JP7704393B2 - Method for detecting base sequences at difficult-to-detect sites in intracellular higher-order structured RNA - Google Patents

Description

The present invention relates to a method for detecting a base sequence located in a difficult-to-detect site in intracellular higher-order structured RNA.

The technology to detect specific base sequences contained in intracellular RNA is one of the fundamental techniques in molecular biology. As well as being a fundamental technology, it is also a technology that can be directly used in applications such as genetic diagnosis and detection of microbial species. Fluorescence in situ hybridization (FISH) is known as one such detection technique.

In the FISH method, 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 base sequence, thereby selectively detecting the target base sequence.

On the other hand, it is known that RNA does not exist in a simple single-stranded state within cells, but forms complex higher-order structures. Therefore, when attempting to selectively detect a specific base sequence in RNA by forming a double strand with a detection probe, some base sequences are easier to detect and others are harder to detect, depending on how they are arranged in the complex higher-order structure of the RNA.

Non-Patent Document 1 reports that the ease of detection of base sequences at various sites in the complex higher-order structure of Escherichia coli 16S rRNA using a detection probe was evaluated into six levels, from Class I to Class VI.

Patent Document 1 discloses a photoresponsive nucleotide analogue having photocrosslinking ability, and discloses that this photoresponsive nucleotide analogue can be used as a photoreactive crosslinking agent.

International Publication No. WO2014/157565

BERNHARD MAXIMILIAN FUCHS, et al. , “Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled "Oligonucleotide Probes", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1998, p. 4973-4982, Vol. 64, No. 12

If there was a way to easily detect the base sequence corresponding to Class VI, which was deemed the most difficult to detect in the complex higher-order structure of E. coli 16S rRNA in Non-Patent Document 1, it would be a major step forward in basic molecular biology technology.

Therefore, the object of the present invention is to provide a means for detecting base sequences in difficult-to-detect sites in intracellular higher-order structured RNA.

The inventors discovered that the above objectives could be achieved by the means described below, and arrived at the present invention.

Therefore, the present invention includes the following (1) and the following:
(1)
A method for photocrosslinking a photocrosslinkable artificial nucleic acid probe with a target base sequence in an RNA molecule by forming a double strand, comprising the steps of:
The method includes a step of forming a duplex between a photo-crosslinkable artificial nucleic acid assist probe and an RNA molecule and photocrosslinking the probe, either simultaneously with or prior to the formation of a duplex between the photo-crosslinkable artificial nucleic acid probe and a target base sequence in the RNA molecule.
(2)
a target base sequence in the RNA molecule is within a region of partial duplex structure;
a partial double-stranded structure continues beyond the target base sequence at one of both ends of the region of the partial double-stranded structure containing the target base sequence,
A region of a partial double-stranded structure extending beyond the target base sequence is designated as region A,
a multi-branched structure of RNA is present at the other of both ends of the region of the partial double-stranded structure containing a target base sequence,
Among the branches due to the multibranched structure of the RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing a target base sequence, the region of one branch is designated as region B and the region of the other branch is designated as region C;
As the photocrosslinkable artificial nucleic acid assist probe, the following:
a photocrosslinkable artificial nucleic acid assist probe A having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the A region;
The method according to (1), comprising: a photo-crosslinkable artificial nucleic acid assisted probe B having a base sequence capable of photo-crosslinking by forming a double strand with the base sequence in the B region; and a photo-crosslinkable artificial nucleic acid assisted probe C having a base sequence capable of photo-crosslinking by forming a double strand with the base sequence in the C region.
(3)
a target base sequence in the RNA molecule is within a region of partial duplex structure;
a multi-branched RNA structure is present at both ends of the region of the partial double-stranded structure containing a target base sequence,
In one of the multibranched structures of RNA at both ends, among the branches due to the multibranched structure of RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing the target base sequence, the region of one branch is designated as region B and the region of the other branch is designated as region C;
In the other of the multibranched structures of RNA at both ends, among the branches due to the multibranched structure of RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing the target base sequence, the region of one branch is designated as region B' and the region of the other branch is designated as region C';
As the photocrosslinkable artificial nucleic acid assist probe, the following:
a photocrosslinkable artificial nucleic acid assist probe B having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the B region;
a photocrosslinkable artificial nucleic acid assist probe C having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the C region;
a photocrosslinkable artificial nucleic acid assist probe B' having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the B' region; and
The method according to (1), comprising a photocrosslinkable artificial nucleic acid assist probe C' having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the C' region.
(4)
The method according to any one of (2) to (3), wherein the RNA multibranched structure is an RNA tri-branched structure or an RNA tetra-branched structure.
(5)
The method according to any one of (1) to (4), wherein the RNA molecule is an intracellular RNA molecule.
(6)
The method according to any one of (1) to (5), which is a method for detecting a target base sequence in an RNA molecule by forming a double strand with a photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule, thereby photocrosslinking the double strand.
(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 FISH method.
(9)
A photocrosslinkable artificial nucleic acid probe,
A photocrosslinkable artificial nucleic acid probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond,
A photocrosslinkable artificial nucleic acid assist probe,
The method according to any one of (1) to (8), which is a photocrosslinkable artificial nucleic acid assisted probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond:

Formula (I):
However, in formula I,
R11 is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or a hydrogen atom;
R12 and R13 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to 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 of formula II or III:

Formula II:
However, in formula II,
R21 is a hydrogen atom or a C1-C3 alkyl group;

Formula III:
However, in formula III,
R31 is a hydrogen atom or a hydroxyl group.

The present invention makes it possible to detect base sequences in difficult-to-detect sites in intracellular higher-order structured RNA.

