accuracy.

[0066] (Method for detecting target RNA) The method for detecting a target RNA disclosed in the present specification can include a step (a) of hybridizing the target RNA with the present probe, a step (b) of forming a crosslinked structure by light irradiation in the hybridization product in which the target RNA and the present probe are hybridized, and a step (c) of removing hybridization products other than the hybridization product having the crosslinked structure after the step (b). According to this method, the present probe can be used to specifically and efficiently hybridize with the target RNA. Furthermore, a crosslinked structure is specifically imparted only to the genuine hybridization product that is genuinely hybridized without mismatch to obtain a genuine crosslinked hybridization product. This genuine crosslinked hybridization product has excellent washing durability and can continue to maintain the hybridized state. As a result of the above, the genuine crosslinked hybridization product can be detected with high sensitivity and high accuracy even in the presence of various impurities.

[0067] (Step (a): Hybridization Step) Step (a) is a step of hybridizing the target RNA with the present probe. The stringency of the conditions in step (a), such as temperature, pH, salt concentration, and time, can be appropriately set depending on the Tm of the present probe, the type of test sample, and the like.

[0068] In this method, the genuine hybridization product is photocrosslinked in the latter step (b), and furthermore, the genuine crosslinked hybridization product can be highly selectively maintained by washing in the latter step (c). Therefore, the step (a) does not necessarily need to be performed with high stringency. Therefore, the hybridization step can be performed, for example, at room temperature as described later, or at a temperature between 25°C and 40°C, or, for example, between 30°C and 40°C.

[0069] In carrying out step (a), a pretreatment may be required to enable hybridization of the target RNA with the present probe, depending on the test sample and detection method used. Pretreatments according to the test sample, detection method, etc. are well known to those skilled in the art, and those skilled in the art can determine the pretreatment method and hybridization conditions based on well-known techniques.

[0070] In step (a), it is necessary to avoid photocrosslinking of the photocrosslinking reactive group of the present probe, and therefore step (a) is carried out in a light-shielded state as necessary depending on the type of the photocrosslinking reactive group.

[0071] The embodiment of step (a) varies depending on the method for detecting nucleic acids to which the present method is applied, such as a detection method using a solid phase such as an array or strip to which the present probe is fixed, or a detection method using CISH or FISH that performs in situ T hybridization.

[0072] In situ T hybridization can be performed on collected cells, cultured cells, or biological tissue sections. Examples of applications of in situ T hybridization include cells collected from tumor patients, cultured cells thereof, or tissue sections thereof, and cells or cultured cells thereof that retain infection by viruses, etc.

[0073] Step (a) may further include hybridization in a liquid phase, hybridization by chromatography using a column or strip on which the probe is immobilized, etc. All of these methods are well known at the time of filing of this application. Those skilled in the art can carry out an appropriate hybridization step based on well-known techniques depending on the type of detection method to be applied.

[0074] (Step (b): Light irradiation step) Step (b) is a step of irradiating the test sample after step (a) with light to crosslink the photocrosslinking reactive group in the true hybridization product with the target RNA to form a true crosslinked hybridization product having a crosslinked structure. In the previous step (a), in addition to the true hybridization product, a nonspecific mishybridization product containing a mismatch but hybridized may also be formed.

[0075] In step (b), the capture sequence containing the second unit hybridizes appropriately to the target sequence to form a crosslinked structure with high specificity between the pyrimidine base at the +1 position of the paired base of the target sequence. If there is a mismatch, it is difficult for the capture sequence and the pyrimidine base to be crosslinked in the target sequence to form a positional relationship that allows crosslinking, and it is difficult to form a crosslinked structure even if appropriate light irradiation is performed. Therefore, a crosslinked structure is formed specifically only in the genuine hybridization product.

[0076] The light irradiation conditions for forming a crosslinked structure are appropriately determined depending on the type of photocrosslinking reactive group used, etc. Although not particularly limited, light irradiation can be performed at a maximum absorption wavelength in the range of, for example, 340 to 550 nm, or, for example, 350 to 450 nm.

