JPH11285386A - Method for detecting higher-order structure of rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect a higher-order structure of an RNA from a result obtained by detecting a sequence B1 B2 in which a probe P1 of a sequence B1 complementary with A1 in a specific sequence A1 A2 of the RNA and a probe P2 of a sequence complementary with A2 are successively hybridized. SOLUTION: A higher-order stricture of an RNA is detected by detecting a successive base sequence B1 B2 formed on the said RNA by successively hybridizing P1 with P2 by reacting a probe P1 having a base sequence B1 capable of hybridizing with A1 out of a specific succeeding base sequence A1 A2 in the RNA with a probe P2 having a base sequence B2 capable of hybridizing with A2 out of the specific succeeding base sequence in the RNA, and judging that the higher-order structure of the specific succeeding base sequence A1 A2 is a single-stranded when the said successive base sequence B1 B2 is detected and the higher-order structure of the specific successive base sequence A1 A2 is a double-stranded when the said successive base sequence B1 B2 is not detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting a higher-order structure of RNA, and c-fos mRN using the method.
A, IL-2 mRNA, and tobacco mosaic virus-L strain (T
MV-L) relates to a probe for detecting higher-order structure of RNA.

[0002]

2. Description of the Related Art In elucidating the function of an RNA molecule, it is important to know not only the base sequence, which is the primary structure, but also the higher-order structures that exist under various physiological conditions. That is, an RNA molecule forms a base pair according to the base sequence in the molecule, forms a stem structure by a double strand in a part of the chain, and forms one base when the base pair is hardly formed in the molecule. It is known to exist as a chain or as a circular single strand (FIG. 24). Therefore,
In order to elucidate the relationship between the structure and function of RNA molecules, it is necessary to detect higher-order structures under specific conditions.
There is a strong demand for a method for detecting the higher-order structure of RNA or its change.

Conventionally, as one of such methods, a method of analyzing the structure of a solid crystal of an RNA molecule and a method of nuclear magnetic resonance absorption (NMR) of an RNA molecule in a solution are known. However, the structural analysis of solid crystals of RNA molecules has the problem that the dynamic motion of RNA molecules in solution and the equilibrium phenomenon cannot be elucidated, and the NMR method measures only relatively small RNA molecules. There was a problem that it was possible.

[0004]

SUMMARY OF THE INVENTION The present invention uses a set of oligonucleotide probes having a specific structure,
It is intended to provide a method for detecting a higher-order structure of RNA. The present invention also provides a probe for detecting the higher-order structure of c-fos mRNA, IL-2 mRNA, and TMV-L RNA.

[0005]

Means for Solving the Problems The present inventors have intensively studied to solve the above-mentioned problems of the prior art, and by using a probe having a specific structure, the higher order We succeeded in finding a method for detecting the structure, and completed the present invention. Further, based on such findings, they have succeeded in developing a probe for detecting the higher-order structure of c-fos mRNA, IL-2 mRNA, and TMV-L RNA.

That is, the present invention relates to a method for detecting a higher-order structure of RNA, which comprises the steps of (1)
Probe P having a base sequence B1 capable of hybridizing with A1 among specific continuous base sequences A1A2 of
1 and a specific continuous base sequence A1 of the RNA
Reacting A2 of A2 with a probe P2 having a base sequence B2 capable of hybridizing, and (2) a continuous sequence formed by continuously hybridizing the probes P1 and P2 on the RNA. (3) (i) when the continuous base sequence B1B2 is detected, the higher order structure of the specific continuous base sequence A1A2 of the RNA is single-stranded; Or (ii) when the continuous base sequence B1B2 is not detected, the specific continuous base sequence A1 of the RNA
It is intended to provide a method comprising the step of judging that the higher-order structure of A2 is double-stranded.

Here, as schematically shown in FIG. 24, in the present invention, the higher-order structure of RNA means that
Mainly, a partial double-strand formation (also referred to as a stem structure) based on the formation of a base pair in a molecule, and a single-strand structure of a portion without the base-pair formation or a cyclic single-strand structure (a loop structure) ). Such a structure is in a specific equilibrium state depending on the state of the solution (temperature, salt concentration, etc.) and varies with the movement of the RNA molecule. The method according to the present invention enables detection of the most likely higher-order structure of RNA under the above-mentioned predetermined conditions (FIG. 1). further,
When such a predetermined condition is changed, as schematically shown in FIG. 2, a change also occurs in the higher-order structure of the RNA in accordance with the change, but the method according to the present invention provides a method for the higher-order structure of the RNA. The change can also be detected.

[0008] Further, the method according to the present invention is characterized in that the two probes detect a hybrid formed by successively hybridizing to RNA.
The detection method is not particularly limited, and includes a specific phenomenon based on the formation of the hybrid.

That is, the present invention provides the above method, wherein in the step (2), the detection is performed by: (a) the probe 1 or 2 is labeled with an energy donor fluorescent molecule; And the other probe is labeled with an energy acceptor fluorescent molecule, and
(b) A method characterized by measuring the fluorescence of the probe 2 due to the transfer of fluorescence energy based on the photoexcitation of the probe 1 or a change thereof.

In the present invention, one preferable method for detecting the above-mentioned hybrid is to use a well-known fluorescence energy transfer phenomenon as schematically shown in FIG. Therefore, in the present invention, if the formation of the hybrid can be confirmed, the fluorescence energy transfer phenomenon (USP) by the combination of various known fluorescent molecules can be confirmed.
4868103, 4996143, EPO229943
Reference)).

The present invention also provides a probe for detecting a higher-order structure of c-fos mRNA using the method according to the present invention.

Another object of the present invention is to provide a probe for detecting the higher-order structure of human IL-2 mRNA.

[0013] The present invention further provides a probe for detecting the higher-order structure of TMV-L RNA.

[0014] Such a probe is used to detect the higher-order structure of each RNA under various conditions. That is, by using a part or all of such a probe, it is possible to detect which position of each RNA molecule has a stem structure or a single-stranded or loop structure. .

Hereinafter, the present invention will be described in more detail in accordance with embodiments of the present invention.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The abbreviations used here, BODI
PY (or Bodipy) is a trademark of Molecular Probes, Inc. CY5, CY3, CY3.5 (or Cy5, Cy3, Cy3.5) is a trademark of Amersham.

The method for detecting the higher-order structure of RNA according to the present invention is, as schematically shown in FIG. 1, a specific base sequence of RNA (indicated as A1A2 in the figure) in a solution. In the case of a single-stranded or loop structure,
Probe according to the present invention (having base sequences B1 and B2)
Can be continuously hybridized, so that a specific phenomenon occurs between the labels F1 and F2 of the probe.

Further, a specific nucleotide sequence of RNA (A in the figure)
1A2) has a double-stranded structure in a solution, it becomes difficult for the probe of the present invention (having the base sequences B1 and B2) to continuously hybridize, and The above specific phenomenon will not occur.

FIG. 3 shows an example utilizing the fluorescence resonance energy transfer (FRET) phenomenon as the specific phenomenon. That is, when the probe according to the present invention continuously hybridizes to RNA, FRET occurs between the fluorescent dyes labeled on each probe, and a change in the fluorescence spectrum is observed.

Further, the method according to the present invention will be described in more detail with reference to the following description of the present invention in which the high image change is monitored when the higher-order structure of RNA changes in solution under various conditions.

(Higher order structure of RNA) RNA according to the present invention
The higher order structure does not exist as a constant structure in a solution, but also includes a structure existing under specific conditions, for example, in a solution, depending on the existing conditions. Specifically, the same RNA
Even so, the most probable higher-order structure varies depending on the specific temperature, salt concentration, composition of the solvent, and the like, and the method according to the present invention can detect such a fluctuating higher-order structure. Is what you do.

As for RNA, its type, structure,
There is no particular limitation on the length and the length. For example, tRN
A, mRNA, rRNA, and synthetic ribonucleotides. Further, RNA does not need to have a nucleic acid structure, and may have other components at the terminal.