FIG. 1 is a partially enlarged view of the overall structure of the prediction model of the higher-order structure of Escherichia coli 16S rRNA disclosed in Non-Patent Document 1. Figure 2A is a CLSM image of E. coli stained with Eco627 illuminated at 385 nm for 0 seconds. Figure 2B is a CLSM image of E. coli stained with Eco627 illuminated at 385 nm for 360 seconds. Figure 2C is a CLSM image of E. coli stained with Eco627 and Eco567(1)-763(8) exposed to light at 385 nm for 0 seconds. Figure 2D is a CLSM image of E. coli stained with Eco627 and Eco567(1)-763(8) illuminated at 385 nm for 360 s. 3A is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 exposed to light at 385 nm for 0 or 360 seconds. Figure 3B is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 and Eco567(1)-763(8) exposed to light at 385 nm for 0 or 360 seconds. FIG. 4 is an explanatory diagram showing the arrangement of a double-stranded region (region A) located to the left of the double-stranded region that is the target region, a double-stranded region (region B) located above the tri-branched structure, and a double-stranded region (region C) located below the tri-branched structure. FIG. 5A is a graph of relative fluorescence intensity based on the values in Table 3. FIG. 5B is a graph of relative fluorescence intensity based on the values in Table 3. FIG. 6 is a graph prepared by quantifying the results of observation of the CLSM images in the same manner as in Example 1. FIG. 7 is an explanatory diagram showing the arrangement of regions to be hybridized by each assist probe for combination number 14 (a combination of Eco567(1), Eco603(4), and Eco745(7)). 8 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combination of the assist probes described above was used, based on the fluorescence intensity when only Eco627 was added. FIG. 9 is an explanatory diagram showing the arrangement of regions to be hybridized by each assist probe for combination number 41 (Eco624(3), Eco650(5), Eco738(6)). 10 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combination of the assist probes described above was used, based on the fluorescence intensity when only Eco627 was added. 11 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combination of assist probes described above was used, based on the fluorescence intensity when only Eco627 was added. 12 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combination of the assist probes described above was used, based on the fluorescence intensity when only Eco627 was added. 13 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combination of the assist probes described above was used, based on the fluorescence intensity when only Eco627 was added.

The present invention will be described in detail below with reference to specific embodiments. The present invention is not limited to the specific embodiments described below.

[Method of photocrosslinking by forming a double strand with a photocrosslinkable artificial nucleic acid probe and a target base sequence in an RNA molecule]
According to the present invention, a method including a step of forming a duplex with a photo-crosslinkable artificial nucleic acid assist probe and an RNA molecule to photocrosslink the probe, either simultaneously with or prior to the formation of a duplex between the photo-crosslinkable artificial nucleic acid probe and a target base sequence in an RNA molecule, enables the photo-crosslinkable artificial nucleic acid probe to form a duplex with a target base sequence in an RNA molecule and be photocrosslinked.

[Detection of RNA structure having a multi-branched structure at one end of a target base sequence]
In a preferred embodiment, the target sequence in the RNA molecule is within a region of partial duplex structure,
a partial double-stranded structure continues beyond the target base sequence at one of both ends of the region of the partial double-stranded structure containing the target base sequence,
A region of a partial double-stranded structure extending beyond the target base sequence is designated as region A,
a multi-branched structure of RNA is present at the other of both ends of the region of the partial double-stranded structure containing a target base sequence,
Among the branches due to the multibranched structure of the RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing a target base sequence, the region of one branch is designated as region B and the region of the other branch is designated as region C;
As the photocrosslinkable artificial nucleic acid assist probe, the following:
a photocrosslinkable artificial nucleic acid assist probe A having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the A region;
The photo-crosslinkable artificial nucleic acid assist probe B has a base sequence capable of photo-crosslinking by forming a double strand with the base sequence in the B region, and the photo-crosslinkable artificial nucleic acid assist probe C has a base sequence capable of photo-crosslinking by forming a double strand with the base sequence in the C region.

[Detection of RNA structure having multiple branched structures at both ends of target base sequence]
In a preferred embodiment, the target sequence in the RNA molecule is within a region of partial duplex structure,
a multi-branched RNA structure is present at both ends of the region of the partial double-stranded structure containing a target base sequence,
In one of the multibranched structures of RNA at both ends, among the branches due to the multibranched structure of RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing the target base sequence, the region of one branch is designated as region B and the region of the other branch is designated as region C;
In the other of the multibranched structures of RNA at both ends, among the branches due to the multibranched structure of RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing the target base sequence, the region of one branch is designated as region B' and the region of the other branch is designated as region C';
As the photocrosslinkable artificial nucleic acid assist probe, the following:
a photocrosslinkable artificial nucleic acid assist probe B having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the B region;
a photocrosslinkable artificial nucleic acid assist probe C having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the C region;
a photocrosslinkable artificial nucleic acid assist probe B' having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the B' region; and
The photocrosslinkable artificial nucleic acid assist probe C' may include a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the C' region.

[Detection of target base sequence using photocrosslinkable artificial nucleic acid probe]
The present inventor has been conducting extensive research into nucleic acid double-strand formation and photocrosslinking technology. When an artificial nucleic acid produced by introducing an artificial nucleotide analogue having a photocrosslinkable artificial base in the same manner as a natural nucleotide is used as a probe, a double strand is formed depending on the base sequence, and a specific target base sequence can be detected by photocrosslinking. The double strand formation by this photocrosslinkable artificial nucleic acid probe becomes irreversible when a photocrosslink is formed, so that it is a very favorable reaction in terms of energy, and the detection sensitivity is high.

In addition, with a photocrosslinkable artificial nucleic acid probe, once double strand formation and photocrosslink formation are achieved, the probe can be easily detected by the detection label that has been attached in advance, so the degree of photocrosslink formation and the resulting detection sensitivity have essentially the same significance.

From this perspective, the present inventors predicted that by using a photocrosslinkable artificial nucleic acid probe, double strand formation and photocrosslinking would enable highly sensitive detection of even target base sequences in RNA molecules with complex higher-order structures. However, even with such a photocrosslinkable artificial nucleic acid probe, it was not possible to obtain sufficiently high sensitivity for a base sequence corresponding to Class VI, which was deemed the most difficult to detect in the complex higher-order structure of Escherichia coli 16S rRNA in Non-Patent Document 1.

