Method for detecting a base sequence at a site difficult to detect in a higher-order structured RNA in a cell

Abstract

To provide means for detecting a base sequence at a difficult-to-detect site in an intracellular higher-order structure RNA.SOLUTION: Provided is a method for photocrosslinking a photocrosslinkable artificial nucleic acid probe by forming a double chain with a target base sequence in an RNA molecule, the method comprising: photocrosslinking a photocrosslinkable artificial nucleic acid assist probe by forming a double chain with an RNA molecule, simultaneously with or prior to double chain formation of the photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting a nucleotide sequence at a site difficult to detect in a higher-order structured RNA in a cell.

Background Art

[0002] A technique for detecting a specific nucleotide sequence contained in RNA in a cell is one of the basic techniques in molecular biology. At the same time as being a basic technique, it is also a technique that can be directly used in applications such as genetic diagnosis and detection of microbial strains. As such a detection technique, the Fluorescence in situ hybridization method (FISH method) is known.

[0003] In the FISH method, a fluorescently labeled nucleic acid is used as a detection probe, which is introduced into cells and allowed to form a double strand with a specific target nucleotide sequence to selectively detect the target nucleotide sequence.

[0004] On the other hand, it is known that RNA does not exist in a simple single-stranded state in cells, but forms a complex higher-order structure. Therefore, when trying to selectively detect a specific nucleotide sequence in RNA by forming a double strand with a detection probe, depending on how it is arranged in the complex higher-order structure of RNA, there are nucleotide sequences that are easily detected and those that are difficult to detect.

[0005] Non-Patent Document 1 reports that for Escherichia coli 16S rRNA, the detectability of nucleotide sequences at various sites in the complex higher-order structure was evaluated in six classes from Class I to Class VI using a detection probe.

[0006] Patent Document 1 discloses a photoreactive nucleotide analog having a photocrosslinking ability, and discloses that this photoreactive nucleotide analog can be used as a photoreactive crosslinking agent.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Document

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] If there is a means to easily detect the base sequence corresponding to Class VI, which is considered to be the most difficult to detect, in the complex higher-order structure of Escherichia coli 16S rRNA in Non-Patent Document 1, it will greatly advance the basic technology in molecular biology.

[0010] Therefore, an object of the present invention is to provide a means for detecting a base sequence at a site difficult to detect in higher-order structure RNA in a cell.

Means for Solving the Problems

[0011] The present inventor has found that the above object can be achieved by the means described below and has reached the present invention.

[0012] Therefore, the present invention includes the following (1). (1) A method for forming a duplex between a photo-crosslinkable artificial nucleic acid probe and a target base sequence in an RNA molecule and performing photo-crosslinking, comprising a step of forming a duplex between a photo-crosslinkable artificial nucleic acid assist probe and the RNA molecule and performing photo-crosslinking simultaneously with or prior to the formation of a duplex between the photo-crosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule. (2) The target base sequence in the RNA molecule is within a region of a partial duplex structure, and a partial duplex structure continues beyond the target base sequence at one of the two ends of the region of the partial duplex structure containing the target base sequence. The region of the partial duplex structure that continues beyond the target base sequence is designated as region A, and there is an RNA multi-branched structure at the other of the two ends of the region of the partial duplex structure containing the target base sequence. Among the branches formed by the RNA multi-branched structure, for the two branches that are close in base sequence to the region of the partial duplex structure containing the target base sequence, one branch region is designated as region B and the other branch region is designated as region C. As the photo-crosslinkable artificial nucleic acid assist probe, the following: A photo-crosslinkable artificial nucleic acid assist probe A having a base sequence capable of forming a duplex with the base sequence in region A and performing photo-crosslinking, A photo-crosslinkable artificial nucleic acid assist probe B having a base sequence capable of forming a duplex with the base sequence in region B and performing photo-crosslinking, and A photo-crosslinkable artificial nucleic acid assist probe C having a base sequence capable of forming a duplex with the base sequence in region C and performing photo-crosslinking, are included in the method according to (1). (3) The target base sequence in the RNA molecule is within a region of a partial duplex structure, and there are RNA multi-branched structures at both ends of the region of the partial duplex structure containing the target base sequence. In the multi-branched structure of one of the RNAs at both ends, among the branches formed by the multi-branched structure of the RNA, of the two branches that are close in nucleotide sequence to the region of the partial double-stranded structure containing the target nucleotide sequence, let the region of one branch be the B region and the region of the other branch be the C region. In the multi-branched structure of the other RNA at both ends, among the branches formed by the multi-branched structure of the RNA, of the two branches that are close in nucleotide sequence to the region of the partial double-stranded structure containing the target nucleotide sequence, let the region of one branch be the B' region and the region of the other branch be the C' region. As the photocrosslinkable artificial nucleic acid assist probe, the following: A photocrosslinkable artificial nucleic acid assist probe B having a nucleotide sequence that can form a double strand with the nucleotide sequence in the B region and can be photocrosslinked. A photocrosslinkable artificial nucleic acid assist probe C having a nucleotide sequence that can form a double strand with the nucleotide sequence in the C region and can be photocrosslinked. B’ A photocrosslinkable artificial nucleic acid assist probe B' having a nucleotide sequence that can form a double strand with the nucleotide sequence in the region and can be photocrosslinked, and C’ A photocrosslinkable artificial nucleic acid assist probe C' having a nucleotide sequence that can form a double strand with the nucleotide sequence in the region and can be photocrosslinked, included in the method according to (1). (4) The method according to any one of (2) to (3), wherein the multi-branched structure of the RNA is a three-branched structure of the RNA or a four-branched structure of the RNA. (5) The method according to any one of (1) to (4), wherein the RNA molecule is an intracellular RNA molecule. (6) A method for detecting a target nucleotide sequence in an RNA molecule by forming a double strand with the target nucleotide sequence in the RNA molecule and photocrosslinking a photocrosslinkable artificial nucleic acid probe, the method according to any one of (1) to (5). (7) The method according to (6), wherein the photocrosslinkable artificial nucleic acid probe is a probe equipped with a detection label. (8) The method according to (7), wherein the photocrosslinkable artificial nucleic acid probe is a probe equipped with a beacon-type fluorescent label for the FISH method. (9) The photocrosslinkable artificial nucleic acid probe is a photocrosslinkable artificial nucleic acid probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond, The photocrosslinkable artificial nucleic acid assist probe is the method according to any one of (1) to (8), wherein a photocrosslinkable artificial nucleic acid assist probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond: Formula (I): JPEG0007704393000001.jpg84170However, in formula I, R11 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group having 2 to 7 carbon atoms, or a hydrogen atom, R12 and R13 are each independently a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group having 2 to 7 carbon atoms, or a hydrogen atom, R14 is a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, an alkylsulfanyl group having 1 to 3 carbon atoms, a nitro group, a fluorine atom, a methyl fluoride group, or a hydrogen atom, The group Y is a group represented by the following formula II or formula III: Formula II: JPEG0007704393000002.jpg85170However, in formula II, R21 is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, Formula III: JPEG0007704393000003.jpg66170However, in formula III, R31 is a hydrogen atom or a hydroxyl group.