In the method according to the present invention, it is necessary that the nucleotide sequence of the RNA of the portion whose higher order structure is to be detected is known. That is, the base sequence of the probe used in the present invention is determined according to the known base sequence.

For the purpose described above, as a method for determining the base sequence of RNA, known base sequence determination methods can be suitably used (for example, the Maxim Gilbert method, the Thie terminator method (Sanger method)) (dideoxy-mediated method).
chain-termination method for DNA sequencing) can be used.

(Detection Method) The method for detecting a higher-order structure of an RNA according to the present invention comprises a set of probes P1 which continuously hybridize to a base sequence A1A2 at a position where a higher-order structure on the RNA is to be detected. P2, each of which is A1,
One having base sequences B1 and B2 complementary to A2 is used.

Now, the base sequence portion A1A of the RNA
2 is a single-stranded or cyclic single-stranded (loop) under predetermined conditions
When a higher-order structure is adopted, a double-stranded (hybrid) can be easily formed by adding the above probe.
Alternatively, when the base sequence portion A1A2 of the RNA has a double-stranded (stem) higher-order structure under a predetermined condition, the double-stranded (stem) structure can be easily removed from the stem structure simply by adding the probe. (Hybrid) is difficult to form.

Accordingly, the formation of a hybrid by such a probe is based on the two base sequences B1 and B2 of the probe.
Are continuously (B1B2) hybrids formed on the RNA. Such a continuous base sequence B1B
2 by any means, the R
The higher-order structure of the NA portion will be detected.

The present invention is not particularly limited with respect to a method for specifically detecting such a continuous base sequence B1B2. It is possible to use various means including known means that specifically cause a specific phenomenon only when the base sequences B1 and B2 continuously hybridize.

Specifically, as summarized below, a label group is bonded to each of the probes P1 and P2 having the base sequences B1 and B2, and the base sequences B1 and B2 are interposed between the label groups.
It is preferable that a specific phenomenon occurs only when the two hybridize continuously.

(1) Fluorescence resonance energy transfer (FRE)
T). That is, the probe according to the present invention is a set of probes, one of which is labeled with a fluorescent molecule having an energy donor property and the other is labeled with a fluorescent molecule having an energy acceptor property. In addition, these probes are continuously RN
When hybridized to A, it has a molecular structure which takes a spatial position such that fluorescence resonance energy transfer occurs efficiently. Therefore, when the energy donor fluorescent molecule is excited by light irradiation to the hybrid of these probes and RNA, the probe moves to another fluorescent molecule having the energy acceptor property and the fluorescence from the energy acceptor fluorescent molecule is observed ( (Fig. 3).

In this case, the fluorescence intensity of the fluorescence from the energy acceptor fluorescent molecule is measured to determine whether or not the above-described fluorescence energy transfer has occurred. Also,
If this energy transfer occurs, it indicates that the two probes can hybridize to RNA in succession. That is, it is known that the RNA has a single-stranded or loop structure. One set of probes according to the present invention is configured to be spatially arranged so that the above-described fluorescence energy transfer occurs efficiently.

Therefore, the use of the method according to the present invention makes it possible to detect the higher-order structure of RNA with extremely high sensitivity.

Further, by quantitatively measuring the fluorescence intensity of the fluorescence from the energy acceptor fluorescent molecule, it is possible to measure the ease with which two probes can continuously hybridize to RNA. In this case, an evaluation of the stability of the higher-order structure in this portion of the RNA under various solution conditions can be obtained.

(2) Fluorescence generated by the formation of excimers and exciplexes is used. That is, a phenomenon that occurs when one dye in the excited state and the other dye molecule in the ground state form a complex is used. Two types 1 labeled with these dyes
The set of probes has a molecular structure that takes a spatial arrangement such that excimers or exciplexes are efficiently formed when hybridized to RNA continuously.
Therefore, by irradiating light to the hybrid of these probes and RNA, the intensity of fluorescence derived from excimers or exciplexes is observed. in this case,
By measuring the fluorescence intensity from these excimers or exciplexes, two types of
It can be seen whether or not it is possible to hybridize to the relevant site of A. That is, it is known whether the portion of the RNA has a single strand, a loop structure, or a double strand. One set of probes according to the present invention is configured to be spatially arranged so that the above-mentioned excimer fluorescence is efficiently generated. Therefore, by using the method according to the present invention, it is possible to detect the higher-order structure of RNA with extremely high sensitivity.

Further, by quantitatively measuring the fluorescence intensity of the excimer fluorescence, it is possible to measure the ease with which two probes can continuously hybridize to RNA. In this case, RNA under various solution conditions
The evaluation of the stability of the higher-order structure in this portion can be obtained.

(Probe) The above-mentioned set of probes used in the present invention has B1 and B2 which are base sequences complementary to the base sequence A1A2 of the portion where the higher-order structure of RNA is to be detected.

When there are a plurality of parts for which the higher-order structure of the RNA is to be detected, similarly, the RNA
It is sufficient to use a plurality of one set of probes complementary to the base sequence. The same applies to the case where the higher-order structure is detected in the entire sequence of RNA. In such cases, for example,
By adding the above set of probes to separately partitioned solutions (specifically, titer plates) and adding RNA to each solution, total RNA can be quickly and easily prepared.
Is detected. Further, by changing the temperature or the like of the solution, a change in the higher-order structure of the total RNA can be detected quickly and easily.

The number of base sequences of each probe is not particularly limited. It can be appropriately selected depending on the number of bases of RNA, higher-order structure, solution conditions, and the like. When the number of base sequences is large, the duplex formed by hybridization is stable, but the information on the higher order structure obtained is small. When the number of base sequences is small, the duplex formed by hybridization becomes unstable. Therefore, it is usually sufficient to use 10 to 40 bases, preferably 15 to 30 bases. Furthermore, the total number of base sequences of each probe is not particularly limited. In general, a hybridized duplex must be sufficiently stable and have a sufficient length without misrecognition.
0 to 50 bases are preferred, and more preferably 20 to 40 bases or more.

(Preparation of Probe) The method for preparing the oligonucleotide sequence portion of the detection probe according to the present invention described above is not particularly limited. Known nucleic acid synthesis methods can be preferably used. In particular, automatic synthesis methods based on various solid phase synthesis methods are preferable, for example, amidite,
Synthetic methods such as triesters are preferably used (Mineo Niwa, Chemistry and Biological Experiment Line, Chemical Synthesis of DNA, Hirokawa Shoten, 1992). Furthermore, a method for introducing a preferable linker portion for binding a fluorescent molecule into a probe can be achieved by using various polypeptide modifying reagents.

In the present invention, in particular, 6- (trifluoroacetylamino) hexyl- (2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite, which is a reagent for amination at the 5′-terminal, is synthesized by the above chemical synthesis. Can be used at the same time. This makes it possible to introduce a hexylamino group (after removing the trifluoroacetyl group) at any position in the oligonucleotide chain.

In addition, by selecting the position of the linker portion and the length of the linker portion, in order to prepare a probe in which the two kinds of fluorescent molecules can have a preferable relative spatial configuration, various probes for estimating the spatial configuration are used. A molecular model, a molecular model computer program, or the like can be used.

A method for bonding a linker moiety to a necessary fluorescent molecule,
Alternatively, there is no particular limitation on the method of bonding the linker moiety to a base at an appropriate position of the oligonucleotide probe, and a chemical synthesis method, an enzyme method, or the like can be used.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the examples.

[0044]

EXAMPLES (1) Synthesis of Fluorescently Labeled Oligo DNA Probe (1-1) Synthesis of Oligo DNA Oligo DNA having the following base sequence was converted to DNA / RNA.
Synthesizer (Perkin Elmer: Model 394 or Per
Septive: Model 18909), and synthesized by the β-cyanoethylamidite method.