[0077] The intensity of the light irradiation is appropriately determined depending on the type of photocrosslinking reactive group used, etc. Although not particularly limited, for example, the intensity is 15 to 20,000 mW / cm 2 For example, 600 to 15,000 mW / cm 2 The irradiation time is not particularly limited, but is, for example, 1 to 600 seconds, or, for example, 10 to 300 seconds, taking into consideration the volatilization of the test sample and tissue damage due to heat generation caused by too long an irradiation time. The integrated light amount of the light irradiation is not particularly limited, but may be sufficient to ensure the crosslinking rate and maintain the recovery efficiency, for example, 15 mJ / cm. 2 ~12,000J / cm 2 The range can be appropriately set.

[0078] The temperature in step (b) is not particularly limited, but taking into consideration heat generation and tissue damage due to light irradiation, it is, for example, 0°C to 60°C, or for example, 0 to 40°C, or for example, 0 to 30°C, or for example, 0 to 20°C, or for example, 0 to 10°C, or for example, 0 to 5°C.

[0079] In addition, since the step (b) utilizes a photoreaction, there are no particular restrictions on the pH, salt concentration, etc., and any pH and salt concentration conditions that allow the hybridization product and other biomaterials to exist stably can be adopted.

[0080] (Step (c): Washing step) Step (c) is a step of maintaining the true cross-linked hybridization products and removing other than the true cross-linked hybridization products. More specifically, the test sample is washed under such high-strength conditions that the hybridization products other than the true cross-linked hybridization products are melted or denatured. This makes it possible to remove non-specific hybridization products, uncross-linked true hybridization products, and the present probe derived from these.

[0081] Step (c) allows for highly specific and reliable retention of genuine cross-linked hybridization products, thus suppressing false negatives.

[0082] Furthermore, according to step (c), in addition to removing nucleic acids (genomic DNA, RNA, etc.) that are contaminants of the test sample, proteins may also be denatured. In this case, the influence of these contaminants on the test results can be suppressed or avoided. As a result, false positive detection results can be suppressed, thereby improving the specificity of the test.

[0083] According to step (c), as described above, false negative and false positive detection results are also suppressed, and as a result, the accuracy with which true positives and true negatives can be detected accurately can be improved.

[0084] To perform high-intensity washing in step (c), a known denaturant known to dissociate hybridization of DNA or RNA can be used, but is not particularly limited thereto. Examples of such denaturants include, but are not particularly limited to, aldehydes such as formamide and dimethylformamide, pyridines, piperidines, dimethylsulfoxide, tetrahydrofuran, and acetonitrile. For example, such a solvent can be appropriately dissolved in water and used as a denaturant solution. When formamide is used, but is not particularly limited thereto, a denaturant solution dissolved in water at, for example, 70% by volume or 80% by volume can be used. Step (c) using such a denaturant is useful, for example, to remove non-specific hybridization products, and is also useful, for example, in the case of ISH, CISH, and FISH using tissue slices.

[0085] Furthermore, a chaotropic denaturant can be used as the denaturant. These may be suitable for denaturing, for example, proteins. Examples of such chaotropic denaturants include urea, thiourea, sodium iodide, potassium iodide, guanidine hydrochloride, and guanidine thiocyanate. These denaturants can be dissolved in a suitable solvent such as water and used as a denaturant solution. Step (c) using such a denaturant is particularly useful when the test sample is derived from a body fluid.

[0086] Moreover, a surfactant can be used as the denaturant. These may be suitable for denaturing, for example, proteins. Although not particularly limited, various surfactants such as SDS, Tween-20, Tween-80, Triton X-100, Triton X-114, NP-40, and Octyl GlTcoside can be used. These surfactants can be dissolved in a suitable solvent such as water and used as a denaturant solution. Such denaturants are particularly useful when the test sample is derived from a body fluid.

[0087] The modifying agent may be used alone or in appropriate combination of two or more kinds, as required.

[0088] The temperature conditions in step (c) are not particularly limited, and may be, for example, 20°C or higher, for example, 30°C or higher, for example, 35°C or higher, for example, 40°C or higher, or for example, 50°C or higher. On the other hand, the upper limit may be, for example, 70°C or lower, or for example, 60°C or lower. The temperature conditions may also be, for example, a temperature higher than room temperature, or may be 40°C or higher and 60°C or lower. For example, the temperature conditions can be imparted to step (c) by adjusting the denaturant solution to the above temperature.

[0089] After step (c), a true cross-linked hybridization product can be obtained from which non-specific hybridization products and other contaminants have been removed. Various known methods can be used to detect a true cross-linked hybridization product.