Advantages of the Invention

[0013] According to the present invention, it is possible to detect a base sequence at a site difficult to detect in the higher-order structure RNA in a cell.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Specific embodiments will be given below to describe the present invention in detail. The present invention is not limited to the specific embodiments given below.

[0016] [Method for forming a double strand with a target base sequence in an RNA molecule and performing photocrosslinking of a photocrosslinkable artificial nucleic acid probe] According to the present invention, a method comprising the step of forming a double strand of a photocrosslinkable artificial nucleic acid assist probe with an RNA molecule and performing photocrosslinking, simultaneously with or prior to the formation of a double strand between the photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule, enables the photocrosslinkable artificial nucleic acid probe to form a double strand with the target base sequence in the RNA molecule and perform photocrosslinking.

[0017] [Detection in an RNA structure having a multi-branched structure at one end of a target base sequence] In a preferred embodiment, the target base sequence in the RNA molecule is within a region of a partial double-strand structure, At one of the two ends of the region of the partial double-strand structure containing the target base sequence, a partial double-strand structure continues beyond the target base sequence, The region of the partial double-strand structure that continues beyond the target base sequence is designated as region A, At the other of the two ends of the region of the partial double-strand structure containing the target base sequence, there is a multi-branched structure of RNA, Of the branches formed by the multi-branched structure of the above RNA, one of the two branches that are close in base sequence to the region of the partial double-strand structure containing the target base sequence is designated as region B, and the other branch region is designated as region C, The above photocrosslinkable artificial nucleic acid assist probe can include the following: A photocrosslinkable artificial nucleic acid assist probe A having a base sequence capable of forming a double strand with the base sequence in region A and performing photocrosslinking, A photocrosslinkable artificial nucleic acid assist probe B having a base sequence capable of forming a double strand with the base sequence in region B and performing photocrosslinking, and A photocrosslinkable artificial nucleic acid assist probe C having a base sequence capable of forming a double strand with the base sequence in region C and performing photocrosslinking.

[0018] [Detection in an RNA structure having a multi-branched structure at both ends of a target base sequence] In a preferred embodiment, the target base sequence in the RNA molecule is within a region of a partial double-stranded structure, and there are multi-branched structures of RNA at both ends of the region of the partial double-stranded structure containing the target base sequence, In one of the multi-branched structures of RNA at both ends, among the branches formed by the multi-branched structure of RNA, of the two branches that are close in base sequence to the region of the partial double-stranded structure containing the target base sequence, one branch region is designated as the B region and the other branch region is designated as the C region, In the other multi-branched structure of RNA at both ends, among the branches formed by the multi-branched structure of RNA, of the two branches that are close in base sequence to the region of the partial double-stranded structure containing the target base sequence, one branch region is designated as the B' region and the other branch region is designated as the C' region, As the above photo-crosslinkable artificial nucleic acid assist probe, the following: A photo-crosslinkable artificial nucleic acid assist probe B having a base sequence capable of forming a double strand with the base sequence in the B region and being photo-crosslinkable, A photo-crosslinkable artificial nucleic acid assist probe C having a base sequence capable of forming a double strand with the base sequence in the C region and being photo-crosslinkable, B’ A photo-crosslinkable artificial nucleic acid assist probe B' having a base sequence capable of forming a double strand with the base sequence in the region and being photo-crosslinkable, and C’ A photo-crosslinkable artificial nucleic acid assist probe C' having a base sequence capable of forming a double strand with the base sequence in the region and being photo-crosslinkable, can be included.

[0019] [Detection of a target base sequence by a photo-crosslinkable artificial nucleic acid probe] The present inventors have intensively studied nucleic acid double-strand formation and photo-crosslinking technology. When an artificial nucleic acid prepared by introducing an artificial nucleotide analog having a photo-crosslinkable artificial base in the same manner as natural nucleotides is used as a probe, it forms a double strand depending on its base sequence and can detect a specific target base sequence by photo-crosslinking. Since the double-strand formation by this photo-crosslinkable artificial nucleic acid probe becomes irreversible due to the formation of photo-crosslinks, it is an energetically very advantageous reaction and the detection sensitivity is high.

[0020] In addition, once double-strand formation and photo-crosslink formation are achieved, it is easy to detect the probe using a detection label that has been previously bound. Therefore, the degree of achievement of photo-crosslink formation and the detection sensitivity thereby have substantially the same meaning.

[0021] From such a viewpoint, the present inventors predicted that by using a photo-crosslinkable artificial nucleic acid probe, even a target base sequence in an RNA molecule having a complex higher-order structure can be detected with high sensitivity if double-strand formation and photo-crosslinking are carried out. However, even when such a photo-crosslinkable artificial nucleic acid probe was used, in Non-Patent Document 1, when it came to the base sequence corresponding to Class VI, which was considered to be the most difficult to detect in the complex higher-order structure of Escherichia coli 16S rRNA, sufficient high sensitivity could not be obtained.

[0022] Therefore, the present inventors analyzed the structure of the site difficult to detect in such a complex higher-order structure RNA and found a tendency that the structure in the vicinity of the target base sequence has a multi-branched structure. The multi-branched structure refers to, for example, a structure in which RNA forms a double-strand structure in the molecule and the double-strand structure branches out into many branches like a road branch at a certain position. The multi-branched structure includes, for example, a three-branched structure and a four-branched structure.

[0023] Then, the inventor came up with the idea that this multi-branched structure might be a constraint and suppress the double-strand formation and photocrosslinking formation between the target base sequence and the photocrosslinkable artificial nucleic acid probe. Furthermore, when the double-strand formation and photocrosslinking formation between the photocrosslinkable artificial nucleic acid probe and the target base sequence were carried out, the inventor added a photocrosslinkable artificial nucleic acid assist probe to assist this process, and by performing the double-strand formation and photocrosslinking formation between the photocrosslinkable artificial nucleic acid assist probe and the corresponding base sequence, it was found that the target base sequence located at a site difficult to detect in RNA with a complex higher-order structure could also be detected with sufficient high sensitivity, thus reaching the present invention.

[0024] According to the study of the inventor, this photocrosslinkable artificial nucleic acid assist probe is required to have a specific positional relationship with respect to the target base sequence.

[0025] Specifically, when there is a multi-branched structure near one end of the double-stranded structure portion containing the target base sequence, among the plurality of branches of the multi-branched structure, it is preferable to use two photocrosslinkable artificial nucleic acid assist probes that are capable of forming double-strands in each of the two branches close to the target base sequence. At the same time, since a double-stranded structure will continue at the other end of the double-stranded structure portion containing the target base sequence, it is preferable to use one photocrosslinkable artificial nucleic acid assist probe that is capable of forming a double-strand with the base sequence in this double-stranded structure simultaneously with the above two photocrosslinkable artificial nucleic acid assist probes.

[0026] Also, specifically, when there are multi-branched structures at both ends of the double-stranded structure portion containing the target base sequence, for each multi-branched structure, among the plurality of branches of the multi-branched structure, it is preferable to use two photocrosslinkable artificial nucleic acid assist probes that are capable of forming double-strands in each of the two branches close to the target base sequence.