F1: 5′-XTCTAGTTGGTCTGTC-3 ′ F2: 5′-XCTTCTAGTTGGTCTG-3 ′ F3: 5′-XATCTTCTAGTTGGTC-3 ′ F4: 5′-XCTCATCTTCTAGTGG-3 ′ F5: CT5CT-7CT 5'-AAGCAGACTTCYTCAT-3'F7: 5'-CAAAGCAGACTYTCTC-3'F8: 5'-CTGCCAAAGCAGYAACTTT-3 'L1: 5'-XATTATTAATTCCATT-3' L2: 5'-XGTAAAACTTAGATGAT-3ATCTATGATCTATGTATCC 3 'L4: 5'-XTAGAAGGCCTGAATA-3' L5: 5'-XCATTCACAATAATAA-3 'L6: 5'-TTTGGG ATTCTYTGTA-3 'L7: 5'-GGCCTTCTTGGYGCAT-3' L8: 5'-ATGAATGTTGTYTTCA-3 'L9: 5'-AATATTTAAAYTAAA-3' L10: 5'-AGATACTATATYTTAGTAG-3TC : 5'-XTCCATCTTTGAGCAA-3 'T3: 5'-XCATGTCTAATAAGTAA-3' T4: 5'-XATACACAATCGTTTG-3 'T5: 5'-XCTTTGTTGTAATACC-3' T6: 5'-XCTCATCAACAACTTC-3 'T7: 5'-XATCTTGCAATGCTAG -3 'T8: 5'-GCTGTTTGTGTYGTAT-3' T9: 5'-ACTAACGTGTTGYGTTC-3 'T10 5'-CTGCCATCTACTTYTGTA-3 'T11: 5'-TGATTTTCTTTGYAAATG-3' T12: 5'-GTTCTTTTTCTGYCATC-3 'T13: 5'-AACATTCTCCAYTGAA-3' T14: 5'-AGAAGCACT ' -(Trifluoroacetylamino) hexyl- (2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite (TFAc hexanolamine linker, Perkin Elmer Japan: Cat No. 400
808), Y is Uni-Link AminoModifier (CLONTECH La
bratories Inc. Code No.CL5190-1).

The obtained reaction products were separated by ion exchange high performance liquid chromatography, and the main peak was collected.
The conditions used for the ion-exchange high-performance liquid chromatograph are as follows: column: TSK-GEL DEAE-2WS manufactured by Tosoh Corporation.
(4.6 mm inner diameter x 250 mm full length), flow rate 0.8 ml / min, temperature 40 ° C, mobile phase HCOOHNH ₄
The slope -20% CH ₃ CN, were detected by absorbance at 260 nm. The HCOOHNH ₄ gradient is as follows: Solution A: 0.2 M HC
OOHNH ₄ , liquid B: prepared by changing the mixing ratio of two liquids of 1M HCOOHNH ₄ . Solution B ratio: 35%
A gradient of -85% / 20 min was used.

The desalted liquid was desalted and freeze-dried.

(1-2) Bodipy493 / 503 of oligo DNA
Labeling The oligo DNAs synthesized above (F1, F2, F3, F
4, L1, L2, L3, L4, L5, T1, T2, T
3, T4, T5, T6, T7) were labeled with Bodipy493 / 503.

NHSS (N-Hydroxysulfosuccinimide, S
oudium salt (2.5 mg) in 30 microliters of sterilized water and EDAC (1-Ethyl-3- (3-dimethylaminopropyl) c)
arbodiimide) was dissolved in 50 microliters of sterile water. These were mixed with a solution of 1 mg of Bodipy493 / 503 propionic acid (Molecular Probes Inc.) in 50 microliters of DMF and mixed at room temperature for 30 minutes.
Allowed to react for minutes. The resulting solution is dried oligo-DN
A was added to a 0.5 M Na ₂ HCO ₃ / NaH ₂ CO ₃ buffer (pH
9.3) It was mixed with a solution dissolved in 300 microliters, and reacted overnight under light shielding. After the reaction solution was subjected to gel filtration to remove unreacted dye, the reaction product was purified by reversed-phase high performance liquid chromatography. The conditions used for reversed-phase high-performance liquid chromatography were Shiseido columns, CAPCELL
Using PAKC18 (6 mm inner diameter x 250 mm full length), flow rate 1 ml / min, temperature 40 ° C, mobile phase CH ₃ CN
Gradient-5 mM TEAA, detected at 260 nm. The CH ₃ CN gradient was as follows: solution A: 5% CH ₃ CN, B
Liquid: prepared by changing the mixing ratio of two solutions of 40% CH ₃ CN. Solution B ratio: 30% -80% / 20 min gradient was used. The absorption spectrum of the collected peak was measured, and the absorption at 260 nm and the absorption of the fluorescent dye (493 n
m) was confirmed. These were lyophilized and stored.

F662D (Bodipy493 / 503-labeled form of F1): 5 ′-(Bodipy493 / 503-X) TCTAGTTTGGTCTTGTC-3 ′ F664D (Bodipy493 / 503-labeled form of F2) 5 ′-(Bodipy493 / 503-X) CTTCTAGTGTGTCTG- 3 'F666D (Bodipy493 / 503-labeled product of F3): 5'-(Bodipy493 / 503-X) ATCTTCTAGTTGGCTC-3 'F669D (Bodipy493 / 503-labeled product of F4): 5'-(Bodipy493 / 503-X) CTCATCTTCTAGTGTG- 3 'L183D (Bodipy493 / 503-labeled L1): 5'-(Bodipy493 / 503-X) ATTATTAATTCCATT-3 'L228D (Bodipy493 / 503-labeled L2): 5'-(Bodipy493 / 503-X) GTAAACTACTAAATGT- 3 'L389D (Bodipy493 / 503-labeled product of L3): 5'-(Bodipy493 / 503-X) GATCCTTTAGTTCC 3 'L536D (Bodipy493 / 503-labeled product of L4): 5'-(Bodipy493 / 503-X) TAGAAGGCCTGATA-3 'L715D (Bodipy493 / 503-labeled product of L5): 5'-(Bodipy493 / 503-X) CATCACACATATAATA- 3 'T62D (Bodipy493 / 503 label of T1): 5'-(Bodipy493 / 503-X) GCCATTGTAGTGTGTA-3 'T2514D (Bodipy493 / 503 label of T2): 5'-(Bodipy493 / 503-X) TCCATCTTTGAGCAA- 3 ′ T3380D (Bodipy493 / 503-labeled T3): 5 ′-(Bodipy493 / 503-X) CATGTCTATAATAAGTAA-3 ′ T4038D (Bodipy493 / 503-labeled T4): 5 ′-(Bodipy493 / 503-X) ATACACAATCGTTTG- 3 'T5281D (Bodipy493 / 503-labeled product of T5): 5'-(Bodipy493 / 503-X) CTTTGTTGTAATAACC- 'T5479D (Bodipy493 / 503-labeled T6): 5'-(Bodipy493 / 503-X) CTCATCAACACTACTTC-3 'T1926D (Bodipy493 / 503-labeled T7): 5'-(Bodipy493 / 503-X) ATCTTGCAATGCTAG-3 (1-3) Cy5 Labeling of Oligo DNA Oligo DNA (F5, F6, F7, F8, L6, L7,
L8, L9, L10, T8, T9, T10, T11, T
12, T13, T14) were labeled with Cy5 dye.

One tube of Cy5 dye (Amersham, FluoroL)
ink Cat. No. PA25001) was dissolved in 100 microliters of sterilized water. This was mixed with the dried oligo DNA at 0.
5M Na ₂ HCO ₃ / NaH ₂ CO ₃ buffer (pH 9.3)
The solution was mixed with a solution dissolved in 200 microliters, and reacted overnight under light shielding. After the reaction solution was subjected to gel filtration to remove unreacted dye, the reaction product was purified by reversed-phase high performance liquid chromatography. The conditions used for the reversed-phase high-performance liquid chromatograph were Shiseido columns, CAPCELLLPAKC1
8 (6mm inside diameter x 250mm full length), flow rate 1m
1 / min, temperature 40 ° C, mobile phase CH ₃ CN gradient −5 mM
TEAA, and was detected by absorption at 260 nm. CH
_{The 3} CN gradient was as follows: solution A: 5% CH ₃ CN, solution B: 40% C
It was made by changing the mixing ratio of the two solutions of H ₃ CN. Solution B ratio: A gradient of 15% -60% / 20 minutes was used. The absorption spectrum of the fractionated peak was measured, and 26
The absorption at 0 nm and the absorption of the fluorescent dye (493 nm) were confirmed. These were lyophilized and stored.

