JPH11127862A - Detection of target nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply detect the presence or absence of the hybrid of a target nucleic acid with a probe nucleic acid by using as a labeling system a plural kinds of labeled substances which are interacted with each other in the coexistence of the hybrid. SOLUTION: This method for detecting a target nucleic acid comprises binding through a linker at least two kinds of labeled substances (e.g. a combination of an electron donor with an electron acceptor) capable of interacting with each other by a phenomenon in which electrons flow in the double spiral structure in the coexistence of the hybrid, thus preparing a labeled unit, adding a probe nucleic acid having a base sequence complementary to the base sequence of a target nucleic acid to a sample, leaving the mixture under a condition capable of forming the hybrid of the target nucleic acid with the probe nucleic acid, mixing the prepared labeled unit with the sample in which the hybrid may be formed, detecting the presence or absence of a change based on the interaction of at least one of the labeled substances in the labeled unit, and thereby detecting the presence or absence of the hybrid in the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a technique for detecting a target nucleic acid having a specific base sequence.

[0002]

2. Description of the Related Art Various techniques for detecting a target nucleic acid having a specific base sequence have been proposed so far, and some of them have been put to practical use. The most typical technique is a solid phase hybridization method using a DNA probe. The problem with the solid-phase hybridization method is that the efficiency of trapping the target nucleic acid or the probe nucleic acid on the solid phase is low, and the process of separating the probe nucleic acid hybridized with the target nucleic acid from the probe nucleic acid not hybridized (B / F separation) is required,
In addition, strictness is required for the washing conditions at the time of B / F separation, and many techniques have been proposed to solve these problems. Examples of such a method include a fluorescence depolarization method that utilizes a change in the polarization characteristic of the fluorescence of a probe labeled with a fluorescent substance when a probe nucleic acid forms a hybrid with a single strand of a target nucleic acid. And a spin elimination method utilizing the fact that the ESR (electron spin resonance) characteristics change.

In recent years, when a probe nucleic acid forms a hybrid with a target nucleic acid, a fluorescent substance bonded to both ends of the probe nucleic acid is arranged at a position three-dimensionally close to each other. An energy transfer method utilizing the fact that energy is transferred to the other fluorescent substance when excited, and as a result the latter fluorescent substance emits light, has been proposed, and there are many patent applications relating to similar techniques.

[0004] In recent years, the double helix structure of nucleic acids has
There is a report that electrons are relatively easy to flow through the connection of nucleic acid base pairs (J. Am. Chem. Soc. 199).
2,114,3656-3660),
For example, when a probe nucleic acid hybridizes with a target nucleic acid, electrons transfer from an electron donor attached to one end of the probe nucleic acid to an electron acceptor attached to the other end, resulting in an electron donor. JP-A-6-153999 discloses a method for detecting the presence of a target nucleic acid by detecting a change in at least one of an electron acceptor and an electron acceptor.

[0005]

Here, in order to accurately detect the presence or absence of a hybrid, and also to accurately detect a mismatch in the hybrid, it is necessary to control the distance between the electron donor and the electron acceptor. preferable. As a specific method for controlling the distance between the electron donor and the electron acceptor, for example, there is a method in which these substances are respectively bound to predetermined positions of the probe nucleic acid. However, in this method, a labeling substance such as an electron donor or an electron acceptor must be bound to each probe nucleic acid corresponding to each base sequence. Since the probe nucleic acid must have a base sequence complementary to the base sequence to be recognized by the target nucleic acid to be detected in the detection, if the target nucleic acid is different, the base sequence of the corresponding probe nucleic acid must be changed each time. Must. There are various methods for binding a labeling substance to a probe nucleic acid, and the degree of difficulty in the binding operation varies depending on the desired position for binding the probe nucleic acid, the chemical structure, and the chemical structure of the labeling substance. If different substances are combined at two or more locations, the difficulty will naturally increase. Performing the synthesis of the probe nucleic acid for each base sequence of the target nucleic acid is a very laborious operation, and the detection cost is also increased accordingly.