[0027] [Position of Photocrosslinkable Artificial Nucleic Acid Probe in Higher-Order Structure RNA] In higher-order structure RNA, the position where the photocrosslinkable artificial nucleic acid probe forms a double strand and forms a photocrosslink is the position of the target base sequence. In a preferred embodiment, the base length of the target base sequence can be, for example, 15 to 20 bases, or 20 to 25 bases. In a preferred embodiment, the target base sequence includes a double-stranded forming portion of, for example, 15 to 20 bases, or 20 to 25 bases among the above base lengths. In a preferred embodiment, the base at the end of the target base sequence close to the center of the multi-branched structure is in the range of a distance, for example, from the 20th to the 30th base, or from the 30th to the 40th base, from the center of the branch of the multi-branched structure in the vicinity. The number of bases indicating the distance from the center of the branch of the multi-branched structure is counted with the first base pair formed between the two strands branched from the multi-branched structure as the first base. For example, if there is a base sequence without base pair formation between the two strands immediately after branching from the multi-branched structure, the first base pair-forming base that appears after that base sequence becomes the first base.

[0028] [Position of the photocrosslinkable artificial nucleic acid assist probe] In higher-order structure RNA, the position where the photocrosslinkable artificial nucleic acid assist probe forms a double strand and the target base sequence for photocrosslink formation is located in any one of the above A region, B region, C region, or in any one of the above B region, C region, B' region, C' region.

[0029] In a preferred embodiment, the base length of the target base sequence located in the A region can be, for example, 15 to 20 bases, or 20 to 25 bases. In a preferred embodiment, the target base sequence located in the A region includes a double-stranded forming portion of, for example, 15 to 20 bases, or 20 to 25 bases among the above base lengths. In a preferred embodiment, the base at the end of the target base sequence located in the A region close to the target base sequence is in the range of a distance, for example, from the 20th to the 30th base, or from the 30th to the 40th base, when the base immediately adjacent to the target base sequence is taken as the first base. The target base sequence located in the A region may include a non-double-stranded structure portion inside.

[0030] In a preferred embodiment, the base length of the target base sequence located in region B can be, for example, 15 to 20 bases, or 20 to 25 bases. In a preferred embodiment, the target base sequence located in region B includes a double-stranded forming portion of, for example, 15 to 20 bases, or 20 to 25 bases among the above base lengths. In a preferred embodiment, the bases at the end near the center of the multi-branched structure of the target base sequence located in region B are in the range of a distance such that they are, for example, the 20th to 30th bases, or the 30th to 40th bases from the center of the branch of the multi-branched structure in the vicinity. The target base sequence located in region B may include a non-double-stranded structure portion inside. In a preferred embodiment, the target base sequence located in region B' can be provided in the same manner as the target base sequence located in region B.

[0031] In a preferred embodiment, the base length of the target base sequence located in region C can be, for example, 15 to 20 bases, or 20 to 25 bases. In a preferred embodiment, the target base sequence located in region C includes a double-stranded forming portion of, for example, 15 to 20 bases, or 20 to 25 bases among the above base lengths. In a preferred embodiment, the bases at the end near the center of the multi-branched structure of the target base sequence located in region C are in the range of a distance such that they are, for example, the 20th to 30th bases, or the 30th to 40th bases from the center of the branch of the multi-branched structure in the vicinity. The target base sequence located in region C may include a non-double-stranded structure portion inside. In a preferred embodiment, the target base sequence located in region C' can be provided in the same manner as the target base sequence located in region B.

[0032] [Photo-crosslinkable artificial nucleoside] In a preferred embodiment, the photo-crosslinkable artificial nucleic acid probe can be a photo-crosslinkable artificial nucleic acid probe in which the photo-crosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond. In a preferred embodiment, the photo-crosslinkable artificial nucleic acid assist probe can be a photo-crosslinkable artificial nucleic acid assist probe in which the photo-crosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond.

[0033] Formula (I): JPEG0007704393000004.jpg85128

[0034] In a preferred embodiment, R11 can be, for example, a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom, and preferably can be a cyano group or a hydrogen atom.

[0035] In a preferred embodiment, R12 and R13 can each independently be a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom, and preferably can be a cyano group or a hydrogen atom.

[0036] In a preferred embodiment, R14 can be a hydroxyl group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a methyl fluoride group, or a hydrogen atom, and preferably can be a nitro group or a hydrogen atom.

[0037] In a preferred embodiment, the group Y can be a group represented by the following formula II or formula III.

[0038] Formula II: JPEG0007704393000005.jpg8788

[0039] In a preferred embodiment, R21 can be, for example, a hydrogen atom or a C1-C3 alkyl group, and preferably can be a methyl group or a hydrogen atom.

[0040] Formula III: JPEG0007704393000006.jpg6752

[0041] In a preferred embodiment, R31 can be, for example, a hydrogen atom or a hydroxyl group.

[0042] [Photo-crosslinking formation] Both the photocrosslinkable artificial nucleic acid probe and the photocrosslinkable artificial nucleic acid assist probe can form a photocrosslink by light irradiation after forming a double strand, respectively. This photocrosslink is formed by the photoreaction of the above-mentioned photocrosslinkable artificial nucleoside introduced into the photocrosslinkable artificial nucleic acid probe and the photocrosslinkable artificial nucleic acid assist probe. The photocrosslinkable artificial nucleoside can form a photocrosslink with a pyrimidine base arranged at a photocrosslinkable position. Examples of the pyrimidine base include thymine (T), cytosine (C), uracil (U), 5-methylcytosine, and 5-hydroxymethylcytosine. In higher-order structured RNA, preferably cytosine (C) and uracil (U) can be mentioned.

[0043] The photocrosslinkable artificial base of the photocrosslinkable artificial nucleoside can form a photocrosslink between the base located only one base on the 5'-terminal side (the base adjacent to the base on the 5'-terminal side of the base at the complementary position) from the base at the position complementary to the photocrosslinkable artificial base in the base sequence of the nucleic acid having a complementary base sequence. That is, in the complementary base sequence, the position only one base on the 5'-terminal side from the base at the position complementary to the photocrosslinkable artificial base is a photocrosslinkable position.

[0044] Since the formation of the photocrosslink by light irradiation is a photoreaction, it can be carried out under a wide range of conditions with respect to temperature, solvent, salt concentration, pH, etc. Therefore, it can be carried out even under physiological conditions and can be preferably used under intracellular conditions. The photocrosslink formation can be carried out under the same conditions as the double-strand formation.

[0045] In a preferred embodiment, the light irradiation can be carried out, for example, by irradiation with light including wavelengths in the range of 340 nm to 390 nm, in the range of 360 nm to 390 nm, such as light including a wavelength of 385 nm. The light irradiation can be carried out, for example, with an irradiation time in the range of 0.1 second to 60 seconds, 1 second to 30 seconds, 5 seconds to 15 seconds. The light irradiation can be carried out, for example, at a temperature in the range of 0°C to 40°C, 0°C to 30°C, 0°C to 20°C, 0°C to 10°C.