[0090] As described above, the target RNA can be detected by a method using a labeling element previously added to the oligonucleotide region of the present probe. In addition, various PCR (reverse transcription PCR, real-time PCR, etc.) can be performed on a region other than the target sequence of the captured target RNA, and the PCR product can be detected. Prior to such a PCR step, the hybridization product can also be recovered using a recovery element previously added to the oligonucleotide region and magnetic particles.

[0091] According to the method described above, it is possible to efficiently and specifically produce only genuine cross-linked hybridization products and to maintain only genuine cross-linked hybridization products, thereby making it possible to easily detect target RNA with high sensitivity and accuracy.

[0092] This method is particularly useful for detecting target RNA in biological samples containing many contaminants, and is also useful for detecting target RNA in in situ T hybridization of tissues, such as CISH or FISH, to accurately evaluate its localization.

[0093] This method can also be carried out as a method for recovering the target RNA as a genuine cross-linked hybridization product. In this case, it is preferable that the oligonucleotide region of this probe is provided with a recovery element for recovering the genuine cross-linked hybridization product. In addition, by carrying out a nucleic acid amplification reaction such as PCR on a region other than the target sequence of the target RNA contained in the recovered genuine cross-linked hybridization product, it is also possible to amplify the region. Identification or detection using such an amplified product is also possible.

[0094] (detection kits, etc.) The detection kit for target RNA disclosed in the present specification includes at least the present probe. The present probe can take various forms as described above. The present detection kit includes, in addition to the present probe, If necessary, reagents and the like used for detection can be appropriately provided. Furthermore, according to the present specification, the kit containing the present probe can also be used as a kit for recovering target RNA. EXAMPLES

[0095] In the following, examples are given as specific examples to more specifically explain the disclosure of the present specification. The following examples are intended to explain the disclosure of the present specification, but are not intended to limit the scope thereof. EXAMPLES

[0096] (Recovery of target RNA from a target RNA solution containing human saliva) In this example, the target RNA used was the SARS-CoV2 N gene (codon optimized) RNA (SEQ ID NO: 1) shown below.

[0097] [ka]

[0098] (Preparing the probe) Of the above sequence, the underlined 19 bases were used as the target sequence to synthesize a probe having a threoninol skeleton and containing a photocrosslinking reactive group (hereinafter referred to as an LTNA-type photocrosslinkable probe). X was a photocrosslinkable nucleoside shown below, and the other bases were nucleosides having an L-threoninol skeleton shown below. Biotin was added to the 3' end as a recovery element.

[0099] 5'-GGTAXTTCTACTACCTGGGC-3' (SEQ ID NO: 2)

[0100] [ka]

[0101] [ka]

[0102] The following primers were used to amplify the bold underlined portion of the target RNA captured by this probe by a nucleic acid amplification reaction.

[0103] Forward primer: 5'-GAACCAACTTGAGAGCAAGATG-3' (SEQ ID NO: 3) Reverse primer: 5'-AGCCTGGGTCACATTGTAAG-3' (SEQ ID NO: 4)

[0104] (Recovery experiment) The ORF region of a plasmid containing a sequence derived from the N2 region of the SARS-CoV2 nucleocapsid (SARS-CoV-2 NTcleocapsid ORF mammalian expression plasmid, Sino Biological) was transcribed and purified to prepare target RNA for probe evaluation. Saliva is often used as a specimen for PCR testing of COVID-19. Saliva samples contain not only proteins and contaminants of nucleic acids derived from other viruses, but also nucleases present in bodily fluids and nucleic acids derived from humans. Therefore, we decided to add a specified amount of human saliva to the target RNA and conduct RNA recovery experiments.

[0105] The probe was dissolved in TE buffer, and 0.6 μl of 1 μM target RNA was mixed with 0.6 μl of 1 μM probe solution, 3.0 μL of 20xSSPE buffer, and 5.4 μl of human saliva, and the total volume was adjusted to 10 μl with TE buffer to prepare a hybridization solution. The mixture was heated at 65°C for 5 minutes to dissociate any RNA that may have been entangled. The mixture was then incubated at 45°C for 90 minutes to form a genuine hybridization product between the target RNA and the probe.

