Fluorescently labeled single-stranded nucleic acid and its uses

Abstract

The purpose of the present invention is to provide a novel fluorescently labeled single-stranded nucleic acid that further reduces the background of exciton oligomers, and to provide a new use of the fluorescently labeled single-stranded nucleic acid. The present invention relates to a labeled single-stranded nucleic acid having at least two pairs of fluorescent atomic groups that show an exciton effect. A labeled single-stranded nucleic acid, characterized in that the luminescence peak wavelength of one of the pair of fluorescent atomic groups (a pair of fluorescent atomic groups A) is shorter than the excitation peak wavelength of the other of the pair of fluorescent atomic groups (a pair of fluorescent atomic groups B), and the pair of fluorescent atomic groups A and the pair of fluorescent atomic groups B have a Forster resonance energy transfer (FRET) effect. It is used as a primer for amplifying a target nucleic acid or a probe for hybridizing with a target nucleic acid.

Description

Technical Field

[0001] The present invention relates to a fluorescently labeled single-stranded nucleic acid and its use. The present invention relates to a fluorescently labeled single-stranded nucleic acid and its use that can further reduce the fluorescence background.

[0002] Cross-references between related applications

[0003] This application claims the benefit of Japanese Patent Application No. 2014-72280, filed on March 31, 2014, the entire contents of which are expressly incorporated herein by reference. Background Art

[0004] In the analysis of cellular life phenomena and the diagnosis of disease factors, detection and diagnosis at the molecular level are required. In order to achieve this detection and diagnosis, it is necessary to detect specific proteins and nucleic acid sequences. In this detection, fluorescence is widely used. Specifically, it is known to use a method of increasing the fluorescence intensity of a fluorescent substance that is bonded to a target substance such as a target protein and a target nucleic acid sequence. As the above-mentioned fluorescent substance, for example, a substance that shows the Forster resonance energy transfer (FRET) effect, a substance that is embedded in a double helix structure and emits fluorescence by irradiation with excitation light is used.

[0005] For example, in the Molecular Beacon method of putting down in writing in non-patent literature 1, use the nucleic acid of different pigments having been imported respectively at 5 ' end, 3 ' end of the nucleotide sequence that takes stem-loop structure independently.When not having hybridization, extinction by FRET effect, if produce specific hybridization, then emit fluorescence.In this method, there is sequence need to take stem-loop structure, need fluorescent pigment to be imported into restrictions such as end.

[0006] Therefore, as an alternative to the above-mentioned prior art extinction mechanism, a method has been proposed that utilizes the exciton effect generated by the juxtaposition of two or more pigment molecules (Non-Patent Documents 2 to 5, Patent Document 1). This method uses a complex labeling substance having the following chemical structure within the same molecule: when single-stranded, it does not exhibit fluorescence due to the exciton effect. However, when these molecules are embedded in nucleic acids or undergo groove binding, the above-mentioned aggregation state is released, generating fluorescence.

[0007] The primer or probe (sometimes referred to as exciton oligomer) obtained by importing the labeling substance into oligonucleotide can be used for amplification and detection of target nucleic acid. The exciton oligomer etc. can realize the switching of fluorescence before and after hybridization with a kind of pigment. In addition, when being utilized in the real-time monitoring of amplification reaction, a sequence-specific fluorescent signal can be given. Therefore, it is possible to overcome the problem of the prior art that nonspecific amplification is also detected when using embedding agents such as SYBR Green I. And then, since fluorophores can be imported into dT or dC, it is also possible to avoid the restriction of sequence.

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 2009-171935 (Japanese Patent No. 4370385)

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2013-183736

[0010] The entire descriptions of Patent Documents 1 and 2 are expressly incorporated herein by reference.

[0011] Non-patent document 1: Tyagi, S., Kramer, FR (1996) Nat. Biotechnol. 14, 303-308.

[0012] Non-patent document 2: Ikeda S, Kubota T, Kino K, Okamoto A., Bioconjug Chem. 2008.19:1719-1725.

[0013] Non-Patent Document 3: Ikeda S, Kubota T, Yuki M, Okamoto A., Angew Chem Int Ed Engl. 2009.48.6480-6484.

[0014] Non-Patent Document 4: Ikeda S, Yuki M, Yanagisawa H, Okamoto A., Tetrahedron Lett. 2009, 51, 7191-7195

[0015] Non-patent document 5: Takeshi Hanami, Diane Delobel, Hajime Kanamori, Yuki Tanaka, Yasumasa Kimura, Ayako Nakasone, Takahiro Soma, Yoshihide Hayashizaki, KengoUsui, Matthias Harbers, PLOS ONE, August 2013, volume8, Issue 8, e70942

[0016] The entire descriptions of Non-Patent Documents 1 to 5 are expressly incorporated herein by reference. Summary of the Invention

[0017] Problems to be solved by the invention

[0018] However, further research by the present inventors has revealed that even when using the aforementioned excitonic oligomers, a certain amount of background light is present in highly sensitive measurements. Furthermore, it has been found that this background light sometimes hinders fluorescence detection in methods that use excitonic oligomers as probes and detect the weak fluorescence generated when they bind to trace amounts of target.