[0046] [Labeling of photocrosslinkable artificial nucleic acid probe] In a preferred embodiment, double-strand formation and photocrosslink formation by the photocrosslinkable artificial nucleic acid probe can be carried out for detecting a target base sequence in an RNA molecule. In this case, the photocrosslinkable artificial nucleic acid probe can be provided with a label for detection. As such a label for detection, known labels for detection can be used. Examples of such labels for detection include FITC, FAM, TAMRA, Rhodamine, Cy3, Cy5, preferably Cy3 and Cy5. In a preferred embodiment, the photocrosslinkable artificial nucleic acid probe can be a probe provided with a beacon-type fluorescent label for the FISH method. In a preferred embodiment, the beacon-type fluorescent label for the FISH method consists of a set of a fluorescent group and a quenching group, and by forming a double strand with the base sequence targeted by the probe, the quenching group is isolated from the fluorescent group, the quenching action of the quenching group does not work, and a configuration is provided in which fluorescence from the fluorescent group can be observed.

[0047] [Addition of photocrosslinkable artificial nucleic acid assist probe] In a preferred embodiment, as described above, double-strand formation and photocrosslink formation by the photocrosslinkable artificial nucleic acid assist probe promote double-strand formation and photocrosslink formation by the photocrosslinkable artificial nucleic acid probe. Double-strand formation and photocrosslink formation by the photocrosslinkable artificial nucleic acid assist probe may be performed prior to double-strand formation and photocrosslink formation by the photocrosslinkable artificial nucleic acid probe, or may be performed simultaneously with double-strand formation and photocrosslink formation by the photocrosslinkable artificial nucleic acid probe. Therefore, the addition of the photocrosslinkable artificial nucleic acid assist probe to the sample to be tested may be performed prior to the addition of the photocrosslinkable artificial nucleic acid probe, may be performed simultaneously with the addition of the photocrosslinkable artificial nucleic acid probe, or may be performed after the addition of the photocrosslinkable artificial nucleic acid probe.

[0048] In a preferred embodiment, since the photocrosslinkable artificial nucleic acid assist probe is for promoting duplex formation and photocrosslinking by the photocrosslinkable artificial nucleic acid probe, typically, the molecules of each photocrosslinkable artificial nucleic acid assist probe can be added based on about the same molar number as the molecules of the photocrosslinkable artificial nucleic acid probe, but it is not limited thereto. In a preferred embodiment, the molar number of the molecules of each photocrosslinkable artificial nucleic acid assist probe may be added at a ratio in the range of, for example, 1 / 10 to 10 / 1, or 1 / 1.5 to 1.5 / 1, relative to the molar number of the molecules of the photocrosslinkable artificial nucleic acid probe.

[0049] In a preferred embodiment, as described above, as the photocrosslinkable artificial nucleic acid assist probe, a photocrosslinkable artificial nucleic acid assist probe that forms a duplex and photocrosslinks with the target base sequence located in region A, a photocrosslinkable artificial nucleic acid assist probe that forms a duplex and photocrosslinks with the target base sequence located in region B, and a photocrosslinkable artificial nucleic acid assist probe that forms a duplex and photocrosslinks with the target base sequence located in region C are preferably used simultaneously as a set. By using these three types of photocrosslinkable artificial nucleic acid assist probe molecules, the promoting effect of duplex formation and photocrosslinking of the photocrosslinkable artificial nucleic acid probe is preferably exerted. In a preferred embodiment, within a range that does not interfere with the promoting effect of these three types of photocrosslinkable artificial nucleic acid assist probe molecules, adding another type of photocrosslinkable artificial nucleic acid assist probe molecule is also within the scope of the present invention.

Example

[0050] The present invention will be described in detail below with reference to examples. The present invention is not limited to the examples illustrated below.

[0051] [Example 1: Examination of the assisting effect of the photocrosslinkable assist probe on the detection by the photocrosslinkable molecular beacon type probe] [Synthesis of probe ODNs] The synthesis of the probe ODNs used in the examples was carried out as follows. The probes used in the examples are a photocrosslinkable molecular beacon-type probe targeting the Eco627 region with a relative fluorescence intensity of 1%, and a photocrosslinkable assist probe for disrupting the higher-order structure of the target (see Table 1). The photocrosslinkable molecular beacon-type probe Eco1437 was modified with a Stem region, a quenching group Dabcyl at the 3'-end, and a fluorescent group Cy3 at the 5'-end, and 3-Cyanovinylcarbazole( CNV D) was added to the Roop region. Both the photocrosslinkable molecular beacon-type probe and the photocrosslinkable assist probe were S-modified ODNs. DNA synthesis was carried out by solid-phase synthesis using a 3400 DNA Synthesizer. A list of the probes used is shown in Table 1 below.

[0052]

Table 1

[0053] The relative fluorescence intensities shown in Table 1 were described as the values shown in Non-Patent Document 1 (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1998, p. 4973-4982, Vol. 64, No. 12).

[0054] Non-Patent Document 1 targeted Escherichia coli 16S rRNA, divided RNA with a higher-order structure into multiple regions, created complementary probes for those regions, detected that the probes hybridized to the corresponding regions of the RNA by fluorescence emission from the fluorescent groups pre-bound to the probes, and disclosed the results of quantifying and comparing the ease of hybridization in each region by fluorescence intensity.

[0055] In Table 1 above, the names of each probe used in this example are listed in the leftmost column, and the sequences of each probe are as shown in the central column of Table 1. Each probe used in this example has a name following the names of the probes described in Non-Patent Document 1 as recognizing the same region, and the relative fluorescence intensities (Relative Probe Fluorescence) shown by each probe used in Non-Patent Document 1 are respectively described in the rightmost column of Table 1 above.

[0056] This relative fluorescence intensity is a relative value with the fluorescence intensity shown by the probe recognizing the Eco1482 region in Non-Patent Document 1 being set as 100%. The larger the numerical value, the more likely the region recognized by each probe is a site that hybridizes easily in the higher-order structure RNA, that is, a site that is easy to detect.

[0057] And as shown in Table 1 above, the region corresponding to the Eco627 probe is a region showing only about 1% fluorescence intensity compared to the Eco1482 region according to the disclosure of Non-Patent Document 1, and it was an extremely difficult region to detect this region. In addition, there are descriptions of classifications of Class II, Class III, Class IV, Class V, and Class VI next to the numerical values of the relative fluorescence intensities in Table 1. In Non-Patent Document 1, these are indicators showing the difficulty of detecting each region according to the values of the relative fluorescence intensities, and Class VI means that it is classified as the most difficult region to detect in the higher-order structure RNA.

[0058] D described in the sequence of Table 1 is CNV D. CNV D is a photocrosslinkable artificial nucleoside having the following structure. CNV D has 3-Cyanovinylcarbazole as the base moiety in the nucleoside and has a serinol (2-amino-1,3-propanediol) structure as the sugar backbone moiety in the nucleoside. CNV Similar to natural nucleosides, D can be introduced into an oligonucleotide sequence. JPEG0007704393000008.jpg8876

[0059] [Preparation for the preparation of observation samples] [Preparation of Escherichia coli glycerol stock] In the examples, Escherichia coli (One Shot® TOP10 Chemically Competent E. coli) was used. E. coli cultured in LB liquid medium heated to 37°C was collected in the logarithmic growth phase. After preparing a glycerol stock (final concentration 20%) of the cultured E. coli competent cells, it was stored at -80°C.