[0106] The hybridization solution was irradiated with 366 nm, 1400 mW light on ice for 5 minutes, and the true hybridization product was photocrosslinked to form a true crosslinked hybridization product. Then, 10 μl of 1 mg / ml streptavidin-coated magnetic particle (Dynabeads M-270 Streptavidin, Thermo Fisher) solution was mixed with the hybridization solution and slowly rotated at room temperature for 15 minutes, and the true crosslinked hybridization product was immobilized on the magnetic particle surface by biotin-avidin binding. Then, the supernatant was removed by magnetic separation, and the magnetic particles were washed once with 10 μl of 6 M urea aqueous solution, once with 10 μl of 0.1 wt% Tween 20 aqueous solution, and finally twice with 20 μl of TE buffer to remove nonspecific adsorbents. Finally, the magnetic particles of Test Example 1 were suspended in 10 μl of TE buffer to recover them.

[0107] (Reverse transcription PCR and electrophoresis of recovered particles) Using the recovered magnetic particles of Test Example 1 as a template, a reverse transcription PCR solution was prepared using RT-PCR QTick MasterMix manufactured by Toyobo Co., Ltd. A forward primer and a reverse primer were used, and 1 μl of recovered particle solution was added to prepare a reaction solution of 50 μl. After the reaction solution was prepared, the reaction was carried out under the conditions of initial denaturation at 90° C. for 30 seconds and reverse transcription at 60° C. for 30 minutes, followed by PCR amplification for 25 cycles of initial denaturation at 94° C. for 1 minute, denaturation at 94° C. for 30 minutes, annealing at 58° C. for 30 seconds, and extension at 72° C. for 30 seconds, and finally extension at 72° C. for 7 minutes.

[0108] Agarose gel electrophoresis was performed to confirm whether the target DNA was amplified by PCR. A 3% agarose gel was set in an electrophoresis apparatus, filled with 1xTBE buffer, and then the PCR reaction solution was poured into the wells and electrophoresed at 100V for 45 minutes. After electrophoresis, the agarose gel was stained by immersing it in Viewa BlTe Stain KANTO manufactured by Kanto Chemical Co., Ltd. for 10 minutes, and decolorized by shaking with pure water for 10 minutes. The results of agarose gel electrophoresis of the reverse transcription PCR product of Test Example 1 are shown in FIG. 1. As a comparative example, electrophoresis was performed using the reverse transcription PCR product of Comparative Example 1 in the same manner as Test Example 1 except that light irradiation was not performed. In addition, electrophoresis was performed using the PCR product of the control example in the same manner as Test Example 1 except that a probe was not used. These results are also shown in FIG. 1.

[0109] (Reverse transcription-real-time PCR of recovered particles) The reverse transcription kit is Prime Script manufactured by Takara Bio Inc. TMUsing an RT reagent Kit (Perfect Real Time), a reverse transcription solution was prepared using the recovered particles as a template. Using a forward primer and a reverse primer, 1 μl of the recovered magnetic particle solution was added to prepare a reaction solution of 10 μl. Reverse transcription reaction was performed at 42 ° C for 15 minutes. For the real-time PCR kit, TB Green Premix Ex Taq II manufactured by Takara Bio Inc. was used, and the reverse transcription reaction solution was added as a template to prepare a PCR solution of 25 μl. Initial denaturation was performed at 95 ° C for 30 seconds, and amplification was performed with denaturation at 95 ° C for 1 minute, annealing at 58 ° C for 30 seconds, and 45 cycles of amplification. The Ct value was calculated from the obtained results according to the analysis software manufactured by Takara. The results are shown in Figure 1.

[0110] As a comparative example, RT real-time PCR was carried out in the same manner as in Test Example 1, except that light irradiation was not performed, and the Ct value was calculated. These results are also shown in FIG.

[0111] As shown in Figure 1, a strong band was confirmed in the magnetic particles (Test Example 1) having a crosslinked hybridization product between the LTNA-type photocrosslinkable probe and the target RNA bound to the surface. On the other hand, the target RNA band was hardly confirmed in the magnetic particles (Comparative Example 1) and the control (negative control) that were collected without light irradiation after hybridization.

[0112] From the above, it was confirmed that the use of a photocrosslinking probe having a threoninol structure makes it possible to recover target RNA even in biological samples.