The inventors therefore analyzed the structure of difficult-to-detect sites in such complex, higher-order RNA structures and discovered a tendency for structures to have multi-branched structures near the target base sequence. A multi-branched structure is, for example, a structure in which RNA forms a double-stranded structure within the molecule, and the double-stranded structure branches into multiple branches from a certain position, like a fork in a road. Examples of multi-branched structures include a three-branched structure and a four-branched structure.

The inventor then came up with the idea that this multi-branched structure may act as some kind of constraint, suppressing the formation of a double strand and photocrosslinking between the target base sequence and the photocrosslinkable artificial nucleic acid probe. Based on this, the inventor discovered that by adding a photocrosslinkable artificial nucleic acid assist probe that assists in the formation of a double strand and photocrosslinking between the photocrosslinkable artificial nucleic acid probe and the target base sequence, and by performing the formation of a double strand and photocrosslinking between the photocrosslinkable artificial nucleic acid assist probe and the corresponding base sequence, it is possible to detect with sufficiently high sensitivity even target base sequences located at difficult-to-detect sites in RNA with a complex higher-order structure, and thus arrived at the present invention.

According to the inventors' research, this photocrosslinkable artificial nucleic acid assist probe is required to have a specific positional relationship with respect to the target base sequence.

Specifically, when there is a multi-branched structure near one end of a double-stranded structure portion containing a target base sequence, it is preferable to use two photo-crosslinkable artificial nucleic acid assisted probes in which, among the multiple branches of the multi-branched structure, two branches close to the target base sequence have a base sequence capable of forming a double strand. At the same time, a double-stranded structure continues at the other end of the double-stranded structure portion containing the target base sequence, so it is preferable to use one photo-crosslinkable artificial nucleic acid assisted probe having a base sequence capable of forming a double strand for the base sequence in this double-stranded structure simultaneously with the two photo-crosslinkable artificial nucleic acid assisted probes.

More specifically, when there are multi-branched structures at both ends of the double-stranded structure portion containing the target base sequence, it is preferable to use, for each multi-branched structure, two photo-crosslinkable artificial nucleic acid assisted probes in which, among the multiple branches of the multi-branched structure, the two branches closest to the target base sequence each have a base sequence capable of forming a double strand.

[Position of photocrosslinkable artificial nucleic acid probe in higher-order structure RNA]
In the 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 strand forming portion of, for example, 15 to 20 bases, or 20 to 25 bases, among the above-mentioned base lengths. In a preferred embodiment, the base at the end close to the center of the multibranch of the target base sequence is in a distance range of, for example, the 20th to 30th bases, or the 30th to 40th bases, from the center of the branch of the multibranch structure nearby. The number of bases indicating the distance from the center of the branch of the multibranch structure is counted by counting the first base pair formed between the two strands branched from the multibranch structure as the first base. For example, if a base sequence that does not form a base pair continues between the two strands immediately after branching from the multibranch structure, the first base pair forming base that appears after that base sequence is the first base.

[Position of Photocrosslinkable Artificial Nucleic Acid Assist Probe]
In the higher-order structure RNA, the photo-crosslinkable artificial nucleic acid assist probe forms a double strand, and the position of the target base sequence to be photo-crosslinked is located in any of the above-mentioned regions A, B, and C, or any of the above-mentioned regions B, C, B', and C'.

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

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 portion of, for example, 15 to 20 bases, or 20 to 25 bases, among the above base lengths. In a preferred embodiment, the end base close to the center of the multi-branched structure of the target base sequence located in region B is located within a distance of, for example, the 20th to 30th base, or the 30th to 40th base, from the center of the branch of the nearby multi-branched structure. The target base sequence located in region B may include a non-double-stranded structure portion therein. In a preferred embodiment, the target base sequence located in region B' can also be provided in the same manner as the target base sequence located in region B.

In a preferred embodiment, the base length of the target base sequence located in the C 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 C region includes a double-stranded 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 close to the center of the multi-branched structure of the target base sequence located in the C region is within a distance of, for example, the 20th to 30th base, or the 30th to 40th base, from the center of the branch of the nearby multi-branched structure. The target base sequence located in the C region may include a non-double-stranded structure portion therein. In a preferred embodiment, the target base sequence located in the C' region can also be provided in the same manner as the target base sequence located in the B region.

[Photocrosslinkable artificial nucleosides]
In a preferred embodiment, the photo-crosslinkable artificial nucleic acid probe may be a photo-crosslinkable artificial nucleic acid probe in which a photo-crosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond. In a preferred embodiment, the photo-crosslinkable artificial nucleic acid assisted probe may be a photo-crosslinkable artificial nucleic acid assisted probe in which a photo-crosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond.

Formula (I):

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 a cyano group or a hydrogen atom.

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 a cyano group or a hydrogen atom.

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 is preferably a nitro group or a hydrogen atom.

In a preferred embodiment, group Y can be a group represented by the following formula II or III:

Formula II:

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

Formula III:

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

[Photocrosslinking formation]
Both the photocrosslinkable artificial nucleic acid probe and the photocrosslinkable artificial nucleic acid assist probe can form a photocrosslink by irradiating light after forming a double strand. This photocrosslink is formed by a photochemical reaction 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 pyrimidine bases include thymine (T), cytosine (C), uracil (U), 5-methylcytosine, and 5-hydroxymethylcytosine, and in higher-order structure RNA, cytosine (C) and uracil (U) are preferable.

The photocrosslinkable artificial base of the photocrosslinkable artificial nucleoside can form a photocrosslink with a base located one base on the 5' end side from the base in a position complementary to the photocrosslinkable artificial base (the base next to the 5' end side of the base in the complementary position) in the base sequence of a nucleic acid having a complementary base sequence. In other words, the position one base on the 5' end side from the base in a position complementary to the photocrosslinkable artificial base in the complementary base sequence is a position capable of photocrosslinking.