[0060] [buffer preparation] Hybridization buffer was prepared as follows. 1.0 M Tris HCl pH 7.2 (final concentration 20 mM), Sodium chloride (final concentration 900 mM), and Sodium dodecyl sulfate (0.01% v / v) were mixed.

[0061] Embedding buffer was prepared as follows. 1x PBS buffer (130 mM Sodium chloride, 10.8 mM Na2HPO4, 4.2 mM NaH2PO4 [pH 7.2]), 0.1% Low-melting-point temp. agarose (0.1% v / v), and Sodium dodecyl sulfate (0.01% v / v) were mixed.

[0062] [Preparation of observation samples] Solutions in which each probe was diluted with Hybridization buffer were mixed with the competent cell solution to a final concentration of 1.0 μM. After allowing this mixed solution to stand on ice for 15 min, a heat shock was applied at 42 °C for 40 sec. After allowing it to stand on ice for 3 min, incubation was carried out at 37 °C for 4.0 h for hybridization. Light irradiation was performed at 4.0 °C (wavelength: 385 nm / irradiation time: 360 sec). The sample solution (5.0 μL) after light irradiation and Embedding buffer (8.0 μL) were mixed on a slide glass, air-dried naturally at r.t. for 10 min, and then a cover glass was placed on top.

[0063] [Observation with a confocal laser microscope] For observation of the FISH samples, a confocal laser microscope (Nikon, C2Si (with use Ti-u)) and Nikon image integration software (NIS-Elements) were used, and a CCD camera attached to the microscope was used for image capture. The observation conditions were set as follows. Excitation wavelength: 561 nm, detection wavelength: 573 - 613 nm Objective lens: x60 (oil immersion lens) Cy3 fluorescence image detection sensitivity (HV) of NIS-Elements: 50, threshold value (Offset): -127 Bright-field detection sensitivity (HV): 128, threshold value (Offset): -127

[0064] [Positional information between each probe and the target] In the examples, Eco627, which has a complex higher-order structure and is the region with the lowest probe affinity, was used as the target, and a combination of a photo-responsive artificial nucleic acid ( CNV D)-containing probe (Table 2.1) was used to CNV verify how much the increase in relative Cy3 fluorescence intensity is affected by the D-containing beacon-type probe. Figure 1 shows the positional information of the target and each normal-type probe that plays a role in disrupting the higher-order structure.

[0065] FIG. 1 is a partially enlarged view of the overall structure of the higher-order structure prediction model of Escherichia coli 16S rRNA disclosed in Non-Patent Document 1. In FIG. 1, "600", "650", and "750" each indicate the number of bases from the 5' end of 16sRNA. The region to which the Eco627 probe hybridizes is shown in FIG. 1. Similarly, the region to which each probe shown in Table 1 hybridizes is shown in FIG. 1 by each probe number. As shown in Non-Patent Document 1, the region to which the Eco627 probe hybridizes is the most difficult region to hybridize in Escherichia coli 16S rRNA, and is a region that is usually almost impossible to detect even if a so-called molecular beacon type probe is used to detect it.

[0066] [Observation results and evaluation] Photoresponsive artificial nucleic acid ( CNV D) By combining the probes CNV We examined how much the D-containing beacon probe affects the increase in relative Cy3 fluorescence intensity. First, we examined how much the increase in relative Cy3 fluorescence intensity would be affected when only the beacon probe Eco627 was added, and when the beacon probe Eco627 and eight normal probes that play a role in disrupting higher-order structures were added. The relative Cy3 fluorescence intensity was measured by quantifying the brightness of images taken with a confocal laser scanning microscope using the image analysis software Image J, and the relative value of the Cy3 fluorescence intensity after each light irradiation time was calculated using Microsoft Office Excel, based on the Cy3 fluorescence intensity at 0 seconds of light irradiation when only the beacon probe Eco627 was added. The light irradiation time was 360 seconds at a wavelength of 385 nm, the same as for the Eco1437 series.

[0067] The results of the obtained observations are illustrated in FIGS. 2A to 2D. FIGS. 2A to 2D show CLSM images (confocal laser scanning microscope images) of E. coli. FIGS. 2A and 2B are CLSM images of E. coli stained with Eco627 irradiated with light at 385 nm for 0 seconds (FIG. 2A) or 360 seconds (FIG. 2B). FIGS. 2C and 2D are CLSM images of E. coli stained with Eco627 and Eco567(1) to 763(8) irradiated with light at 385 nm for 0 seconds (FIG. 2C) or 360 seconds (FIG. 2D). The scale bar is 20 μm.

[0068] As observed from the CLSM images of FIGS. 2A to 2D, as a result of FISH, aggregation of fluorescence emission by Cy3 was observed in the range defined as cells in the bright-field image (PC), suggesting that the probe was taken up into the cells. In addition, when eight types of normal probes responsible for disrupting the higher-order structure were added to the beacon-type probe Eco627, an increase in the relative Cy3 fluorescence intensity was confirmed by light irradiation, suggesting that the probe bound to the target RNA. Quantification of the fluorescence intensity was performed by quantifying the luminance of the Cy3 fluorescence image obtained by a confocal microscope using Image J and analyzing it using Excel. The graphs created by quantification are shown in FIGS. 3A to 3B.

[0069] FIG. 3A is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 irradiated with light at 385 nm for 0 seconds or 360 seconds. FIG. 3B is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 and Eco567(1) to 763(8) irradiated with light at 385 nm for 0 seconds or 360 seconds.

[0070] In FIGS. 3A and 3B, the relative fluorescence intensity on the vertical axis of the graph is shown by this relative value, with the light irradiation time at 0 seconds when only the photocrosslinkable molecular beacon-type probe Eco627 was added being set as "1". This is to enable easy understanding of to what extent the detection of the complementary region by hybridization of the photocrosslinkable molecular beacon-type probe Eco627 is sensitized by photocrosslinking, and further to what extent it is sensitized by the photocrosslinkable assist probe.

[0071] As shown in FIG. 3A, the relative Cy3 fluorescence intensity 360 seconds after light irradiation when only the photocrosslinkable molecular beacon-type probe Eco627 was added increased 7-fold compared to after 0 seconds of light irradiation. Although the 7-fold increase in fluorescence intensity is a large increase, it is smaller than expected as sensitization by photocrosslinking. This means that the region targeted by the photocrosslinkable molecular beacon-type probe Eco627 has a complex higher-order structure and is a difficult region to detect. That is, as evaluated as Class VI, which was the most difficult to detect in Non-Patent Document 1, it was confirmed that it is an extremely difficult region to detect.