F677A (Cy5 label of F5): 5′-GCAGACTTCTC (Y-Cy5) ATCT-3 ′ F679A (Cy5 label of F6): 5′-AAGCAGACTTC (Y-Cy5) TCAT-3 ′ F681A (F7) 5′-CAAAAGCAGACT (Y-Cy5) TCTC-3 ′ F684A (Cy5 label of F8): 5′-CTGCAAGCAG (Y-Cy5) ACTT-3 ′ L198A (Cy5 label of L6): 5'-TTTGGGATTCT (Y-Cy5) TGTA-3 'L243A (Cy5 label of L7): 5'-GGCCTTCTTGGG (Y-Cy5) GCAT-3' L404A (Cy5 label of L8): 5'-ATGAATGTGTGT (Y -Cy5) TTCA-3 'L550A (L5 Cy5 label) 5'-AATTATTTAAA (Y-Cy5) TAAA-3 'L730A (Cy5 label of L10): 5'-AGACTACTATAT (Y-Cy5) TTAA-3' T77A (Cy5 label of T8): 5'-GCTGTTTGTGGT (Y -Cy5) GTAT-3 'T2529A (Cy5 label of T9): 5'-ACTAACGTTGTG (Y-Cy5) GTTC-3' T3396A (Cy5 label of T10): 5'-CTGCATCTACTT (Y-Cy5) TGTA-3 'T4053A (Cy5 label of T11): 5'-TGATTTTCTTTTG (Y-Cy5) AATG-3' T5296A (Cy5 label of T12): 5'-GTTCTTTCTCTG (Y-Cy5) CATC-3 'Cy5A of T13A (T13) Labeled product): 5'-AACATTCTCC A (Y-Cy5) TGAA-3 'T1941A (Cy5 label of T14): 5'-AGAAGCCTTT (Y-Cy5) CAGA-3' (2) Preparation of human IL-2 RNA Plasmid DNA having human IL-2 cDNA , PTCGF-II (ATCC
# 39673, Proc.Natl.Acad.Sci.USA, 81,2543-2547,198
4) The IL-2 cDNA fragment excised with the restriction enzyme pst I was
At the pst I digestion site of the NA synthesis vector pBluescript KS (+)
The ligation was performed using a ligation kit (Takara) such that the same cDNA was located downstream of the T3 promoter. The obtained recombinant plasmid was introduced into competent cells (Takara) of Escherichia coli JM109 strain, and the obtained transformant of the same Escherichia coli was cultured. From 100 ml of the same culture, Plasmid Midi Kit (QIAG
46.2 μg of plasmid DNA was extracted and purified using EN).

The recombinant plasmid was linearized by digestion with the restriction enzyme SmaI, the protein present in the plasmid solution was digested with proteinase K, and then denatured and removed using phenol / chloroform.

Using this purified gene fragment (0.66 μg) as a template, the composition of each base constituting RNA is A: C: G: U = 3.5: 1.8:
A 1.4: 3.2 T3 RNA polymerase solution was prepared using an in vitro transcription reaction kit (Megascript T3 Kits, Ambion), and the polymerase reaction was performed at 37 ° C. for 6 hours.
IL-2 RNA was synthesized using L-2 cDNA as a template. After completion of the reaction, the template DNA was digested with DNase I (same kit, Ambion), and the protein in the transcription reaction solution was denatured and removed using phenol / chloroform. To the obtained RNA solution, 2-fold volume of isopropanol was added, and human IL-2 RNA was collected as a precipitate by centrifugation (14 krpm, 7 minutes), and low-molecular substances such as nucleotides, which were unreacted enzyme reaction substrates, were removed. .
RNA precipitate (139 μg) rinsed once with 70% ethanol
Dissolve in Nase free water (same kit, Ambion) and add 5
A μg / μl RNA solution was prepared and used for subsequent hybridization experiments.

(3) Preparation of TMV-L genomic RNA TMV-L genomic RNA is a c384 nucleotide having a total sequence of 6384 bases of TMV-L.
A containing plasmid: pLFW3 (Proc. Natl. Acad. Sci. US
A., 83, 5043-5047, 1986) by replacing the Pm promoter with a T7 promoter (Bio / Tech., 11,930-932, 1993;
Molecular pathology of tobacco mosaic virus reveal
ed by biologically active cDNAs, p.149-186.In: Gene
tic Engineering with Plant Viruses.Wilson, TMAan
d Davis, JW (Eds.), CRC Press, Boca Raton, FL.) as a template. That is, pTLW3 was cut linearly with the restriction enzyme MluI, and genomic RNA was synthesized by allowing T7 RNA polymerase to act on the T7 promoter located immediately before the cDNA of TMV-L.

For the synthesis, an in vitro transcription reaction kit for T7 promoter (MEGAscript T7Kits, Ambion) was used. The cap structure (m7G (5 ') p
Since pp (5 ') G: Cap Analog) is not added and the end that has been cut linearly with Mlu I is not blunt, the synthesized RNA is 3 bases (CGC ) Extra sequences are added.

Extraction and purification of the synthesized TMV-L RNA was performed using an in vitro transcription reaction kit (MEGAscriptT7 Kits, Ambion
The method was the same as the method of the attached reagent and kit. That is, after in vitro transcription reaction, DNase I was added to the reaction solution at 0.1 U / μl.
l, and react at 37 ° C for 30 minutes.
Was disassembled. Then, Ammonium Acetate Stop Solution
(5 M Ammonium Acetate, 100 mM EDTA) was added so that the final concentration of each was 0.5 M Ammonium Acetate, 10 mM EDTA, and the reaction of DNase I was stopped. After the reaction was stopped, phenol / chloroform and chloroform were extracted, followed by isopropyl alcohol precipitation to recover TMV-L RNA.

(4) Preparation of Human c-fosRNA RNA of the human c-fos gene was prepared by an in vitro transcription RNA synthesis reaction using a single-stranded DNA containing the c-fos gene DNA as a template.

A plasmid containing the coding region (2100 bases) of the human c-fos gene: pSPT-c-fos (RIKEN Gene ban)
k) was recombined downstream of a pBluescript plasmid vector having a T3 promoter: pBlue-c-fo
s built. This pBlue-c-fos plasmid is
M109 strain was transformed, and the transformed E. coli JM109 strain was mass-cultured to obtain pBlue-c-fos
The plasmid was amplified in large quantities. Then, E. coli JM1
The pBlue-c-fos plasmid was extracted and purified from the 09 strain. The purified pBlue-c-fos plasmid was replaced with the restriction enzyme SmaI.
And used as a template for an in vitro transcribed RNA synthesis reaction. C-fos by in vitro transcription RNA synthesis reaction
RNA was synthesized by allowing T3 RNA polymerase to act on a T3 promoter located upstream of c-fos DNA contained in the template DNA. For synthesis, for synthesis,
In vitro transcription reaction kit for T3 promoter (MEGA
scriptT3 Kits, Ambion). Isopropanol was added to the obtained RNA solution twice in volume, and human c-fos RNA was recovered as a precipitate by centrifugation (14 krpm, 7 minutes), and low-molecular substances such as nucleotides, which were unreacted enzyme reaction substrates, were removed. . RNA precipitate rinsed once with 70% ethanol (13
9 μg) was dissolved in RNase free water (same kit, Ambion).