Because the formation of photocrosslinks by irradiation with light is a photochemical reaction, it can be carried out under a wide range of conditions, including temperature, solvent, salt concentration, and pH. Therefore, it can be carried out under physiological conditions and can be suitably used in intracellular conditions. Photocrosslinks can be formed under the same conditions as double-strand formation.

In a preferred embodiment, the light irradiation can be performed by irradiation with light having a wavelength in the range of 340 nm to 390 nm, 360 nm to 390 nm, for example, light having a wavelength of 385 nm. The light irradiation can be performed for an irradiation time in the range of 0.1 seconds to 60 seconds, 1 second to 30 seconds, or 5 seconds to 15 seconds. The light irradiation can be performed at a temperature in the range of 0°C to 40°C, 0°C to 30°C, 0°C to 20°C, or 0°C to 10°C, for example.

[Labeling of photocrosslinkable artificial nucleic acid probe]
In a preferred embodiment, the double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid probe can be performed for the detection of a target base sequence in an RNA molecule. In this case, the photocrosslinkable artificial nucleic acid probe can be provided with a detection label. As such a detection label, a known detection label can be used. As such a detection label, for example, FITC, FAM, TAMRA, Rhodamine, Cy3, Cy5, preferably Cy3 and Cy5 can be mentioned. 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 is composed of a set of a fluorescent group and a quenching group, and is configured such that the quenching group is isolated from the fluorescent group by the probe forming a double strand with the target base sequence, the quenching action of the quenching group does not work, and the fluorescence from the fluorescent group can be observed.

[Addition of photocrosslinkable artificial nucleic acid assist probe]
In a preferred embodiment, as described above, the double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid assist probe promotes the double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid probe. The double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid assist probe may be performed prior to the double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid probe, or may be performed simultaneously with the double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid probe. Therefore, the photocrosslinkable artificial nucleic acid assist probe may be added to the sample to be examined prior to the addition of the photocrosslinkable artificial nucleic acid probe, may be added simultaneously with the addition of the photocrosslinkable artificial nucleic acid probe, or may be added after the addition of the photocrosslinkable artificial nucleic acid probe.

In a preferred embodiment, the photocrosslinkable artificial nucleic acid assist probe is intended to promote double strand formation and photocrosslinking by the photocrosslinkable artificial nucleic acid probe, so typically, the molecules of each photocrosslinkable artificial nucleic acid assist probe can be added in approximately the same molar amount as the molecules of the photocrosslinkable artificial nucleic acid probe, but this is not limited thereto. In a preferred embodiment, the number of moles of each photocrosslinkable artificial nucleic acid assist probe may be added in a ratio of, for example, 1/10 to 10/1, or 1/1.5 to 1.5/1, relative to the number of moles of the molecules of the photocrosslinkable artificial nucleic acid probe.

In a preferred embodiment, as described above, it is preferable to simultaneously use three types of probe molecules as a set, namely, a photo-crosslinkable artificial nucleic acid assist probe that forms a double strand and photo-crosslinks with a target base sequence located in the A region, a photo-crosslinkable artificial nucleic acid assist probe that forms a double strand and photo-crosslinks with a target base sequence located in the B region, and a photo-crosslinkable artificial nucleic acid assist probe that forms a double strand and photo-crosslinks with a target base sequence located in the C region. By using these three types of photo-crosslinkable artificial nucleic acid assist probe molecules, the promotion effect of the photo-crosslinkable artificial nucleic acid probe in double strand formation and photo-crosslinking is preferably exhibited. In a preferred embodiment, it is also within the scope of the present invention to add another type of photo-crosslinkable artificial nucleic acid assist probe molecule within a range that does not interfere with the promotion effect of these three types of photo-crosslinkable artificial nucleic acid assist probe molecules.

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

[Example 1: Examination of the assist effect of a photocrosslinkable assist probe on detection using a photocrosslinkable molecular beacon 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 were 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 in the stem region with the quenching group Dabcyl at the 3' end and in the 5' end with the fluorescent group Cy3, and 3-cyanovinylcarbazole ( ^CNV D) was added to the loop region. Both the photocrosslinkable molecular beacon type probe and the photocrosslinkable assist probe were S-modified ODN. 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.

The relative fluorescence intensities shown in Table 1 are the values given in Non-Patent Document 1 (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1998, p. 4973-4982, Vol. 64, No. 12).

Non-Patent Document 1 discloses that, for Escherichia coli 16S rRNA, RNA with a higher-order structure is divided into multiple regions, complementary probes are created for the regions, and hybridization of the probe to the corresponding region of the RNA is detected by fluorescence emission from a fluorescent group that has been pre-bound to the probe, and the ease of hybridization in each region is quantified and compared based on the fluorescence intensity.

In the above Table 1, the leftmost column lists the names of the probes used in this example, and the sequences of the probes are as shown in the center column of Table 1. The names of the probes used in this example are modeled after the names of the probes described in Non-Patent Document 1 as recognizing the same region, and the relative fluorescence intensity (Relative Probe Fluorescence) of each probe used in Non-Patent Document 1 is listed in the rightmost column of the above Table 1.

This relative fluorescence intensity is a relative value with the fluorescence intensity shown by the probe that recognizes the Eco1482 region in Non-Patent Document 1 taken as 100%, and the larger the value, the more likely the region recognized by each probe is to hybridize in the higher-order structure RNA, i.e., the easier it is to detect.

As shown in Table 1 above, the region corresponding to the Eco627 probe, according to the disclosure of Non-Patent Document 1, exhibits only about 1% of the fluorescence intensity of the Eco1482 region, making it extremely difficult to detect. Note that next to the relative fluorescence intensity values in Table 1, the classifications Class II, Class III, Class IV, Class V, and Class VI are listed, but in Non-Patent Document 1, these are indicators showing the difficulty of detecting each region according to the value of relative fluorescence intensity, and Class VI means that it is classified as the most difficult region to detect in higher-order structure RNA.