[0113] In addition, the results of real-time PCR also showed that there was a difference of 9.19 in the Ct values ​​between Test Example 1 and Comparative Example 1, indicating that Test Example 1 recovered approximately 625 times the amount of RNA as Comparative Example 1.

[0114] From the above, it was found that by using a photocrosslinkable probe having a threoninol skeleton, it is possible to suppress or avoid the effects of hybridization inhibition by contaminating nucleic acids or proteins, even in biological samples, and to obtain large amounts of genuine crosslinked hybridization products.

[0115] Furthermore, a DNA-type photocrosslinkable probe was prepared in which the nucleotides other than X in the LTNA-type photocrosslinkable probe were replaced with deoxyribonucleotides. The previously prepared LTNA-type photocrosslinkable probe and DNA-type photocrosslinkable probe were subjected to the same procedure as in Test Example 1. The results are shown in FIG.

[0116] As shown in Figure 2, when the LTNA-type photocrosslinking probe was used, a sufficiently clear band was confirmed on the electrophoretic gel, whereas when the DNA-type photocrosslinking probe was used, the band was significantly fainter. This shows that the LTNA-type probe, which is nuclease-resistant and has high affinity with the target RNA, allowed for the recovery of a larger amount of target RNA than the DNA-type probe, which is decomposed by nuclease and has low affinity with RNA.

[0117] In addition, the Ct values ​​of the real-time RT-PCR were 13.61 for the DNA-type photocrosslinking probe and 23.20 for the LTNA-type photocrosslinking probe, confirming a difference of 9.59. In other words, when the LTNA-type photocrosslinking probe was used, the DNA concentration in the recovered magnetic particle solution was 2.0 times higher than when the DNA-type photocrosslinking probe was used. 9.59 From the above, it was found that the efficiency of recovering target RNA from biological samples is significantly improved by using LTNA-type photocrosslinkable probes with excellent RNA affinity and nuclease resistance. EXAMPLES

[0118] (Detection of target RNA by CISH against target RNA in tissue sections) In this example, CISH targeting miR-124a was performed using mouse brain frozen sections. From the sequence of miR-124a, the following sequences were used as target sequences to synthesize the various probes shown below. The 5' end was modified with digoxigenin (DIG) for color development with alkaline phosphatase.

[0119] (Preparing the probe) Target RNA sequence (miR-124a): 5'-UUAAGGCACGCGGUGAAUGCCA-3' (SEQ ID NO:5)

[0120] Test Example 2-1 (LTNA-type photocrosslinkable probe): 5'-GGXATTCACCGCGTGCCTTA-3' (SEQ ID NO: 6) X was a photocrosslinked nucleoside represented by the following formula, and the other bases were nucleosides having an L-threoninol skeleton as shown below.

[0121] [ka]

[0122] [ka]

[0123] Test Example 2-2 (SNA-type photocrosslinkable probe): 5'-GGXATTCACCGCGTGCCTTA-3' (SEQ ID NO:6) X was the same as above, and the other bases were nucleosides having a serinol skeleton as shown below.

[0124] [ka]

[0125] Comparative Example 3-1 (DNA-type non-photocrosslinkable probe): 5'-GGCATTCACCGCGTGCCTTA-3' (SEQ ID NO:7) All bases were derived from deoxyribonucleotide units.

[0126] Comparative Example 3-2 (DNA-type photocrosslinkable probe): 5'-GGXATTCACCGCGTGCCTTA-3' (SEQ ID NO:6) X was the same as above, and the other bases were units derived from deoxyribonucleotides.

[0127] Comparative Example 3-3 (RNA-type photocrosslinkable probe): 5'-GGXATTCACCGCGTGCCTTA-3' (SEQ ID NO:6) X was the same as above, and the other bases were units derived from ribonucleotides.

[0128] Comparative Example 3-4 (LNA-type photocrosslinkable probe): 5'-GGXATTCACCGCGTGCCTTA-3' (SEQ ID NO:6) X was the same as above, and the other bases were units derived from the bridged artificial nucleic acid LNA.