(5) Preparation of RNA of Xenopus Transcription Elongation Factor Gene (XELF1-α) RNA of Xenopus transcription elongation factor gene (XELF1-α) is prepared by using a single-stranded DNA containing XELF1-α gene DNA as a template. It was prepared by an in vitro transcribed RNA synthesis reaction.

An in vitro transcription reaction kit (MEGAscript
Plasmid attached to T3 kit (Ambion): linea
The rized TRIPLEscript plasmid was treated with the restriction enzyme EcoRI to cut out XELF1-α DNA having a length of about 1800 bases.
Plasmid: pBluescript II KS (+) (STRAT)
AGEL) to obtain a plasmid containing XELF1-α DNA: pBlue XELF1-α (about 4760 bases). The obtained pBlue XELF1-α was cleaved linearly with a restriction enzyme SmaI and used as a template for an in vitro transcribed RNA synthesis reaction. XELF1 by in vitro transcribed RNA synthesis reaction
The synthesis of -αRNA is based on the XELF1-αD contained in the template DNA.
This was performed by allowing T3 RNA polymerase to act on the T3 promoter located upstream of NA. For synthesis, T3
In vitro transcription reaction kit for promoter (MEGAscri
pt T3 Kits, Ambion). Synthesized XELF1-
For extraction and purification of α, use QIAGEN Total RNA Kits (QIAGEN
And the method was in accordance with the method of the kit.

(6) Extraction of mRNA mixture from cells Cos7 cells were treated with guanidine hydrochloride to disrupt the cells. The cell lysate was treated with Oligod (T) latex beads (Roche) to affinity-purify polyARNA.

(7) Measurement of Fluorescence Spectrum The fluorescence spectrum of the aqueous solution containing each fluorescently labeled oligo DNA probe was measured under the following conditions.

Fluorescence spectrophotometer: Hitachi F4500 Excitation wavelength: 480 nm Fluorescence measurement wavelength: 500-750 nm Temperature: room temperature (7-1) When target RNA is IL-2 RNA The fluorescence spectrum of the fluorescently labeled oligo DNA probe is 1 × SSC
(15 mM Sodium citrate, 150 mM NaCl; pH 7.0) at a concentration of 30
Measured at 0 nM. To measure the fluorescence spectrum of a sample in which each of the fluorescent-labeled oligo DNA probes and IL-2 RNA were mixed, the mixture was mixed in 1 × SSC at a molar ratio of 1: 1 (the concentration of each of the probe and RNA was 300 nM). 15 at room temperature
After standing for minutes, the fluorescence spectrum was measured.

(7-2) The target RNA is TMV-LR
In the case of NA, the fluorescence spectrum of the fluorescence-labeled oligo DNA probe is 1
Measured at a concentration of 330 nM in xSSC buffer. When measuring the fluorescence spectrum of a sample obtained by mixing each fluorescently labeled oligo DNA probe and TMV-L RNA, the molar ratio was 1: 1 (the concentration of the probe and RNA was 330 n each).
M), the mixture was mixed in a 1 × SSC buffer, and allowed to stand at room temperature for 30 minutes or more, and then the fluorescence spectrum was measured.

(7-3) The target RNA is c-fos R
In the case of NA, the fluorescence spectrum of the fluorescence-labeled oligo DNA probe is 1
Measurements were taken at a concentration of 250 nM in xSSC buffer. When measuring the fluorescence spectrum of a sample obtained by mixing each fluorescent-labeled oligo DNA probe and c-fosRNA, the molar ratio was 1: 1 (the concentration of the probe and that of the RNA were 250 n each).
M), mixed in 1 × SSC buffer and left at room temperature for 30 minutes, and then the fluorescence spectrum was measured.

(8) High Performance Liquid Chromatograph (HPL)
C) Each fluorescently labeled oligo DNA probe and RNA (c-fosRN)
A or IL-2 RNA or xelf1-αRNA) at a molar ratio of 1: 1 (probe and RNA concentrations are 250 nM each)
At 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0
And left at room temperature for 30 minutes. Then, the fluorescence-labeled oligo DNA not bound to the fluorescence-labeled oligo DNA probe bound to RNA was analyzed by HPLC under the following conditions.
Separated from the probe.

Column: TSKgel DEAE-NPR (4.6 mm inner diameter x 3.5 cm total length) (Tosoh) Flow rate: 1 ml / min Temperature: 25 degrees Mobile phase: Solution A: 20 mM Tris-HCl, pH 9.0 Solution B: 20 mM Tris-HCl, 0.5 M NaCl pH 9.0 A linear gradient of NaCl concentration was prepared by mixing solution A and solution B. 0.125 M-0.5 M / 10 min. Detection was performed by ultraviolet absorption (260 nm) and fluorescence intensity. The detection conditions based on the fluorescence intensity are as follows: the excitation wavelength is 475 nm / the fluorescence measurement wavelength is 515 nm when the fluorescent dye labeled on the oligo DNA probe is Bodipy493 / 503, and the excitation wavelength is 650 nm when the fluorescent dye labeled on the oligo DNA probe is Cy5. / Fluorescence detection wavelength 667nm
It is.

(9) Search for secondary structure of IL-2 RNA IL-2 RNA is a single-stranded RNA having a total length of 801 bases. The following five sets of fluorescently labeled oligo DNA probes were used to examine the secondary structure of this RNA. Fluorescently labeled oligo D
NA probe is oligo D labeled with Bodipy493 / 503
One set of two types, one type of NA and one type of oligo DNA labeled with Cy5, was used in the experiment. Each of these five sets of probes has a nucleotide sequence at which two types of probes can continuously hybridize at a specific 30 base site of IL-2 RNA.

(1). L183D probe and L198A probe The L183D probe is a probe labeled with Bodipy493 / 503 having a nucleotide sequence complementary to the 183 bases to 197 bases of IL-2 RNA, and the L198A probe is IL-2 RNA
This probe has a nucleotide sequence complementary to the site of 198 bases to 212 bases and is labeled with Cy5.

(2). L228D probe and L243A probe The L228D probe is a probe that has a nucleotide sequence complementary to the 228 bases to 242 bases site of IL-2 RNA and is labeled with Bodipy493 / 503.
Cy has a base sequence complementary to the base of 3 bases-257 bases and Cy
5 is a probe labeled.

(3). L389D probe and L404A probe The L389D probe has a nucleotide sequence complementary to the 389 base-403 base site of IL-2 RNA and is labeled with Bodipy493 / 503.
Cy has a base sequence complementary to the base of 4 bases-418 bases.
5 is a probe labeled.

(4). L536D probe and L550A probe The L536D probe is a probe labeled with Bodipy493 / 503 having a nucleotide sequence complementary to the 536 bases-549 bases of IL-2 RNA.
0 bases-Cy has a base sequence complementary to 563 bases
5 is a probe labeled.

(5). L715D probe and L730A probe The L715D probe is a probe that has a nucleotide sequence complementary to the 715 to 729 bases of IL-2 RNA and is labeled with Bodipy493 / 503.
0 base-Cy has a base sequence complementary to the site of 744 bases.
5 is a probe labeled.

For each of the five sets of probes, one set of two probes was mixed at a molar ratio of 1: 1 and the fluorescence spectrum was measured. Further, to this solution, IL-2 RNA was added at a molar ratio of 1: 1 with respect to the probe and reacted at room temperature, and then the fluorescence spectrum was measured.
Changes in the spectrum were observed (FIGS. 4-8). as a result,
When the combination of (2) (L228D and L243A) was used, the addition of IL-2RNA decreased the fluorescence intensity of Bodipy493 / 503 (fluorescence having a peak around 515 nm) and the fluorescence intensity of Cy5 (wavelength 670 nm). fluorescence with a peak at nm). This is because the two types of probes are IL-2RN
The two fluorescent dyes (Bodipy493 / 503) were hybridized successively to each other at sites 228-257 of A.
And Cy5), the distance between Bodipy493 / 503 and C
Indicates that resonance energy transfer occurred during y5.
Combination of (1) (L183D and L197A), and (3)
A similar change in the fluorescence spectrum was observed when the combination (L389D and L403A) was used, but the amount of change was smaller than that of the combination (2). Also,
Combination of (4) (L536D and L550A), and
When the combination (5) (L715D and L730A) was used, the fluorescence spectrum hardly changed.