D described in the sequences in 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 a serinol (2-amino-1,3-propanediol) structure as the sugar backbone moiety in the nucleoside. ^CNV D can be introduced into an oligonucleotide sequence in the same manner as natural nucleosides.

[Preparation of observation sample]
[Preparation of Escherichia coli glycerol stock]
In the examples, Escherichia coli (One Shot (registered trademark) TOP10 Chemically Competent E. coli) was used. E. coli cultured in LB liquid medium heated to 37°C was harvested in the logarithmic growth phase. Glycerol stocks (final concentration 20%) of the cultured E. coli competent cells were prepared and stored at -80°C.

[Buffer preparation]
A 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.

The 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.

[Preparation of observation sample]
Each probe was diluted with Hybridization buffer and mixed with competent cell solution to a final concentration of 1.0 μM. This mixed solution was left on ice for 15 min, then heat-shocked at 42 ° C for 40 sec, left on ice for 3 min, then incubated at 37 ° C for 4.0 h, and hybridized. Light irradiation was performed at 4.0 ° C (wavelength: 385 nm / irradiation time: 360 sec). The sample solution (5.0 μL) and Embedding buffer (8.0 μL) after light irradiation were mixed on a slide glass, and the mixture was naturally dried at rt for 10 min, and then a cover glass was placed on it.

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

[Positional information of each probe and target]
In the examples, Eco627, which has a complex higher-order structure and is the region with the lowest probe affinity, was targeted, and the effect of the combination of a photoresponsive artificial nucleic acid ( ^CNV D)-containing probe (Table 2.1) on the increase in relative Cy3 fluorescence intensity was examined with ^{a CNV} 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.

Figure 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 Figure 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 Figure 1. Similarly, the region to which each probe shown in Table 1 hybridizes is shown in Figure 1 by its respective 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.

[Observation results and evaluation]
The effect of the combination of photoresponsive artificial nucleic acid ( ^CNV D)-containing probes on the increase in relative Cy3 fluorescence intensity was examined for ^{the CNV} D-containing beacon probe. First, the effect of the increase in relative Cy3 fluorescence intensity was examined when only the beacon probe Eco627 was added, and when the beacon probe Eco627 and eight normal probes that play a role in breaking down the higher-order structure were added. The relative Cy3 fluorescence intensity was calculated by quantifying the brightness of the image taken by 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 the Eco1437 series.

The results of the observations are shown in Figures 2A to 2D. Figures 2A to 2D show CLSM images (confocal laser microscope images) of E. coli. Figures 2A and 2B are CLSM images of E. coli stained with Eco627 irradiated with light at 385 nm for 0 seconds (Figure 2A) or 360 seconds (Figure 2B). Figures 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 (Figure 2C) or 360 seconds (Figure 2D). The scale bar is 20 μm.

As can be seen from the CLSM images in Figures 2A to 2D, the FISH results showed that Cy3 fluorescence was aggregated in the area defined as a cell in the bright field image (PC), suggesting that the probe was taken up into the cell. In addition, when the beacon probe Eco627 and eight types of normal probes that play a role in disrupting higher-order structures were added, an increase in relative Cy3 fluorescence intensity was confirmed by light irradiation, suggesting that the probe bound to the target RNA. Fluorescence intensity was quantified by quantifying the brightness of the Cy3 fluorescence image obtained by confocal microscopy using Image J and analyzing it using Excel. Graphs created by quantification are shown in Figures 3A to 3B.

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

In Figures 3A and 3B, the relative fluorescence intensity on the vertical axis of the graph is shown as a relative value, with the light irradiation time of 0 seconds when only the photocrosslinkable molecular beacon probe Eco627 was added being set as "1." This is to make it easy to understand the extent to which the detection of the complementary region by hybridization of the photocrosslinkable molecular beacon probe Eco627 is made more sensitive by photocrosslinking, and furthermore, the extent to which it is made more sensitive by the photocrosslinkable assist probe.

As shown in Figure 3A, when only the photocrosslinkable molecular beacon probe Eco627 was added, the relative Cy3 fluorescence intensity after 360 seconds of light irradiation increased to 7 times compared to 0 seconds of light irradiation. Although the 7-fold increase in fluorescence intensity is a large increase, it is smaller than expected as a high sensitivity due to photocrosslinking. This means that the region targeted for detection by the photocrosslinkable molecular beacon probe Eco627 has a complex higher-order structure and is difficult to detect. In other words, it was confirmed that this is an extremely difficult region to detect, as evaluated as Class VI, which is considered to be the most difficult to detect in Non-Patent Document 1.

In contrast, as shown in FIG. 3B, when the photocrosslinkable molecular beacon probe Eco627 and eight kinds of photocrosslinkable assist probes (Eco567(1) to Eco763(8) shown in Table 1) were added, the relative Cy3 fluorescence intensity after 360 seconds of light irradiation increased to 39 times compared to 0 seconds after light irradiation with only the photocrosslinkable molecular beacon probe Eco627. This shows that by combining the photocrosslinkable molecular beacon probe Eco627 and the photocrosslinkable assist probe, it is possible to achieve surprisingly high sensitivity and make it possible to detect areas that have complex higher-order structures and are difficult to detect. The reason for this is unclear, but the inventor believes that the photocrosslinkable assist probe changes the higher-order structure near the target area of the photocrosslinkable molecular beacon probe Eco627, creating a state in which photocrosslinking of the photocrosslinkable molecular beacon probe Eco627 can be formed.

[Example 2: Examination of combination of photocrosslinkable assist probes and assist effect]
[Region in higher order structured 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 probe Eco 627. Therefore, in order to efficiently promote this change in higher-order structure, the combination of photocrosslinkable assist probes and the assist effect were examined.