[0072] In contrast, as shown in FIG. 3B, when the photocrosslinkable molecular beacon-type probe Eco627 and 8 kinds of photocrosslinkable assist probes (Eco567(1) to Eco763(8) shown in Table 1) were added, the relative Cy3 fluorescence intensity 360 seconds after light irradiation increased 39-fold compared to after 0 seconds of light irradiation of only the photocrosslinkable molecular beacon-type probe Eco627. From this, it was found that by combining the photocrosslinkable molecular beacon-type probe Eco627 and the photocrosslinkable assist probe, it is possible to achieve surprisingly high sensitization and enable detection even for regions with complex higher-order structures that are difficult to detect. Although the reason for this is unclear, the present inventor considers that the photocrosslinkable assist probe may have changed the higher-order structure near the target region of the photocrosslinkable molecular beacon-type probe Eco627 to create a state in which photocrosslinking of the photocrosslinkable molecular beacon-type probe Eco627 can be formed.

[0073] [Example 2: Examination of the combination of photocrosslinkable assist probes and the assist effect] [Region in higher-order structure RNA] As described above, from the results of Example 1, the present inventors considered that the photocrosslinkable assist probe changed the higher-order structure near the target region of the photocrosslinkable molecular beacon-type probe Eco627. Therefore, in order to efficiently promote this higher-order structure change, the combination of photocrosslinkable assist probes and the assist effect were examined.

[0074] The higher-order structure RNA shown in Fig. 1 is said to have a complex higher-order structure that cannot actually be illustrated. However, the present inventors modeled and considered the essence of the structure near the target region of the photocrosslinkable molecular beacon-type probe Eco627 of the higher-order structure RNA shown in Fig. 1 as the following structure. That is, in Fig. 1, there is a double-stranded region that is the target region of Eco627, there is a double-stranded region (A region) located on the left side of the double-stranded region that is the target region, there is a three-branched structure located on the right side of the target region, there is a double-stranded region (target region) in the left branch of the three-branched structure, there is a double-stranded region (B region) located above the three-branched structure, and there is a double-stranded region (C region) located below the three-branched structure. The arrangement of this A region, B region, and C region is shown in Fig. 4 below.

[0075] Based on the positional information of the complementary regions of each assist probe, the present inventors hypothesized that hybridization and photocrosslinking by the assist probe to the above three regions would bring about a change in the higher-order structure that facilitates hybridization and photocrosslinking in the target region, and further examination was carried out.

[0076] [Combination of assist probes] From the assist probes shown in Table 1, combinations of three probes were extracted, and the assist effects were examined for 56 combinations. These combinations are shown in Table 2 below. In Table 2, each probe is indicated by the probe number shown in Table 1. For example, "1.2" in the second row of the leftmost column of Table 2 means the combination of Eco567(1) and Eco645(2) described in Table 1, and "3" in the first row of the second column from the left in Table 2 means Eco614(3) in Table 1, and "1" in the second row of the second column from the left in Table 2 means that the combination of Eco567(1), Eco645(2), and Eco614(3) in Table 1 is set as combination number "1".

[0077]

Table 2

[0078] [Experiment, Observation, and Evaluation] In Example 1, a comparative experiment was conducted between the case where only the photocrosslinkable molecular beacon type probe Eco627 was added and the case where the photocrosslinkable molecular beacon type probe Eco627 and eight types of photocrosslinkable assist probes were all added. In this Example 2, experiments were conducted for the case where only the photocrosslinkable molecular beacon type probe Eco627 was added and the case where any one of the combination numbers 1 to 56 of the above photocrosslinkable assist probes and the photocrosslinkable molecular beacon type probe Eco627 were added. Experiments, observations, and evaluations were carried out in the same manner as in Example 1 except for adding the combinations. The results are shown in Table 3 below.

[0079] Table 3 shows the combinations of each probe and the relative Cy3 fluorescence intensity detected by that combination. For example, the combination described as combination 1 (1.2.3) is combination number 1, indicating that the photocrosslinkable assist probes (1), (2), and (3) described in Table 1 were combined, and showing that the obtained relative fluorescence intensity was 21.0.

[0080]

Table 3

[0081] Graphs of relative fluorescence intensities based on the values in Table 3 are shown in FIGS. 5A and 5B.

[0082] In Table 3, FIGS. 5A and 5B, results of 56 combinations are shown as combinations of probes. Among the 56 combinations, the combinations that gave a larger increase in relative Cy3 fluorescence intensity are as follows.

[0083] Among these combinations, the combination that gave the highest relative Cy3 fluorescence intensity was a combination having one photocrosslinkable assist probe having a complementary region for each of the three regions of the above-described Region A, Region B, and Region C. In the combination that gave the highest relative Cy3 fluorescence intensity, an increase of 35.5-fold in relative Cy3 fluorescence intensity was observed. This increase of 35.5-fold was a relative Cy3 fluorescence intensity comparable to the case where 8 types of probes were added. From this result, the hypothesis that hybridization and photocrosslinking with the assist probe to the above three regions bring about a change in the higher-order structure that facilitates hybridization and photocrosslinking in the target region has been confirmed. That is, it has become clear that disrupting the higher-order structure of the three regions is a necessary condition for fluorescent detection of the target.

[0084] Furthermore, when examining the results in more detail, the combination No. 14 (combination of Eco567(1), Eco603(4), and Eco745(7)) that gave the largest increase in relative Cy3 fluorescence intensity is considered to disrupt the structures of Region A with Eco603(4), Region B with Eco745(7), and Region C with Eco567(1), respectively. In particular, according to the hypothesis of the present inventors, Eco745(7) was expected to play a role in disrupting the three-branched structure, and it has been clarified from the above experimental results that it is actually an important probe.

[0085] [Excerpt and detailed evaluation of combinations with large increases in relative fluorescence intensity] Combinations with large increases in relative fluorescence intensity were excerpted, and experiments, observations, evaluations, and considerations were conducted in the same manner as in Example 1. Graphs created by quantifying the results of observing CLSM images using the same method as in Example 1 are shown in Fig. 6. Fig. 6 is a graph of the relative fluorescence intensity of E. coli stained with Eco627, with respect to the case where only Eco627 was added, for each of the described combinations of assist probes used.

[0086] From Fig. 6, it was found that the relative fluorescence intensity of combination number 14 (combination of Eco567(1), Eco603(4), and Eco745(7)) showed the largest increase. For this combination number 14 (combination of Eco567(1), Eco603(4), and Eco745(7)), the arrangement of the regions to which each assist probe hybridizes is shown in Fig. 7.

[0087] [Excerpt and detailed evaluation of combinations with small increases in relative fluorescence intensity] Combinations with small increases in relative fluorescence intensity were excerpted, and experiments, observations, evaluations, and considerations were conducted in the same manner as in Example 1. Graphs created by quantifying the results of observing CLSM images using the same method as in Example 1 are shown in Fig. 8. Fig. 8 is a graph of the relative fluorescence intensity of E. coli stained with Eco627, with respect to the case where only Eco627 was added, for each of the described combinations of assist probes used.

[0088] As shown in Fig. 8, the relative fluorescence intensity resulted in 5.1 times for the lowest combination 41 (3.5.6). Among them, even for the relatively high combination 16 (1.5.6), it was 9.4 times.