Each of the four probes L228D, L243A, L715D, and L730A was mixed with IL-2 RNA at a molar ratio of 1: 1.
After that, the probe bound to IL-2 RNA and the probe not bound were separated by HPLC (FIG. 9). A free probe was found at a retention time of 5 minutes, and a retention time of 7.
At 5 minutes, IL-2 RNA and the probe hybridized with IL-2 RNA are eluted, respectively. As a result of the experiment, L228
D, L243A, L715D, L730A are 35%, 16%, 20%, 2 respectively
% Of the probe eluted at the position of IL-2 RNA. That is, the ratio of the probe hybridized with the IL-2 RNA is 35%, 16%, 20%, and 2%, respectively.

These results show that the product of the amount of change in the fluorescence spectrum and the ratio of the hybridization of each of the two types of probes to the target RNA is correlated. That is, from the measurement of the fluorescence spectrum of the mixture of the two kinds of fluorescently labeled oligo DNA probes and the target RNA, it is determined how much the specific site of the target RNA is different from other nucleic acids having complementary base sequences mixed in the solution. It is possible to know whether or not it has the property of easily forming a hybrid.

(10) Search for Secondary Structure of TMV-LRNA TMV-LRNA is a single-stranded RNA having a total length of 6384 bases. The following seven sets of fluorescently labeled oligo DNA probes were used to examine the secondary structure of this RNA. The fluorescence-labeled oligo DNA probe was used in the experiment as a set of two types of oligo DNA labeled with Bodipy493 / 503 and one type of oligo DNA labeled with Cy5. The following seven combinations were used. All of these 7 sets of probes are TMV-
At each specific 30-32 base site in the LRNA,
The probe has a base sequence capable of continuously hybridizing the kinds of probes.

(1). T62D probe and T77A probe The T62D probe is a probe labeled with Bodipy493 / 503 with a nucleotide sequence complementary to the 62-76 base region of TMV-LRNA, and the T77A probe is 77 bases of TMV-LRNA.
This probe has a base sequence complementary to a 91 base site and is labeled with Cy5.

(2). T2514D probe and T2529A probe The T2514D probe is a probe labeled with Bodipy493 / 503 with a nucleotide sequence complementary to the 2514 to 2528 base region of TMV-LRNA, and the T2529A probe is for TMV-LRNA.
Has a nucleotide sequence complementary to the site of 2529 bases-2543 bases, and
This is a probe labeled with y5.

(3). T3380D probe and T3396A probe The T3380D probe is a probe labeled with Bodipy493 / 503 with a nucleotide sequence complementary to the 3380 base-3395 base site of TMV-LRNA, and the T3396A probe is TMV-LRN.
This probe has a nucleotide sequence complementary to the site of 3396 bases to 3411 bases of A and is labeled with Cy5.

(4). T4038D probe and T4053A probe The T4038D probe is a probe labeled with Bodipy493 / 503 having a nucleotide sequence complementary to the 4038 base-4052 base site of TMV-LRNA, and the T4053A probe is TMV-LRNA.
This is a probe labeled with Cy5, having a nucleotide sequence complementary to the site of bases 4053 to 4068.

(5). T5281D probe and T5296A probe The T5281D probe is a probe labeled with Bodipy493 / 503 having a nucleotide sequence complementary to the 5281 base-5295 base region of TMV-LRNA, and the T5296A probe is 5% of TMV-LRNA.
Having a nucleotide sequence complementary to the site of 296 bases to 5310 bases,
This is a probe labeled with y5.

(6). T5479D probe and T5494A probe The T5479D probe is a probe labeled with Bodipy493 / 503 and has a nucleotide sequence complementary to the 5479 base-5493 base region of TMV-LRNA, and the T5494A probe is TMV-LRNA.
The probe is a Cy5-labeled probe having a base sequence complementary to the site of 5494 bases to 5508 bases.

(7). T1926D probe and T1941A probe The T1926D probe is a probe labeled with Bodipy493 / 503 having a nucleotide sequence complementary to the 1926 base-1940 base site of TMV-LRNA, and the T1941A probe is one of TMV-LRNA.
Has a nucleotide sequence complementary to the site of 941 bases-1955 bases,
This is a probe labeled with y5.

For each of the seven sets of probes, two kinds of one set of probes were mixed at a molar ratio of 1: 1 and the fluorescence spectrum was measured. Further, TMV-LRNA was added to the solution at a molar ratio of 1: 1 with respect to the probe and reacted at room temperature, and then the fluorescence spectrum was measured.
A change in the spectrum was observed (FIGS. 10 to 16). As a result, when the probe combination of (1) was used, a spectrum change was observed in which the fluorescence intensity of Bodipy493 / 503 decreased and the fluorescence intensity of Cy5 increased when TMV-LRNA was added. When the probe combination of (5) was used, a change similar to that when the probe combination of (1) was used was observed, but the amount of change was smaller than that when (1) was used. Also, (2) and (3)
(4) When the probe combinations (6) and (7) were used, almost no change in spectrum was observed.

The probe combination of (1) and XELF1
When the change in fluorescence spectrum was measured by mixing with -αRNA, no change in spectrum was observed when TMV-LRNA was added. That is, the change in the fluorescence spectrum that occurred when the probe combination of (1) and TMV-LRNA were mixed,
It was a specific change when NA was used.

From these results, under the conditions tested in this example (room temperature, physiological salt concentration, etc.), the site of 62-91 bases was replaced with another complementary base sequence mixed in the solution. It can be seen that it has the property that it is easy to form a hybrid with the nucleic acid that it has, while the site of 1926-1955 bases has the property that it does not make a hybrid.

(11) Search for secondary structure of c-fosRNA c-fosRNA is a single-stranded RNA having a total length of 2094 bases. First, the following fluorescently labeled oligo DNA probes were prepared. The fluorescence-labeled oligo DNA probe was used in the experiment as a set of two types of oligo DNA labeled with Bodipy493 / 503 and one type of oligo DNA labeled with Cy5.

(1) F664D probe and F679A probe The F664D probe is a probe having a nucleotide sequence complementary to the site of 664 bases to 678 bases of c-fosRNA and labeled with Bodipy493 / 503, and the F679A probe is c-fosRNA. Of 679
Cy5 having a base sequence complementary to the site of base-693 bases
Is a probe labeled with.

The two types of probes were used in a molar ratio of 1: 1.
And the fluorescence spectrum was measured.

Further, c-fosRNA was added to this solution at a molar ratio of 1: 1 to the probe and reacted at room temperature. Then, the fluorescence spectrum was measured and the change in the spectrum was observed ( (FIG. 17). As a result, c-fosRN
When A is added, the fluorescence intensity of Bodipy493 / 503 decreases and Cy5
A change in the spectrum in which the fluorescence intensity of was increased. Using the same probe, an excess of mR from Cos7 cells
When NA (250 times by weight) was added, no change was observed in the fluorescence spectrum. In addition, c-
When the equimolar amount of fosRNA was added to the probe, the fluorescence spectrum changed. Therefore, 679-693
Under the conditions (room temperature, physiological salt concentration, etc.) tested in this example, the base site has a structure that easily forms a hybrid with another nucleic acid having a complementary base sequence mixed in the solution. It turns out that it is taking in the aqueous solution.

Next, in order to search in more detail the structure around the 664-693 base site, the following three sets of fluorescently labeled oligo DNA probes in which a part of the base sequence site was shifted upstream and downstream were used. used. Each of these probes is located at a specific 30 base site of c-fosRNA,
Two types of probes have a base sequence that enables continuous hybridization.