The higher-order structure RNA shown in Figure 1 is said to have a complex higher-order structure that cannot actually be illustrated, but the inventors have modeled the essence of the structure near the target region of the photocrosslinkable molecular beacon probe Eco627 of the higher-order structure RNA shown in Figure 1 into the following structure. That is, in Figure 1, there is a double-stranded region that is the target region of Eco627, a double-stranded region (A region) located to the left of the double-stranded region that is the target region, a three-branched structure located to the right of the target region, a double-stranded region (target region) in the left branch of the three-branched structure, a double-stranded region (B region) located above the three-branched structure, and a double-stranded region (C region) located below the three-branched structure. The arrangement of the A region, B region, and C region is shown in the following Figure 4.

Based on the positional information of the complementary regions of each assist probe, the inventor 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 would facilitate hybridization and photocrosslinking in the target region, and conducted further investigations.

[Assist probe combination]
From the assist probes shown in Table 1, combinations of three probes were extracted, and the assist effect was 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 description of the combination of Eco567(1) and Eco645(2) in Table 1, "3" in the first row of the second column from the left end of Table 2 means Eco614(3) in Table 1, and "1" in the second row of the second column from the left end of Table 2 means that the combination of Eco567(1), Eco645(2), and Eco614(3) in Table 1 is set to combination number "1".

[Experiments, Observations and Evaluations]
In Example 1, a comparative experiment was carried out between the case where only the photocrosslinkable molecular beacon probe Eco627 was added and the case where both the photocrosslinkable molecular beacon probe Eco627 and all eight kinds of photocrosslinkable assist probes were added, whereas in this Example 2, an experiment was carried out between the case where only the photocrosslinkable molecular beacon probe Eco627 was added and the case where any combination of the photocrosslinkable assist probes with combination numbers 1 to 56 was added together with the photocrosslinkable molecular beacon probe Eco627. Except for the addition of the combination, the experiment, observation and evaluation were carried out in the same manner as in Example 1. The results are shown in Table 3 below.

Table 3 shows the combinations of each probe and the relative Cy3 fluorescence intensity detected by the combination. For example, the combination described as Combination 1 (1.2.3) is combination number 1, which indicates the combination of the photocrosslinkable assist probes (1), (2), and (3) described in Table 1, and indicates that the relative fluorescence intensity obtained was 21.0.

Graphs of relative fluorescence intensity based on the values in Table 3 are shown in Figures 5A and 5B.

Table 3, Figures 5A and 5B show the results of 56 combinations of probes. Among the 56 combinations, the combinations that produced the largest increase in relative Cy3 fluorescence intensity are as follows:

Among these combinations, the combination that gave the highest relative Cy3 fluorescence intensity was a combination that had one photocrosslinkable assist probe having a complementary region for each of the three regions A, B, and C described above. In the combination that gave the highest relative Cy3 fluorescence intensity, a 35.5-fold increase in relative Cy3 fluorescence intensity was observed. This 35.5-fold increase was the same level of relative Cy3 fluorescence intensity as when eight types of probes were added. This result confirmed the hypothesis that hybridization and photocrosslinking by the assist probe to the above three regions brings about a change in the higher-order structure that facilitates hybridization and photocrosslinking in the target region. In other words, it became clear that disrupting the higher-order structure of the three regions is a necessary condition for fluorescent detection of the target.

Furthermore, when the results were examined in more detail, it was found that combination number 14 (a combination of Eco567(1), Eco603(4), and Eco745(7)), which produced the greatest increase in relative Cy3 fluorescence intensity, is thought to be caused by Eco603(4) disrupting the structure of region A, Eco745(7) disrupting region B, and Eco567(1) disrupting region C. In particular, Eco745(7) was predicted to disrupt the three-branched structure according to the inventor's hypothesis, and the above experimental results made it clear that it is in fact an important probe.

[Extraction and detailed evaluation of combinations with large increases in relative fluorescence intensity]
Combinations that showed a large increase in relative fluorescence intensity were selected, and experiments, observations, evaluations, and studies were performed in the same manner as in Example 1. The results of the observation of CLSM images were quantified in the same manner as in Example 1, and a graph is shown in Figure 6. Figure 6 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combinations of assist probes described above were used, with the result of adding only Eco627 as the reference.

Figure 6 shows that combination number 14 (a combination of Eco567(1), Eco603(4), and Eco745(7)) showed the greatest increase in relative fluorescence intensity. Figure 7 shows the arrangement of the regions to which each assist probe hybridizes for combination number 14 (a combination of Eco567(1), Eco603(4), and Eco745(7)).

[Selection and detailed evaluation of small combinations of increases in relative fluorescence intensity]
Combinations with small increases in relative fluorescence intensity were selected and experiments, observations, evaluations, and studies were performed in the same manner as in Example 1. The results of the observation of CLSM images were quantified in the same manner as in Example 1, and a graph is shown in Figure 8. Figure 8 is a graph of the relative fluorescence intensity of E. coli stained with Eco627 when the combinations of assist probes described above were used, with the result of adding only Eco627 as the reference.

As shown in Figure 8, the lowest relative fluorescence intensity was 5.1 times for combination 41 (3.5.6). The relatively high intensity was 9.4 times for combination 16 (1.5.6).

What these combinations have in common is that they contain two assist probes for each of the three regions A, B, and C mentioned above. Taking combination 41 (Eco624(3), Eco650(5), Eco738(6)), which had the lowest increase rate, as an example, it contains two assist probes for region B, Eco650(5) and Eco738(6). For Eco650(5) and Eco738(6), the region that is assisted by hybridization is the same, while the assist effect is almost nonexistent for region C, so it is thought that the overall efficiency of assist is lower than other combinations. In addition, since Eco650(5) and Eco738(6) are almost complementary strands, it is thought that a complementary strand-forming interaction occurs between Eco650(5) and Eco738(6), resulting in a decrease in the proportion of Eco650(5) and Eco738(6) that effectively bind to the RNA structure. It is thought that the increase rate of the relative Cy3 fluorescence intensity is low for the same reason, not only for complementary strands, but also for combinations in which the assist probes overlap by several bases.

Figure 9 shows the arrangement of the regions to be hybridized by each assist probe for combination number 41 (Eco624 (3), Eco650 (5), Eco738 (6)).