An object of the present invention is to solve the above-mentioned problems in a method for detecting a hybrid of a target nucleic acid and a probe nucleic acid by forming a labeling system from at least two kinds of labeling substances described above.

[0007]

The detection method of the present invention is a method for detecting the presence or absence of a hybrid of a target nucleic acid and a probe nucleic acid in a sample. Preparing a labeling unit in which a first and a second labeling substance capable of interacting with each other by a phenomenon in which electrons flow in a helical structure are connected via a linker; a base sequence complementary to the base sequence of the target nucleic acid Adding to the sample a probe nucleic acid having the formula: and then subjecting the sample to conditions under which the target nucleic acid and the probe nucleic acid can form a hybrid; and a sample in which the hybrid may be formed And the labeling unit are mixed, and the presence or absence of a change based on the interaction of at least one of the first labeling substance and the second labeling substance of the labeling unit is determined. Out, and having a step, of detecting the presence or absence of hybrid in the sample.

According to the method of the present invention, instead of allowing the probe nucleic acid to act on the target nucleic acid in a state in which the labeling substance is previously bound, a hybrid substance of the probe nucleic acid and the target nucleic acid is formed and then linked with a linker. According to the present invention, a complicated operation of selecting and binding a labeling substance for each probe nucleic acid is omitted. As a result, the operation can be simplified and the measurement efficiency can be simplified.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The labeling substance used in the present invention is used for detecting a hybrid of a target nucleic acid and a probe nucleic acid in a state of being bound with a linker. As the labeling substance, at least two kinds of substances that can form a labeling system capable of producing a detectable change in accordance with electron transfer between these substances can be used. For example, a combination of an electron donor and an electron acceptor can be used. Examples of the electron donor and the electron acceptor include various dyes having these functions. As a specific example, an electron-donating dye (donor) and an electron-accepting dye (acceptor) are bonded to both ends of a linker, and the transfer of electrons from the donor to the acceptor is caused by photoexcitation of the donor dye. Alternatively, there is a method of detecting by a light emission phenomenon of an acceptor dye. Furthermore, TEMPO (2,2,6,6-tetram)
ethyl-piperidinyloxy; free
A method of capturing and detecting the movement of electrons by using a spin trapping agent such as a radical trap may be used.

The linker in the present invention forms a skeleton of a labeling unit for adding a plurality of labeling substances to the detection system in a linked state, and does not cause electron transfer via the linker itself. Moreover, in a state where the labeling substance is bound to the double helix structure of the hybrid, a plurality of labeling substances can be linked at appropriate intervals to such an extent that a change required for detection is caused by electron transfer through the hybrid. Those having various materials and structures are used.

[0011] The change due to electron transfer between the labeling substances used in the present invention can be detected between a state in which the labeling unit is not bound to the hybrid and a state in which the labeling unit is bound to the hybrid. It is sufficient if it is sufficient, and it is more preferable to use a combination of labeling substances that enables electron transfer only after the labeling unit binds to the hybrid in order to enhance detection accuracy.

The labeling unit formed by linking a labeling substance with a linker is preferably soluble in a solvent in which the target nucleic acid, the probe nucleic acid, and the hybrid of the target nucleic acid and the probe nucleic acid are soluble. For example,
An aqueous buffer similar to that used when handling biomolecules such as nucleic acids can be suitably used.

The structure of the linker is not particularly limited as long as it satisfies the above conditions. For example, an oligomer or a polymer comprising a single or a plurality of constituent elements can be used. Elements constituting the linker include amino acids, phosphoric acids, nucleotides, sugars, esters, glycols, imides, and the like, and are preferably water-soluble compounds in consideration of the reaction conditions with the hybrid. In particular, a water-soluble peptide having an α-helical structure is more preferable because it can maintain the length stably and can control the direction (orientation) of the labeling substance to some extent.

Various bonding modes can be used for bonding the linker and the labeling substance, and examples thereof include an amide bond, an ester bond, and a thioether bond.
The preferred length of the linker is 15 double-stranded DNA.
About 50 ° corresponding to に 20 base pairs.