(2) F662D probe and F677A probe The F662D probe is a probe having a nucleotide sequence complementary to the site at 662 bases to 676 bases of c-fosRNA and labeled with Bodipy493 / 503, and the F677A probe is c-fosRNA. Of 677
Cy5 having a base sequence complementary to the site of base-691 bases
Is a probe labeled with.

(3) F666D probe and F681A probe The F666D probe is a probe having a nucleotide sequence complementary to the site of 666 to 680 bases of c-fosRNA and labeled with Bodipy493 / 503, and the F681A probe is c-fosRNA. Of 681
Cy5 has a base sequence complementary to the site of base-695 bases, and
Is a probe labeled with.

(4) F669D Probe and F684A Probe The F669D probe is a probe having a nucleotide sequence complementary to the 669 base-683 base portion of c-fosRNA and labeled with Bodipy493 / 503, and the F684A probe is c-fosRNA. Of 684
Cy5 has a base sequence complementary to the site of base-698 bases, and
Is a probe labeled with.

For each of the three sets of probes, one set of two probes was mixed at a molar ratio of 1: 1 and the fluorescence spectrum was measured. Further, c-fosRNA was added to this solution at a molar ratio of 1: 1 with respect to the probe and reacted at room temperature, and then the fluorescence spectrum was measured.
Changes in the spectrum were observed (FIGS. 18-20). As a result, the amount of change in the fluorescence spectrum was large when the probe combination of (1) and (2) was used, and was small when the probe combination of (3) and (4) shifted by 2-5 bases was used. (FIG. 21).

For each of the four sets of eight fluorescently labeled oligo DNA probes used in the above experiment, c-fosRN was used.
After mixing with A at a molar ratio of 1: 1, c-fo
The probe bound to the sRNA and the probe not bound were separated (FIG. 22). A free probe was detected at a retention time of 3 to 4 minutes, and c-fosRNA and c-fosR were detected at a retention time of 7 minutes.
The probe hybridized with NA is eluted. The formation rate of the hybrid of each probe was determined from the area ratio of each peak (FIG. 23). F662D probe,
F664D probe, F666D probe, F669D probe, F677A
All five types of probes formed hybrids with a high efficiency of 40% -80%, whereas the F679A probe
%, And the F681A probe and the F684A probe hardly formed a hybrid. These results indicate that the probe combination (3) (F666D probe and F68
1A probe) and probe combination of (4) (F669
This shows a good correlation with the result that the fluorescence spectrum of the D probe and the F684A probe) did not change much.

From these results, it can be seen that one set of two types of fluorescently labeled oligo DNA probes and the target R
By measuring the fluorescence spectrum of the mixture in an aqueous solution of NA, the conditions of the sample (room temperature, salt concentration, etc.)
It can be seen that the difference in the ease of formation of a hybrid at a specific 30 bases site in the RNA having a total length of 2000 bases in 2-5 base units can be known.

[0100]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 16 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA tomRNA sequence NTCTAGTTGG TCTGTC 16 SEQ ID NO: 2 Sequence length: 16 Sequence type: Number of nucleic acid strands: double-stranded Topology: linear Sequence type: cDNA tomRNA sequence NCTTCTAGTT GGTCTG 16 SEQ ID NO: 3 Sequence length: 16 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear sequence Type: cDNA tomRNA sequence NATCTTCTAG TTGGTC 16 SEQ ID NO: 4 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence NCTCATCTTC TAGTTG 16 SEQ ID NO: 5 Sequence Length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence GCAGACTTCT CNATCT 16 SEQ ID NO: 6 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Two Main strand Topology: Linear Sequence type: cDNA tomRNA sequence AAGCAGACTT CNTCAT 1 6 SEQ ID NO: 7 Sequence length: 16 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNAtomRNA sequence CAAAGCAGAC TNTCTC 16 SEQ ID NO: 8 Sequence length: 16 sequence type : Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence CTGCAAAGCA GNACTT 16 SEQ ID NO: 9 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence NATTATTAAT TCCATT 16 SEQ ID NO: 10 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA tomRNA sequence NGTAAAACTT AAATGT 16 SEQ ID NO: 11 Sequence Length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence NGATCCCTTT AGTTCC 16 SEQ ID NO: 12 Sequence length: 15 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA tomRNA sequence NTAGAAGGCC TGAT A 15 SEQ ID NO: 13 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNAtomRNA sequence NCATTCAACA TAATAA 16 SEQ ID NO: 14 Sequence length: 16 Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence TTTGGGATTC TNTGTA 16 SEQ ID NO: 15 Sequence length: 16 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Type Sequence type: cDNA tomRNA sequence GGCCTTCTTG GNGCAT 16 Sequence number: 16 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence ATGAATGTTG TNTTCA 16 SEQ ID NO: 17 Sequence length: 15 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA tomRNA sequence AATATTTAAA NTAAA 15 SEQ ID NO: 18 Sequence length: 16 Sequence type: number of nucleic acid strand : Double strand Topology: Linear Sequence type: cDNA tomRNA sequence AGATACTAT A TNTTAA 16 SEQ ID NO: 19 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA tomRNA sequence NGCCATTGTA GTTGTA 16 SEQ ID NO: 20 Sequence length: 16 Sequence Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNAtomRNA sequence NTCCATCTTT GAGCAA 16 SEQ ID NO: 21 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Double strand Topology: Straight Linear Sequence type: cDNA tomRNA sequence NCATGTCTAA TAAGTAA 17 SEQ ID NO: 22 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence NATACACAAT CGTTTG 16 SEQ ID NO: 23 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence NCTTTGTTGT AATACC 16 SEQ ID NO: 24 Sequence length: 16 Sequence type: Nucleic acid Strand Number: double-stranded Topology: linear Sequence type: cDNA tomRNA sequence NCTCATCAAC AACTTC 16 SEQ ID NO: 25 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence NATCTTGCAA TGCTAG 16 SEQ ID NO: 26 Sequence length: 16 Sequence Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNAtomRNA sequence GCTGTTTGTG TNGTAT 16 SEQ ID NO: 27 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Straight Linear Sequence type: cDNA tomRNA sequence ACTAACGTGT GNGTTC 16 Sequence number: 28 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence CTGCATCTAC TTNTGTA 17 Sequence number: 29 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA tomRNA Sequence TGATTTTTCTT TGNAATG 17 SEQ ID NO: 30 Sequence length: 16 Sequence type: Nucleic acid Chain Number: double-stranded topology Linear Sequence type: cDNA tomRNA sequence GTTCTTTTCT GNCATC 16 SEQ ID NO: 31 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA tomRNA sequence AACATTCTCC ANTGAA 16 Sequence number : 32 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA tomRNA sequence AGAAGCCTTT TNCAGA 16.

[0101]

The method according to the present invention is a method for detecting a higher-order structure of an RNA, wherein (1) the RNA and the RN
A1 of specific continuous base sequence A1A2 of A
P1 having a base sequence B1 capable of hybridizing with a probe, and a specific continuous base sequence A of the RNA
Base sequence B2 hybridizable with A2 of 1A2
Reacting with a probe P2 having
Detecting a continuous base sequence B1B2 formed by continuously hybridizing the probes P1 and P2 on the RNA; and (3) (i) detecting the continuous base sequence B1B2 In the case, the higher-order structure of the specific continuous base sequence A1A2 of the RNA is single-stranded, or (ii) when the continuous base sequence B1B2 is not detected, the specific continuous base sequence A1 of the RNA is not detected.
It is intended to provide a method comprising the step of judging that the higher-order structure of 1A2 is double-stranded.

Here, the higher-order structure of RNA in the present invention refers to partial double-strand formation (also called stem structure) based on the formation of base pairs in a molecule in a solution.
And a single-stranded structure of a portion not forming the base pair, or a cyclic 1
This refers to a main chain structure (referred to as a loop structure). Such a structure is in a specific equilibrium state depending on the state of the solution (temperature, salt concentration, etc.) and varies with the movement of the RNA molecule. The method according to the present invention is characterized in that R
It enables detection of the most likely higher-order structure of NA. Furthermore, when such a predetermined condition is changed, the higher-order structure of the RNA also changes according to the change. However, the method according to the present invention enables the detection of the change in the higher-order structure of the RNA. It is.