[Example 3: Study on the combination of four photocrosslinkable assist probes]
[Select combination number for adding a fourth assist probe]
In Example 2, a combination of three assist probes was examined in examining combinations of photo-crosslinkable assist probes. In Example 3, a combination of four photo-crosslinkable assist probes was examined as follows.

In the study, we first selected combination number 14 (a combination of Eco567 (1), Eco603 (4), and Eco745 (7)), which showed the greatest increase in relative Cy3 fluorescence intensity in the study of Example 2, and combination 41 (Eco624 (3), Eco650 (5), and Eco738 (6)), which showed the smallest increase in relative Cy3 fluorescence intensity. Then, based on combination numbers 14 and 41, we further examined what effect would be caused by adding one assist probe.

Regarding combination number 14 in Example 2, the assist probe has already been added to each of the A, B, and C regions, and it is believed that the RNA higher-order structure has been disrupted to the extent that is thought to be necessary for Eco627 to hybridize and photocrosslink to the target region. If an assist probe were further added in this state, it was thought that the relative Cy3 fluorescence intensity might actually decrease due to interactions between the assist probes, and therefore this point was investigated.

Combination number 41 in Example 2 contained one assist probe for region A (Eco614 (3)) and two assist probes for region B (Eco650 (5), Eco738 (6)). It was thought that in combination number 41 in Example 2, the negative cooperativity between these two assist probes for region B prevented an increase in relative Cy3 fluorescence intensity. Therefore, it was examined whether the addition of an assist probe for region C to combination number 41 in Example 2 would result in an increase in relative Cy3 fluorescence intensity due to positive cooperativity between the probes. Furthermore, it was examined whether the addition of assist probes for regions A and B would further reduce the relative Cy3 fluorescence intensity due to negative cooperativity.

The experiments and evaluation were carried out in the same manner as in Example 1.

[Study on 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. The results of the observation of CLSM images were quantified in the same manner as in Example 1, and a graph is shown in Figure 10. Figure 10 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the respective combinations of assist probes described above were used, with the fluorescence intensity when only Eco627 was added being taken as the reference.

[Study on 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. The results of the observation of CLSM images were quantified in the same manner as in Example 1, and a graph is shown in Figure 11. Figure 11 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the respective combinations of assist probes described above were used, with the fluorescence intensity when only Eco627 was added being taken as the reference.

[Evaluation of experiments combining four photocrosslinkable assist probes]
For combination 14 (Eco567(1), Eco603(4), Eco745(7)), the addition of the probes reduced the relative Cy3 fluorescence intensity due to negative cooperativity between the probes. When Eco645(2) was added, the intensity was 27.6 times (22.2% reduction), whereas when Eco614(3) was added, the intensity was 16.5 times (53.5% reduction), and when Eco763(8) was added, the intensity was 17.8 times (49.9% reduction). The reason for this reduction rate is that Eco645(2) is partially complementary to Eco745(7), but is close to the target in position and is a probe that plays a role in disrupting the T-shaped structure, so it is thought that there was not much reduction in the relative Cy3 fluorescence intensity due to negative cooperativity.

For combination 41 (Eco624 (3), Eco650 (5), Eco738 (6)), the addition of probes positively promoted the cooperativity of each probe, resulting in an increase in the relative Cy3 fluorescence intensity. For this combination as well, the results suggest that not only the three regions but also the positional relationship with the target are important. If we were to make a prediction focusing only on the three regions, it would be thought that the structure of the three regions could be disrupted by adding the probes Eco567 (1) and Eco763 (8) for the C region, resulting in an increase in the relative Cy3 fluorescence intensity. However, the experimental results showed that when Eco645 (2) and Eco745 (7) were added, the relative Cy3 fluorescence intensity was increased by 25.6 and 23.9 times, respectively. These Eco645 (2) and Eco745 (7) are probes that are close to the target and play the role of disrupting the three-branched structure. This suggests that the presence or absence of a probe that plays a role in disrupting the three-branch structure, not only in the three regions but also in the close positional relationship with the target, is important.

[Example 4: Study on the combination of two photocrosslinkable assist probes]
The effect of removing one probe was examined for combination number 14 (Eco567(1), Eco603(4), Eco745(7)) and combination number 41 (Eco624(3), Eco650(5), Eco738(6)) in Example 2. For combination number 14, which showed a large increase in relative fluorescence intensity, it was examined which of the three regions predicted to be necessary for disrupting the higher-order structure of the target was important to disrupt, and whether hybridization efficiency with the target would be significantly improved if two regions were disrupted. For combination number 41, which showed a small increase in relative fluorescence intensity, it was examined whether an increase in relative Cy3 fluorescence intensity would be obtained if one of the probes Eco650(5) and Eco738(6), which are complementary strands and have negative cooperativity, was removed.

The experiments and evaluation were carried out in the same manner as in Example 1.

[Study on reducing the assist probe from combination number 14]
An experiment was carried out by reducing one assist probe from combination number 14 of Example 2. The results of the observation of CLSM images were quantified in the same manner as in Example 1, and a graph is shown in Figure 12. Figure 12 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when only Eco627 was added, when the respective combinations of assist probes described above were used.

[Study on reducing the assist probe from combination number 41]
An experiment was carried out by reducing one assist probe from combination number 41 of Example 2. The results of the observation of CLSM images were quantified in the same manner as in Example 1, and a graph is shown in Fig. 13. Fig. 13 is a graph showing the relative fluorescence intensity of E. coli stained with Eco627 when the combination of assist probes described above was used, based on the fluorescence intensity when only Eco627 was added.

[Evaluation of experiments combining two photocrosslinkable assist probes]
For combination number 14, a decrease in the relative Cy3 fluorescence intensity was observed by reducing one assist probe. For each combination, the decrease was 12.9 times for Eco567(1)/Eco614(3), 18.1 times for Eco567(1)/Eco745(7), and 11.5 times for Eco745(7)/Eco603(4). From these results, it was found that when it becomes impossible to disrupt the higher-order structure of one of the three regions, the relative Cy3 fluorescence intensity is reduced compared to when all three regions are disrupted.