[0015] The binding mode of the labeling substance bound to the linker with the hybrid is such that at least one labeling substance is stably bound to the hybrid to the extent necessary for measurement, and as a result, at least two labeling substances are bound to each other. The binding mode is not particularly limited as long as it is a binding mode in which electron transfer via the heavy helical structure is possible. In general, the mode of binding between a substance and a double helix structure is intercalation, in which the substance is inserted between base pairs of the double helix, and groove binding, in which the substance enters a groove formed by the double helix structure. Are known. Also in the present invention, these intercalations or groove bindings are desirable as a binding mode between the labeling substance and the hybrid having a double helix structure, in consideration of the stability of the binding and the like.

When the target nucleic acid is a double-stranded nucleic acid, a hybrid is formed between one of the single-stranded nucleic acids and the probe nucleic acid, and then reacted with a linker to which a labeling substance is bound. In the case where is detected by non-specifically binding to a double strand contained in a reaction system other than the hybrid and hinders detection, the target nucleic acid may be used as a single strand. When the target nucleic acid is a single-stranded nucleic acid such as RNA, it is not necessary to consider such a problem.

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 [1] Synthesis of peptide bound to ruthenium complex and rhodium complex (1) Synthesis of Ru (phen) ₂ phen ' ²⁺ The synthesis of ruthenium complex Ru (phen) ₂ phen' ²⁺ was described in A. M. Pyle et al. (J. Am. Chem. So
c. Synthesis was performed with reference to 1989, 111, 3051-3058. Here, phen indicates 1,10-phenanthroline,
en 'represents 5-amino-1,10-phenanthroline. (2) Synthesis of Peptide Having Rhodium Complex at Terminal by Solid Phase Method Peptide having a rhodium complex [Rh (phi) ₂ phen] at a terminal is described by Niranjan Y. Sardesa
i et al. (Bioconjugate Chem. 199).
5, 6, 302-312). Where p
hi indicates phenanthrenequinone diimine. According to this document, a peptide having a sequence consisting of the following 24 amino acids (SEQ ID NO: 1) is synthesized on a resin by the Fmoc solid-phase peptide synthesis method, and a rhodium complex is added to the terminal amino group via glutaric acid. Combined. NH ₂ -Ala-Lys-Leu-Gln-Lys-Ala-Ala-Leu-Ala-Leu-Ala
-Gln-Lys-Ile-Ala-Ala-Lys-Leu-Val-Ala-Ala-Leu-Lys-G
ly-resin In addition, the amino acids constituting the peptide were separated from the hydrophobic amino acid and the hydrophilic amino acid so that the peptide became amphiphilic. (3) Excision of Peptide from Resin Peptide extraction from peptide was also performed by Nira
njan Y. This was performed according to Sardesai et al. After cutting out, high performance liquid chromatography (HP
LC), and the desired product was recovered by freeze-drying. The coupling of the cleaved peptide with the ruthenium complex to the C-terminal carboxylic acid group was performed by performing a DCC coupling reaction in DMF (dimethylformamide). Here, DCC indicates dicyclohexylcarbodiimide. The coupling reaction was carried out under the condition of a large excess of the ruthenium complex and the DCC-related reagents (DCC and 1-hydroxybenzotriazole), purification was carried out using HPLC using a reversed-phase column, and then this was recovered by freeze-drying. (4) Formation of α-helix of synthesized peptide The circular dichroism spectrum of the labeled unit obtained by linking the synthesized rhodium complex and ruthenium complex with a peptide is 1
mM phosphate buffer (pH 7.0) / 145 mM NaCl
As a result of measurement in / 5 mM KCl, a negative peak was observed at around 220 nm, and it was confirmed that the peptide constituting the labeling unit had an α-helix structure in the phosphate buffer. [2] Synthesis of oligonucleotide A single-stranded DNA having the following base sequence complementary to a part of M13mp18 DNA (single-stranded) as a target DNA is synthesized by a conventional method, and the purity is confirmed by HPLC. Used as nucleic acid. 5'-GTTGTAAAACGACGGCCAGT-
3 ′ (SEQ ID NO: 2) [3] Hybridization reaction between oligonucleotide and M13mp18 DNA 0.2 μM of the above 20-mer probe nucleic acid and M13mp18 DNA (manufactured by Takara Shuzo Co., Ltd.) at each concentration were added to a 1 mM phosphate buffer (pH 7). .0) / 145 mM NaCl / 5 mM
The mixture was heated to 80 ° C. in KCl, and then gradually cooled to lower the temperature to room temperature to produce a hybrid having a partially double-stranded structure. Next, the previously synthesized labeled unit was added to the obtained hybrid at a final concentration of 0.2 μm.
M was added. [4] Measurement of fluorescence intensity Measurement of fluorescence intensity was performed by using a spectrofluorometer F-
4010. Using an artificial quartz cell for the measurement, fixing the concentration of the probe nucleic acid and the labeling unit,
The concentration of the target nucleic acid is 0.05 μM from 0 M to 0.25 μM
The change in the fluorescence intensity of the ruthenium complex at each concentration was measured while changing the amount by M. The excitation wavelength of the ruthenium complex was 450 nm, and the fluorescence intensity at 600 nm was measured. FIG. 1 shows the fluorescence intensity ratio at each concentration (the intensity ratio when the fluorescence intensity when the target nucleic acid is not contained (0M) is 1). FIG. 1 shows that the target nucleic acid M13mp
It can be seen that the fluorescence from the ruthenium complex weakens as the concentration of 18DNA increases. This is because electrons flow inside the double-stranded DNA as a result of the binding of the labeling unit to the hybrid. This means that the hybrid of the probe nucleic acid and the target nucleic acid has been detected by the labeling unit.