[0089] What these combinations have in common is that, with respect to the three regions of the above-described Region A, Region B, and Region C, for each region, two assist probes are included. Taking the combination 41 (Eco624(3), Eco650(5), Eco738(6)) with the lowest increase rate as an example, two, namely Eco650(5) and Eco738(6), are included as assist probes for Region B. Since the regions affected by the assist by hybridization of Eco650(5) and Eco738(6) are the same region, while there is almost no assist effect on Region C, it is considered that the overall assist efficiency is lower than that of other combinations. In addition, since Eco650(5) and Eco738(6) are almost complementary strands, a complementary-strand-forming interaction between Eco650(5) and Eco738(6) also occurs, and as a result, it is also considered that the proportion of Eco650(5) and Eco738(6) that effectively binds to the RNA structure has decreased. Not only regarding the relationship of complementary strands, but also for combinations in which the assist probes overlap by several bases, it is considered that the increase rate of the relative Cy3 fluorescence intensity has decreased for the same reason.

[0090] Figure 9 shows the arrangement of the regions to which each assist probe hybridizes for combination number 41 (Eco624(3), Eco650(5), Eco738(6)).

[0091] [Example 3: Examination of Combining Four Photo-Crosslinkable Assist Probes] [Selection of Combination Numbers for Adding a Fourth Assist Probe] In Example 2, when examining the combination of photo-crosslinkable assist probes, the combination of three assist probes was examined. In Example 3, the examination of combining four photo-crosslinkable assist probes was carried out as follows.

[0092] In the study, first, in the study of Example 2, combination number 14 (the combination of Eco567(1), Eco603(4), and Eco745(7)) where the increase in relative Cy3 fluorescence intensity was the largest, and combination 41 (Eco624(3), Eco650(5), Eco738(6)) where the increase in relative Cy3 fluorescence intensity was the smallest were selected. Then, based on these combination numbers 14 and 41, further examination was made of what effects would be brought about by adding one more assist probe.

[0093] Regarding combination number 14 in Example 2, assist probes have already been added to all of regions A, B, and C respectively, and it is considered that the RNA higher-order structure has collapsed to the extent necessary for Eco627 to hybridize and photocrosslink to the target region. When an assist probe was further added in this state, it was considered that due to the interaction between the assist probes, rather, the relative Cy3 fluorescence intensity might decrease, so examination was conducted on this point.

[0094] Regarding combination number 41 in Example 2, one assist probe for region A (Eco614(3)) and two assist probes for region B (Eco650(5), Eco738(6)) were included. In combination number 41 of Example 2, it was also considered that due to the negative cooperativity between these two assist probes for region B, an increase in relative Cy3 fluorescence intensity could not be obtained. Therefore, for combination number 41 in Example 2, it was examined whether an increase in relative Cy3 fluorescence intensity could be obtained due to the positive cooperativity between the probes by adding an assist probe for region C. Further, it was also examined whether the relative Cy3 fluorescence intensity would further decrease due to negative cooperativity by adding assist probes for regions A and B.

[0095] The experiment and evaluation were conducted in the same manner as in Example 1.

[0096] [Examination of adding an assist probe to combination number 14] An experiment was conducted in which one more assist probe was added to combination number 14 of Example 2. A graph created by quantifying the results of observing the CLSM images in the same manner as in Example 1 is shown in FIG. 10. FIG. 10 is a graph of the relative fluorescence intensity of E. coli stained with Eco627 when using each of the described assist probe combinations, with the case of adding only Eco627 as a reference.

[0097] [Examination of adding an assist probe to combination number 41] An experiment was conducted in which one more assist probe was added to combination number 41 of Example 2. A graph created by quantifying the results of observing the CLSM images in the same manner as in Example 1 is shown in FIG. 11. FIG. 11 is a graph of the relative fluorescence intensity of E. coli stained with Eco627 when using each of the described assist probe combinations, with the case of adding only Eco627 as a reference.

[0098] [Evaluation of an experiment combining four photocrosslinkable assist probes] Regarding combination 14 (Eco567(1), Eco603(4), Eco745(7)), a decrease in relative Cy3 fluorescence intensity was observed due to the negative cooperativity between the probes by adding a probe. When Eco645(2) was added, it was 27.6 times (a 22.2% decrease), while when Eco614(3) was added, it was 16.5 times (a 53.5% decrease), and when Eco763(8) was added, it was 17.8 times (a 49.9% decrease). As the reason for such a decrease rate, Eco645(2) forms a partial complementary strand with Eco745(7), and since it is also close to the target and plays a role in disrupting the T-shaped structure, it is considered that the decrease in relative Cy3 fluorescence intensity due to negative cooperativity did not occur as much.

[0099] Regarding combination 41 (Eco624(3), Eco650(5), Eco738(6)), by adding probes, the cooperativity of each probe worked positively, and an increase in relative Cy3 fluorescence intensity was obtained. From this result, it was also suggested that not only the three regions but also the positional relationship with the target are important. If we only focus on predicting the three regions, it is considered that by adding probes Eco567(1) and Eco763(8) to region C, the structure of the three regions can be disrupted and an increase in relative Cy3 fluorescence intensity can be obtained. However, from the experimental results, when Eco645(2) and Eco745(7) were added, relative Cy3 fluorescence intensities of 25.6 times and 23.9 times were obtained respectively. These Eco645(2) and Eco745(7) are also close to the target in terms of positional relationship and play a role in disrupting the three-branched structure. Therefore, it was suggested that not only the three regions but also the presence or absence of probes that play a role in disrupting the three-branched structure with a close positional relationship to the target are important.

[0100] [Example 4: Examination of Combining Two Photo-Crosslinkable Assist Probes] Regarding combination number 14 (Eco567(1), Eco603(4), Eco745(7)) and combination number 41 (Eco624(3), Eco650(5), Eco738(6)) in Example 2, we examined what kind of effects would occur by reducing one probe. For combination number 14 with a large increase in relative fluorescence intensity, among the three regions predicted to be necessary for disrupting the higher-order structure of the target, we examined which region's higher-order structure is important to disrupt and whether the hybridization efficiency with the target would be significantly improved if two regions were disrupted. Regarding combination number 41 with a small increase in relative fluorescence intensity, we examined whether an increase in relative Cy3 fluorescence intensity could be obtained when either one of the probes Eco650(5) and Eco738(6), which are complementary strands and have negative cooperativity, was removed.

[0101] The experiments and evaluations were conducted in the same manner as in Example 1.

[0102] [Study on reducing assist probes from combination number 14] An experiment was conducted to reduce one assist probe from combination number 14 in Example 2. A graph created by quantifying the results of observing the CLSM images in the same manner as in Example 1 is shown in Fig. 12. Fig. 12 is a graph of the relative fluorescence intensity of E. coli stained with Eco627 when using the described combinations of assist probes, with the case of adding only Eco627 as the reference.

[0103] [Study on reducing assist probes from combination number 41] An experiment was conducted to reduce one assist probe from combination number 41 in Example 2. A graph created by quantifying the results of observing the CLSM images in the same manner as in Example 1 is shown in Fig. 13. Fig. 13 is a graph of the relative fluorescence intensity of E. coli stained with Eco627 when using the described combinations of assist probes, with the case of adding only Eco627 as the reference.