For combination number 41, an increase in the relative Cy3 fluorescence intensity was obtained by removing one type of assist probe. Comparing the increase rate for each combination, the increase was 18.6-fold when Eco650 (5) was removed, and 19.1-fold when Eco738 (6) was removed. In contrast, when Eco614 (3) was removed, that is, in the case of the combination of Eco650 (5) and Eco738 (6), the increase was only 9.6-fold, which was not as great. From these results, it was found that an increase in the relative Cy3 fluorescence intensity can be obtained by removing either one of the probes, Eco650 (5) or Eco738 (6), which are complementary strands and have negative cooperativity.

The results of examining the combination of two photocrosslinkable assist probes showed that in order to disrupt the complex higher-order structure of the target and detect the target fluorescently, it is necessary to change the higher-order structure of all three regions. Furthermore, these results showed that in the two combinations, of the three regions A, B, and C, region B is the most important, followed by region C, and then region A.

The present invention provides a means for detecting base sequences in difficult-to-detect sites in intracellular higher-order structured RNA. The present invention is an industrially useful invention.

Claims (7)

A method for photocrosslinking a photocrosslinkable artificial nucleic acid probe with a target base sequence in an RNA molecule by forming a double strand, comprising the steps of:
The method includes a step of forming a double strand of a photo-crosslinkable artificial nucleic acid assist probe with an RNA molecule to photocrosslink the RNA molecule simultaneously with or prior to the formation of a double strand of a photo-crosslinkable artificial nucleic acid probe with a target base sequence in the RNA molecule,
a target base sequence in the RNA molecule is within a region of partial duplex structure;
a partial double-stranded structure continues beyond the target base sequence at one of both ends of the region of the partial double-stranded structure containing the target base sequence,
A region of a partial double-stranded structure extending beyond the target base sequence is designated as region A,
a multi-branched structure of RNA is present at the other of both ends of the region of the partial double-stranded structure containing a target base sequence,
Among the branches due to the multibranched structure of the RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing a target base sequence, the region of one branch is designated as region B and the region of the other branch is designated as region C;
As the photocrosslinkable artificial nucleic acid assist probe, the following:
a photocrosslinkable artificial nucleic acid assist probe A having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the A region;
The method includes a photo-crosslinkable artificial nucleic acid assist probe B having a base sequence capable of photo-crosslinking by forming a double strand with a base sequence in the B region, and a photo-crosslinkable artificial nucleic acid assist probe C having a base sequence capable of photo-crosslinking by forming a double strand with a base sequence in the C region,
A photocrosslinkable artificial nucleic acid probe,
A photocrosslinkable artificial nucleic acid probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond,
A photocrosslinkable artificial nucleic acid assist probe,
A photocrosslinkable artificial nucleic acid assisted probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond:

Formula (I):
However, in formula I,
R11 is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or a hydrogen atom;
R12 and R13 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to 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 of formula II:

Formula II:
However, in formula II,
R21 is a hydrogen atom or a methyl group . A method for photocrosslinking a photocrosslinkable artificial nucleic acid probe with a target base sequence in an RNA molecule by forming a double strand, comprising the steps of:
The method includes a step of forming a double strand of a photo-crosslinkable artificial nucleic acid assist probe with an RNA molecule to photocrosslink the RNA molecule simultaneously with or prior to the formation of a double strand of a photo-crosslinkable artificial nucleic acid probe with a target base sequence in the RNA molecule,
a target base sequence in the RNA molecule is within a region of partial duplex structure;
a multi-branched RNA structure is present at both ends of the region of the partial double-stranded structure containing a target base sequence,
In one of the multibranched structures of RNA at both ends, among the branches due to the multibranched structure of RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing the target base sequence, the region of one branch is designated as region B and the region of the other branch is designated as region C;
In the other of the multibranched structures of RNA at both ends, among the branches due to the multibranched structure of RNA, of two branches close in base sequence to the region of the partial double-stranded structure containing the target base sequence, the region of one branch is designated as region B' and the region of the other branch is designated as region C';
As the photocrosslinkable artificial nucleic acid assist probe, the following:
a photocrosslinkable artificial nucleic acid assist probe B having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the B region;
a photocrosslinkable artificial nucleic acid assist probe C having a base sequence capable of photocrosslinking by forming a double strand with the base sequence in the C region;
The method includes a photo-crosslinkable artificial nucleic acid assist probe B' having a base sequence capable of photo-crosslinking by forming a double strand with the base sequence in the B' region, and a photo-crosslinkable artificial nucleic acid assist probe C' having a base sequence capable of photo-crosslinking by forming a double strand with the base sequence in the C' region,
A photocrosslinkable artificial nucleic acid probe,
A photocrosslinkable artificial nucleic acid probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond,
A photocrosslinkable artificial nucleic acid assist probe,
A photocrosslinkable artificial nucleic acid assisted probe in which a photocrosslinkable artificial nucleoside represented by the following formula (I) is introduced into a base sequence via a phosphodiester bond:

Formula (I):
However, in formula I,
R11 is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or a hydrogen atom;
R12 and R13 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to 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 of formula II:

Formula II:
However, in Formula II,
R21 is a hydrogen atom or a methyl group . The method according to any one of claims 1 to 2, wherein the RNA multibranched structure is a RNA tri-branched structure or an RNA tetra-branched structure. The method according to any one of claims 1 to 3, wherein the RNA molecule is an intracellular RNA molecule. 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 a photocrosslinkable artificial nucleic acid probe and a target base sequence in the RNA molecule, and photocrosslinking the double strand. The method according to claim 5, wherein the photocrosslinkable artificial nucleic acid probe is a probe equipped with a detection label. The method according to claim 6, wherein the photocrosslinkable artificial nucleic acid probe is a probe equipped with a beacon-type fluorescent label for the FISH method.