[0129] (CISH) Frozen sections of mouse brain tissue were thawed at room temperature, fixed with 4% PFA, and digested with PK according to standard methods. Prehybridization was performed with TE buffer containing 40% formamide at 55°C for 30 minutes. A hybridization solution was prepared with 40% formamide and a probe concentration of 1 μg / ml. 50 μl of the hybridization solution heated at 80°C for 3 minutes was dropped onto the sections, covered with a cover glass, and left to stand at 55°C for 1 hour for hybridization.

[0130] After hybridization, the cover glass was removed and the tissue sections were irradiated with 366 nm light for 60 seconds. The sections were then immersed in 80% formamide solution warmed to 55°C for 5 minutes three times, followed by two washes with TBS buffer. The sections were then treated with alkaline phosphatase-labeled anti-DIG antibody, and stained with NBT / BCIP to compare the color development. The results are summarized in Figure 3.

[0131] The evaluation criteria in FIG. 3 are as follows: Criterion 1 (Signal) A:20.1~, B:10.1~20.0, C:~10.0 Criterion 2 (Noise) A:~10.0, B:10.1~20.0, C:20.1~ Judgment criteria (S / N) A:2.01~, B:1.01~2.00, C:~1.00 Degradation resistance Throughout the series of operations, when a stable signal was detected from the probe solution, it was marked as A; when the signal significantly decreased, it was marked as C; and when it was intermediate between the two, it was marked as B.

[0132] As shown in Figure 3, when a photocrosslinkable probe having an L-threoninol skeleton (Test Example 2-1) and a photocrosslinkable probe having a serinol skeleton (Test Example 2-2) were used, both low background and signal intensity were achieved, the S / N ratio was high compared to the results for other probes, and a clear detection signal was obtained for the target miR.

[0133] In contrast, none of the probes of Comparative Examples 3-1 to 3-4 were able to provide sufficient results.

[0134] That is, when the DNA-type non-photocrosslinkable probe was used (Comparative Example 3-1), no signal was observed at all.

[0135] When the DNA-type photocrosslinkable probe (Comparative Example 3-2) was used, a weak signal was observed only in the area where miR-124a is thought to be highly expressed (the hippocampus), and no signal was observed in other areas.

[0136] When an RNA-type photocrosslinkable probe (Comparative Example 3-3) was used, it had a higher affinity for miR than the DNA-type photocrosslinkable probe (Comparative Example 3-2), and thus a good S / N ratio and a strong signal in the hippocampus were confirmed; however, the RNA was easily decomposed and the stability of the probe solution was low, making it unsuitable for practical use.

[0137] When the LNA-type photocrosslinkable probe (Comparative Example 3-4) was used, in addition to the color development specific to miR-124a, a very strong nonspecific signal was observed in the area where miR-124a is not normally expressed.

[0138] From the above, by using a photocrosslinkable probe using a specific skeleton, the influence of contaminants derived from biological samples can be suppressed or avoided, and detection sensitivity as well as detection accuracy can be improved.

Claims

1. an oligonucleotide region defining a capture sequence that specifically hybridizes to a target RNA; The oligonucleotide region is a probe having a first unit derived from a nucleoside represented by the following formula (1) and / or (2) and at least one second unit including a photocrosslinking reactive group that photocrosslinks pyrimidine bases in the target RNA. 【Chemistry 15】 【Chemistry 16】 (In formula (1) and formula (2), each B independently represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group.)

2. The probe according to claim 1 , wherein the second unit is a unit derived from one or two types of nucleosides selected from the group consisting of units represented by the following formulas (3) and (4): 【Chemistry 17】 【Chemistry 18】

3. The probe according to claim 1 or 2, wherein the oligonucleotide region has the first unit as all remaining units other than the second unit.

4. The probe of claim 1 , comprising a label element at the 3′ end and / or 5′ end of the oligonucleotide region for detecting the target RNA.

5. 1. A method for detecting a target RNA, comprising: (a) hybridizing the target RNA with the probe; A step (a) of hybridizing a probe comprising the target RNA and an oligonucleotide region defining a capture sequence that specifically hybridizes to the target RNA, the oligonucleotide region having a first unit represented by the following formula (1) and / or (2) and at least one second unit containing a photocrosslinking reactive group that photocrosslinks pyrimidine bases in the target RNA; (b) forming a crosslinked structure by light irradiation in a hybridization product in which the target RNA and the probe are hybridized; (c) removing the hybridization products other than the hybridization products having the cross-linked structure after the step (b); A method of providing.

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