Further, c-fos mR
It becomes possible to obtain a probe for detecting the higher-order structure of NA, IL-2 RNA and TMV-L RNA.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a method for detecting a higher-order structure of RNA by a method according to the present invention. (a) shows the case where hybridization is possible, and (b) shows the case where hybridization is impossible.

FIG. 2 is a diagram schematically showing a method for detecting a change in the higher-order structure of RNA by the method according to the present invention.

FIG. 3 is a diagram schematically showing a method for detecting a higher-order structure of RNA using the FRET phenomenon by the method according to the present invention.

FIG. 4 shows a pair of two types of fluorescently labeled oligo DNA probes (L183D and L198A) and IL-2 RNA at a molar ratio of 1: 1.
FIG. 4 is a diagram showing a fluorescence spectrum when mixed in FIG.
(A) shows the fluorescence spectrum when only the probe is used. (B) shows a fluorescence spectrum after adding IL-2 RNA to the probe.

FIG. 5 shows a pair of two types of fluorescently labeled oligo DNA probes (L228D and L243A) and IL-2 RNA at a molar ratio of 1: 1.
FIG. 4 is a diagram showing a fluorescence spectrum when mixed in FIG.
(A) shows the fluorescence spectrum when only the probe is used. (B) shows a fluorescence spectrum after adding IL-2 RNA to the probe.

FIG. 6 shows a pair of two types of fluorescently labeled oligo DNA probes (L389D and L404A) and IL-2 RNA at a molar ratio of 1: 1.
FIG. 4 is a diagram showing a fluorescence spectrum when mixed in FIG.
(A) shows the fluorescence spectrum when only the probe is used. (B) shows a fluorescence spectrum after adding IL-2 RNA to the probe.

FIG. 7 shows a pair of two types of fluorescently labeled oligo DNA probes (L536D and L550A) and IL-2 RNA at a molar ratio of 1: 1.
FIG. 4 is a diagram showing a fluorescence spectrum when mixed in FIG.
(A) shows the fluorescence spectrum when only the probe is used. (B) shows a fluorescence spectrum after adding IL-2 RNA to the probe.

FIG. 8 shows a pair of two types of fluorescently labeled oligo DNA probes (L715D and L730A) and IL-2 RNA at a molar ratio of 1: 1.
FIG. 4 is a diagram showing a fluorescence spectrum when mixed in FIG.
(A) shows the fluorescence spectrum when only the probe is used. (B) shows a fluorescence spectrum after adding IL-2 RNA to the probe.

FIG. 9 shows fluorescently labeled oligo DNA probes and IL-2
It is a figure which shows the HPLC chart at the time of isolate | separating the sample which mixed RNA by molar ratio 1: 1 by high performance liquid chromatography (HPLC). The upper chart shows the elution pattern when detected by fluorescence intensity, and the lower chart shows the elution pattern when detected by ultraviolet absorption value (260 nm).

FIG. 10 shows a set of two fluorescently labeled oligo DNs.
It is a figure which shows the fluorescence spectrum when A probe (T62D and T76A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 11 shows a set of two fluorescently labeled oligo DNs.
It is a figure which shows the fluorescence spectrum when A probe (T2514D and T2529A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 12 shows a set of two fluorescently labeled oligo DNs.
It is a figure which shows the fluorescence spectrum when A probe (T3380D and T3396A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 13 shows a pair of two types of fluorescently labeled oligo DNs.
It is a figure which shows the fluorescence spectrum when A probe (T4038D and T4053A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 14 is a set of two types of fluorescently labeled oligo DNs
It is a figure which shows the fluorescence spectrum when A probe (T5281D and T5296A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 15 is a set of two fluorescently labeled oligo DNs
It is a figure which shows the fluorescence spectrum when A probe (T5479D and T5494A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 16 shows a pair of two types of fluorescently labeled oligo DN.
It is a figure which shows the fluorescence spectrum when A probe (T1926D and T1941A) and TMV-LRNA are mixed at a molar ratio of 1: 1. RNA (-) represents the fluorescence spectrum of the probe alone. RNA (+) represents the fluorescence spectrum after adding TMV-LRNA to the probe.

FIG. 17 shows a set of two fluorescently labeled oligo DNs.
It is a figure which shows the fluorescence spectrum when A probe (F664D and F679A) and c-fosRNA are mixed at a molar ratio of 1: 1.

FIG. 18 shows a set of two fluorescently labeled oligo DNs.
It is a figure which shows the fluorescence spectrum when A probe (F662D and F677A) and c-fosRNA are mixed at a molar ratio of 1: 1.

FIG. 19 is a set of two types of fluorescently labeled oligo DNs
It is a figure which shows the fluorescence spectrum when A probe (F666D and F681A) and c-fosRNA are mixed at a molar ratio of 1: 1.

FIG. 20 shows two sets of fluorescently labeled oligo DNs
It is a figure which shows the fluorescence spectrum when A probe (F669D and F684A) and c-fosRNA are mixed at a molar ratio of 1: 1.

FIG. 21 shows fluorescence intensity (I ₅₁₅ ) at ₅₁₅ nm and 670 n in the fluorescence spectra of FIGS. 17 to 20.
m (I ₆₇₀ / I ₅₁₅ ) to the fluorescence intensity (I ₆₇₀ )
It is plotted for each spectrum. For each probe combination, F664D / F679A has the spectrum shown in FIG. 17, F662D / F677A has the spectrum shown in FIG. 18, F666D / F681A has the spectrum shown in FIG.
/ F684A is the spectrum of FIG. Also, "-c
"-fosRNA" is the value of the fluorescence spectrum of the probe alone.

FIG. 22 shows fluorescently labeled oligo DNA probes and
HPLC when c-fosRNA mixed at a molar ratio of 1: 1 was separated by high performance liquid chromatography (HPLC)
FIG. The elution pattern when detected by fluorescence intensity is shown.

FIG. 23 is a diagram showing an analysis result of the HPLC chart of FIG. 19; The ratio of the component bound to c-fosRNA is shown for each probe.
The ratio of the two components of the HPLC chart (early eluting peak → free probe, late peak → probe bound to c-fosRNA) was determined from the area ratio.

FIG. 24 is a diagram schematically showing a higher-order structure of RNA as referred to in the present invention, which shows a single-stranded structure, a loop structure, or a double-stranded structure.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Akihiko Tsuji 5000 Hiraguchi, Hamakita City, Shizuoka Prefecture Inside Molecular Biophotonics Research Institute Co., Ltd.

Claims (2)

[Claims] 1. A method for detecting a higher-order structure of an RNA, comprising: (1) a probe having the RNA and a base sequence B1 capable of hybridizing with A1 among specific continuous base sequences A1A2 of the RNA; Reacting P1 with a probe P2 having a base sequence B2 capable of hybridizing with A2 in the specific continuous base sequence A1A2 of the RNA; (2) reacting the probes P1 and P2 on the RNA; Detecting a continuous base sequence B1B2 formed by continuously hybridizing, and (3) (i) when the continuous base sequence B1B2 is detected, a specific continuous base of the RNA The higher-order structure of the sequence A1A2 is single-stranded, or (ii) the continuous base sequence B1B2
If no is detected, a method comprising determining that the higher-order structure of the specific continuous base sequence A1A2 of the RNA is a double strand. 2. The method according to claim 1, wherein the method comprises:
In the step (2), (a) the probe 1
Or 2, one of the probes is labeled with an energy donor fluorescent molecule, and the other probe is labeled with an energy acceptor fluorescent molecule, and (b) the fluorescent energy transfer based on photoexcitation of the probe 1. A method comprising measuring changes in the fluorescence of probe 1 and probe 2.