Example 2 A change in fluorescence intensity at each target nucleic acid concentration was examined in the same manner as in Example 1 except that the probe nucleic acid having the following sequence was used. 5'-GTTGTAAAAGGACGGCCAGT-
3 ′ (SEQ ID NO: 3) This sequence differs from the sequence of Example 1 by one base,
13mp18 DNA is designed to be mismatched. The results obtained are shown in FIG. As shown in the figure, no large quenching phenomenon as observed in Example 1 was observed, and no significant change was observed in the fluorescence intensity even when the concentration of the target nucleic acid was increased. This result can be attributed to the mismatch between the probe nucleic acid and the target nucleic acid, preventing the formation of an accurate double strand and the transfer of electrons through the hybrid.

Example 3 A change in fluorescence intensity at each target nucleic acid concentration was examined in the same manner as in Example 1 except that a probe nucleic acid having the following sequence was used. 5'-TATATAAAATADATATADATA-
3 ′ (SEQ ID NO: 4) The results obtained are shown in FIG. As shown in the figure, as in Example 2, no quenching phenomenon was observed, and no significant change was observed even when the concentration of the target nucleic acid was increased. This result can be attributed to the fact that the probe nucleic acid had low complementarity to the target nucleic acid, so that an accurate double strand was not formed and no electron transfer occurred.

Example 4 Detection of mRNA Using a Labeling Unit [1] Synthesis of Oligonucleotide A 22-mer probe nucleic acid having the following base sequence complementary to a part of the base sequence of human β2-adrenergic receptor mRNA was prepared. It was synthesized by the method. 5'-ATGCTGGCCGTGACGCACAGCA
-3 '(SEQ ID NO: 5) [2] from human β2 adrenergic receptor cDNA using T ₇ RNA polymerase synthesizes the human β2 adrenergic receptor mRNA by combining a conventional method of mRNA, and purified after DNase treatment. [3] Hybridization reaction between probe nucleic acid and mRNA 0.2 μm of the 22-mer oligonucleotide synthesized above
M and 0.M of human β2 adrenergic receptor mRNA.
2 μM in 1 mM phosphate buffer (pH 7.0) / 145 m
The mixture was heated to 80 ° C. in M NaCl / 5 mM KCl, and then gradually cooled to lower the temperature to room temperature to produce a hybrid having a partially double-stranded nucleic acid.