[0104] [Evaluation of experiments combining two photocrosslinkable assist probes] Regarding combination number 14, a decrease in relative Cy3 fluorescence intensity was observed by reducing one assist probe. For each combination, they were Eco567(1)·Eco614(3): 12.9 times, Eco567(1)·Eco745(7): 18.1 times, and Eco745(7)·Eco603(4): 11.5 times, respectively. From this result, it was found that when the higher-order structure of one of the three regions could not be disrupted, the relative Cy3 fluorescence intensity decreased compared to the case of disrupting all three regions.

[0105] Regarding combination number 41, by reducing the number of assist probes by one type, an increase in relative Cy3 fluorescence intensity was obtained. When comparing the increase rates in combinations, it increased 18.6-fold when Eco650(5) was excluded, and 19.1-fold when Eco738(6) was excluded. On the other hand, when Eco614(3) was excluded, that is, in the case of the combination of Eco650(5) and Eco738(6), the increase was only 9.6-fold and not as significant. From these results, it was found that by excluding either one of the probes Eco650(5) and Eco738(6), which are complementary strands and exhibit negative cooperativity, an increase in relative Cy3 fluorescence intensity can be obtained.

[0106] From the results of examining the combination of two photocrosslinkable assist probes, it was found that in order to disrupt the complex higher-order structure of the target and detect the target by fluorescence, it is necessary to change the higher-order structures of all three regions. Also, from these results, it was found that among the A region, B region, and C region, the importance of the three regions in the two types of combinations is that the B region is the most important, followed by the C region, and then the A region.

Industrial Applicability

[0107] The present invention provides a means for detecting a base sequence at a site difficult to detect in higher-order structured RNA in cells. The present invention is an industrially useful invention.

Claims

【Claim 1】 A method for forming a duplex with a target base sequence in an RNA molecule and performing photocrosslinking on the photocrosslinkable artificial nucleic acid probe, comprising: a step of forming a duplex between the photocrosslinkable artificial nucleic acid assist probe and the RNA molecule and performing photocrosslinking simultaneously with or prior to the formation of the duplex between the photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule; the target base sequence in the RNA molecule is within a region of a partial duplex structure; at one of the two ends of the region of the partial duplex structure containing the target base sequence, a partial duplex structure continues beyond the target base sequence; the region of the partial duplex structure that continues beyond the target base sequence is defined as region A; at the other of the two ends of the region of the partial duplex structure containing the target base sequence, there is a multi-branched structure of the RNA; among the branches formed by the multi-branched structure of the RNA, for the two branches that are close in base sequence to the region of the partial duplex structure containing the target base sequence, one branch region is defined as region B and the other branch region is defined as region C; as the photocrosslinkable artificial nucleic acid assist probe, the following are included: a photocrosslinkable artificial nucleic acid assist probe A having a base sequence capable of forming a duplex with the base sequence in region A and performing photocrosslinking; a photocrosslinkable artificial nucleic acid assist probe B having a base sequence capable of forming a duplex with the base sequence in region B and performing photocrosslinking; and a photocrosslinkable artificial nucleic acid assist probe C having a base sequence capable of forming a duplex with the base sequence in region C and performing photocrosslinking; the photocrosslinkable artificial nucleic acid probe is a photocrosslinkable artificial nucleic acid probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond; the photocrosslinkable artificial nucleic acid assist probe is a method, which is a photocrosslinkable artificial nucleic acid assist probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond: Formula (I): However, in formula I, R11 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group having 2 to 7 carbon atoms, or a hydrogen atom; R12 and R13 are each independently a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group having 2 to 7 carbon atoms, or a hydrogen atom; R14 is a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, an alkylsulfanyl group having 1 to 3 carbon atoms, a nitro group, a fluorine atom, a methyl fluoride group, or a hydrogen atom; the group Y is a group represented by the following formula II: Formula II: However, in Formula II, R21 is a hydrogen atom or a methyl group. **Claim 2** A method for forming a duplex with a target base sequence in an RNA molecule and performing photocrosslinking on the photocrosslinkable artificial nucleic acid probe, the method comprising: a step of forming a duplex between the photocrosslinkable artificial nucleic acid assist probe and the RNA molecule and performing photocrosslinking simultaneously with or prior to the formation of the duplex between the photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule; the target base sequence in the RNA molecule is within a region of a partial duplex structure, there are RNA multi-branched structures at both ends of the region of the partial duplex structure containing the target base sequence, in one of the RNA multi-branched structures at both ends, among the branches formed by the RNA multi-branched structure, out of the two branches that are close in base sequence to the region of the partial duplex structure containing the target base sequence, one branch region is defined as the B region and the other branch region is defined as the C region; in the other RNA multi-branched structure at both ends, among the branches formed by the RNA multi-branched structure, out of the two branches that are close in base sequence to the region of the partial duplex structure containing the target base sequence, one branch region is defined as the B' region and the other branch region is defined as the C' region; as the photocrosslinkable artificial nucleic acid assist probe, the following: a photocrosslinkable artificial nucleic acid assist probe B having a base sequence capable of forming a duplex with the base sequence in the B region and performing photocrosslinking, a photocrosslinkable artificial nucleic acid assist probe C having a base sequence capable of forming a duplex with the base sequence in the C region and performing photocrosslinking, a photocrosslinkable artificial nucleic acid assist probe B' having a base sequence capable of forming a duplex with the base sequence in the B' region and performing photocrosslinking, and a photocrosslinkable artificial nucleic acid assist probe C' having a base sequence capable of forming a duplex with the base sequence in the C' region and performing photocrosslinking; the photocrosslinkable artificial nucleic acid probe is a photocrosslinkable artificial nucleic acid probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond, the photocrosslinkable artificial nucleic acid assist probe is a method wherein a photocrosslinkable artificial nucleic acid assist probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into the base sequence by a phosphodiester bond: Formula (I): However, in Formula I, R11 is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group having 2 to 7 carbon atoms, or a hydrogen atom. R12 and R13 are each independently a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom, R14 is a hydroxyl group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a methyl fluoride group, or a hydrogen atom, The group Y is a group represented by the following formula II: Formula II: However, in formula II, R21 is a hydrogen atom or a methyl group. **Claim 3** The method according to any one of claims 1 to 2, wherein the multi-branched structure of the RNA is a 3-branched structure of the RNA or a 4-branched structure of the RNA. **Claim 4** The method according to any one of claims 1 to 3, wherein the RNA molecule is an intracellular RNA molecule. **Claim 5** The method according to any one of claims 1 to 4, which is a method for detecting a target base sequence in an RNA molecule by forming a double strand with the target base sequence in the RNA molecule and photocrosslinking a photocrosslinkable artificial nucleic acid probe. **Claim 6** The method according to claim 5, wherein the photocrosslinkable artificial nucleic acid probe is a probe provided with a detection label. **Claim 7** The method according to claim 6, wherein the photocrosslinkable artificial nucleic acid probe is a probe provided with a beacon-type fluorescent label for the FISH method.

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