When the fluorescence intensity of this hybrid was measured in the same manner as in Example 1, the fluorescence intensity of the ruthenium complex was greatly reduced as compared with the case where no mRNA was present. This is because the labeling unit having the metal complex bound to the hybrid as in Example 1
This is because electrons flow inside the NA. This means that mRNA having a specific base sequence was detected.

Example 5 A change in fluorescence intensity at each target nucleic acid concentration was examined in the same manner as in Example 4 except that a probe nucleic acid having the following sequence was used. 5'-CGATAAATCAGCATTGCTATTT
-3 '(SEQ ID NO: 6) The sequence of this probe nucleic acid is not complementary to the nucleotide sequence of human β2-adrenergic receptor mRNA. When the probe nucleic acid having this base sequence was used, the quenching phenomenon as in Example 4 was not observed, and the same fluorescence intensity was measured as when measured using only the labeling unit. This result is because a double-stranded nucleic acid was not formed because the base sequence of the probe nucleic acid was not complementary to the target nucleic acid, and the quenching phenomenon of the ruthenium complex due to electron transfer through the double-stranded nucleic acid did not occur. You can say that.

Example 6 [1] Synthesis of Peptide Linked with Ruthenium Complex, Pyrene and TEMPO (1) Synthesis of Ru (phen) ₂ phen ″ ²⁺ The ruthenium complex Ru (phen) ₂ phen ″ ²⁺
A. M. Pyle et al. (J. Am. Chem. Soc. 1)
989, 111, 3051-3058. Here, phen indicates 1,10-phenanthroline, and phen ''
Is 5- (aminoglutaryl) -1,10-phenanthroline [5- (aminoglutaryl) -1,10
-Phenanthroline]. Also, ph
The synthesis of en '' is described in Niranjan Y. Sardes
ai et al. (Bioconjugate Chem. 199)
5, 6, 302-312). (2) Synthesis of peptide A peptide having the following amino acid sequence (SEQ ID NO: 7) was synthesized according to the Fmoc solid-phase synthesis method as in Example 1. NH ₂ -Ala-Lys-Leu-Gln-Lys-Ala-Ala-Leu-Ala-Leu-Ala
-Gln-Lys-Ile-Ala-Ala-Lys-Leu-Val-Ala-Ala-Leu-Lys-G
ly-resin However, since it is necessary to introduce TEMPO into the side chain at the specific site of the peptide (the amino group of the second Lys from the N-terminal), lysine (Lys) closest to the N-terminal is defined as The amino group is changed to Boc (tert-butoxy)
Those protected with a carbonyl group are used, and the amino group of the other lysine is ClZ (carbobyl).
Those protected by an enzyme chloride group were used. The synthesis was performed with the content of the peptide in the resin being 0.2 mmol. (3) Peptide and 1-pyrenebutyric acid (1-pyreneb)
231 mg (0.8 mmol) of 1-pyrenebutyric acid in DMF
Dissolve in 0.8 ml and add 1-hydroxybenzotriazole (1-hydroxybenzotriazole).
e: HoBt) in 1.6 ml of a 0.5 M DMF solution and 101 mg of diisopropylcarbodiimide (0.8 mmol)
l) was added and the mixture was stirred at room temperature for 30 minutes. H of this pyrene
The oBt ester was added to the deprotected peptide on the resin and allowed to react for 10 minutes. After confirming that the reaction was completely completed by a ninhydrin reaction or the like, the resultant was washed with DMF. (4) Removal of Boc group as a protecting group and cleavage of peptide from resin Removal of peptide from resin was performed using trifluoroacetic acid according to a conventional method. By excision with trifluoroacetic acid, the Boc group protecting the amino group of lysine was removed, and the amino group of lysine was deprotected at this stage. At this stage, the ClZ group which is a protecting group is not eliminated. The resulting product was purified by HPLC and collected by freeze-drying. (5) Binding of TEMPO to peptide After dissolving the obtained peptide in a small amount of DMSO (dimethyl sulfoxide), 4-isothiocyanate TEM
21 mg (0.1 mmol) of PO was added and reacted overnight. After completion of the reaction, the DMSO solution was used for the next reaction without purification, in particular. (6) Binding of Ruthenium Complex to Peptide The terminal amino group of the peptide to which pyrene and TEPO are bound and the previously synthesized ruthenium complex Ru (phen) ₂ p
The amino group of hen '' ²⁺ was coupled by a conventional DCC coupling reaction. The reaction was performed under the condition of a large excess of the ruthenium complex and the DCC-related reagent. After the reaction, purification was performed by HPLC to obtain a peptide as a labeling unit to which pyrene, TEMPO and a ruthenium complex were bound. (7) Removal of ClZ group ClZ group which was not removed by trifluoroacetic acid, which was a protecting group, was removed by TMSOTf / TFA and deprotected according to a conventional method. (8) α-Helix formation of the synthesized peptide The circular dichroic acid speckle of the synthesized labeling unit is 1
mM phosphate buffer (pH 7.0) / 145 mM NaC
As a result of measurement in 1/5 mM KCl, a negative peak appeared around 220 nm. This peak is a typical peak of a peptide having an α-helical structure, and it was confirmed that the peptide constituting the skeleton of the labeling unit formed an α-helical structure in this buffer. [2] Probe nucleic acid and target nucleic acid A reaction was carried out in the same manner as in [3] of Example 1, except that the labeling unit was changed to the one obtained by the above operations (1) to (7). [3] Measurement of ESR spectrum ES of sample obtained by each reaction of the above [2]
R was performed using an apparatus manufactured by JEOL Ltd. For the measurement,
A flat cell made of artificial quartz was used. After preparing the sample, the sample was irradiated with excitation light of 450 nm, which is the excitation wavelength of the ruthenium complex, and used for measurement. The settings of the ESR and the light irradiation device are as follows.

[0024]

[Table 1] 2 and 3 show the ESR signal intensity ratio and the line width with respect to the concentration of the target nucleic acid. As can be seen from FIGS. 2 and 3, it can be seen that as the concentration of the target nucleic acid is increased, the ESR intensity ratio decreases without changing the line width. Since no change occurs in the line width, it can be seen that this change in ESR intensity is not due to a chemical change. Therefore, as a result of electrons flowing from the ruthenium complex bonded to the terminal of the peptide to the TEMPO bonded to the opposite side via the peptide, it was confirmed that the spin of TEMPO was eliminated.

Example 7 The ESR intensity of each sample was measured in the same manner as in Example 6 except that the following sequence was used as the probe nucleic acid sequence. 5'-GTTGTAAAAGGACGGCCAGT-
3 ′ (SEQ ID NO: 8) This sequence differs from the sequence of Example 6 by one base,
It is designed to mismatch with M13mp18 DNA. As a result of measuring the ESR intensity of each sample, the intensity change of the ESR signal depending on the concentration of the target nucleic acid as observed in Example 6 was not observed, and only the probe nucleic acid was used in the reaction system (the target nucleic acid contained No), the same strength as in the case. This can be said that in the case of a hybrid of a mismatched probe nucleic acid and target nucleic acid, an accurate double strand was not formed and electrons did not move.

[0026]

The method for detecting a target nucleic acid according to the present invention has an advantage that it is not necessary to perform complicated synthesis such as labeling of a probe nucleic acid for each base sequence. Further, in the method of binding a labeling substance to a probe nucleic acid, there may be a case where a restriction can be imposed on a substance that can be used as a label in these matchings. Not,
The selection range of the labeling substance can be expanded. As a result, the target nucleic acid can be easily detected simply by synthesizing the probe nucleic acid for each base sequence.

[0027]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 24 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ala Lys Leu Gln Lys Ala Ala Leu Ala Leu Ala Gln Lys Ile 1 5 10 Ala Ala Lys Leu Val Ala Ala Leu Lys Gly 15 20 SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTTGTAAAAC GACGGCCAGT 20 SEQ ID NO: 3 Sequence length: 20 Sequence Type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTTGTAAAAG GACGGCCAGT SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TATATAAAAT ATATATATAT 20 SEQ ID NO: 5 Sequence length: 22 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATGCTGGCCG TGACGCACAG CA 22 SEQ ID NO: 6 Sequence length: 22 Sequence type : Acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGATAAATCA GCATTGCTAT TT 22 SEQ ID NO: 7 Sequence length: 24 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Lys Leu Gln Lys Ala Ala Leu Ala Leu Ala Gln Lys Ile Ala Ala Lys 1 5 10 15 Leu Val Ala Ala leu Lys Gly 20 SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other Nucleic acid of synthetic DNA sequence GTTGTAAAAG GACGGCCAGT

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the fluorescence intensities measured in Examples 1 to 3 as a fluorescence intensity ratio where the fluorescence intensity without adding a target nucleic acid is 1; , × indicate the results obtained in Example 2, and Δ indicate the results obtained in Example 3.

FIG. 2 is a diagram showing a time-dependent change in ESR signal intensity obtained in Examples 6 and 7 as an intensity ratio where the intensity when a target nucleic acid is not added is set to 1; ■ indicates the results obtained in Example 7, respectively.

FIG. 3 is a diagram showing a change over time in the line width of an ESR signal obtained in Examples 6 and 7, in which Δ represents the result obtained in Example 6, and Δ represents the result obtained in Example 7. Are respectively shown.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Mitsuhiko Shioya 2-4-1 Tatsumigaoka, Okazaki City, Aichi Prefecture

Claims (13)

[Claims] 1. A method for detecting the presence or absence of a hybrid of a target nucleic acid and a probe nucleic acid in a sample, wherein the interaction is caused by a phenomenon that electrons flow through the double helix structure in the presence of the hybrid. First possible
And preparing a labeling unit in which a second labeling substance is bound via a linker; adding a probe nucleic acid having a base sequence complementary to the base sequence of the target nucleic acid to the sample; Placing the target nucleic acid and the probe nucleic acid under conditions capable of forming a hybrid; and mixing the sample in which the hybrid is likely to be formed with the labeling unit; Detecting the presence or absence of a change based on the interaction of at least one of the labeling substance and the second labeling substance, and detecting the presence or absence of the hybrid in the sample. 2. The detection method according to claim 1, wherein the electron transfer occurs only after the labeling substance of the labeling unit binds to the double helix structure. 3. The method according to claim 2, wherein the labeling unit comprises the target nucleic acid,
The detection method according to claim 1, wherein the probe nucleic acid and the hybrid are soluble in a soluble solvent. 4. The method according to claim 3, wherein the solvent is an aqueous buffer.
The detection method described in 1. 5. The detection method according to claim 1, wherein the linker is an oligomer or a polymer composed of a water-soluble constituent. 6. The detection method according to claim 5, wherein the constituent elements of the linker include at least one of phosphate, nucleotide, sugar, glycol, amino acid and imide. 7. The detection method according to claim 5, wherein the linker is a peptide having an α-helix structure. 8. The detection method according to claim 1, wherein the binding between at least one of the labeling substances and the double helix structure is by intercalation. 9. The detection method according to claim 1, wherein the binding between at least one of the labeling substances and the double helix structure is by groove binding. 10. The method according to claim 1, wherein the target nucleic acid is a single-stranded nucleic acid. 11. The detection method according to claim 10, wherein the single-stranded nucleic acid is RNA. 12. The method according to claim 1, wherein the RNA is mRNA.
2. The detection method according to 1. 13. A method for detecting the presence or absence of a mismatch in a nucleic acid hybrid having a double helix structure, wherein the method occurs when there is no mismatch in the double helix structure.
A step of preparing a labeling unit in which a first and a second labeling substance which can interact with each other by a phenomenon in which electrons flow in a double helix structure are connected via a linker; a base sequence of a target nucleic acid contained in the sample Mixing a probe nucleic acid having a base sequence that is likely to be complementary to the sample with the sample, and then hybridizing the probe nucleic acid and the target nucleic acid in the sample; and A sample containing a hybrid is mixed with the labeling unit, and the presence or absence of a change based on the interaction of at least one of the first and second labeling substances is detected, whereby a mismatch in the hybrid is detected. Detecting the presence / absence of the sample.