[0019] Therefore, an object of the present invention is to provide a novel fluorescently labeled single-stranded nucleic acid having a further reduced background of the above-mentioned excitonic oligomer, and to provide a novel use of the fluorescently labeled single-stranded nucleic acid.

[0020] Conventional exciton oligomers are labeled single-stranded nucleic acids with two fluorescent dyes (thiazole orange or its analogs) incorporated into them. In their single-stranded state, they emit virtually no fluorescence due to the exciton effect of the two fluorescent dyes forming an exciton complex. However, they exhibit the following properties: upon hybridization with target DNA, the two dyes separate, eliminating the exciton effect and allowing the fluorescent dye's inherent fluorescence to be realized.

[0021] However, the inventors' research revealed that the extinction mechanism of fluorescence generated by the exciton effect is not perfect, and the inherent fluorescence of the fluorochrome cannot be completely extinguished. Consequently, background fluorescence from single-chain structures, while minimal, still exists. Consequently, they conducted extensive research to further reduce background fluorescence using exciton oligomers as their basic framework. As a result, they discovered that combining fluorescence switching generated by the exciton effect with the FRET effect could further reduce background fluorescence, leading to the completion of the present invention.

[0022] Solutions to Problems

[0023] The present invention is as follows.

[0024] [1] A labeled single-stranded nucleic acid, characterized in that it is a labeled single-stranded nucleic acid having at least two pairs of fluorescent atomic groups that exhibit an exciton effect,

[0025] The emission peak wavelength of one of the pair of fluorescent atomic groups (hereinafter referred to as the pair of fluorescent atomic groups A) is shorter than the excitation peak wavelength of the other of the pair of fluorescent atomic groups (hereinafter referred to as the pair of fluorescent atomic groups B).

[0026] The pair of fluorescent atomic groups A and the pair of fluorescent atomic groups B have a Forster resonance energy transfer (FRET) effect.

[0027] [2] The labeled single-stranded nucleic acid according to [1], wherein the base having the pair of fluorescent atomic groups A and the base having the pair of fluorescent atomic groups B are contained in the labeled single-stranded nucleic acid at a distance such that the pair of fluorescent atomic groups A and the pair of fluorescent atomic groups B have a FRET effect.

[0028] [3] The labeled single-stranded nucleic acid according to [2], wherein

[0029] The distance between the base having the pair of fluorescent atomic groups A and the base having the pair of fluorescent atomic groups B is 1 to 11 bases.

[0030] [4] The labeled single-stranded nucleic acid according to any one of [1] to [3], wherein

[0031] The base having a pair of fluorescent atomic groups exhibiting the exciton effect has a structure represented by the following formula (16), (16b), (17), or (17b).

[0032] [Chemical Formula 1]

[0033]

[0034] [Chemical Formula 2]

[0035]

[0036] [Chemical Formula 3]

[0037]

[0038] [Chemical Formula 4]

[0039]

[0040] In formulas (16), (16b), (17), and (17b),

[0041] B is an atomic group having a natural nucleic acid base (adenine, guanine, cytosine, thymine or uracil) skeleton or an artificial nucleic acid base skeleton,

[0042] E is (i) an atomic group having a deoxyribose backbone, a ribose backbone, or a structure derived from either of them, or (ii) an atomic group having a peptide structure or a peptoid structure,

[0043] Z 11 and Z 12 They are fluorescent atomic groups showing exciton effect, which can be the same or different.

[0044] L1 , L 2 and L 3 are connecting segments (cross-linking atoms or atomic groups), the main chain length (number of main chain atoms) is arbitrary, the main chain may contain or not contain C, N, O, S, P and Si, the main chain may contain or not contain single bonds, double bonds, triple bonds, amide bonds, ester bonds, disulfide bonds, imino groups, ether bonds, thioether bonds and thioester bonds, L 1 , L 2 and L 3 They can be the same or different from each other.

[0045] D is CR, N, P, P=O, B or SiR, R is a hydrogen atom, an alkyl group or any substituent,

[0046] b is a single bond, a double bond or a triple bond,

[0047] Alternatively, in the above formulas (16) and (16b), L 1 and L 2 is the above-mentioned connecting fragment, L 3 , D and b do not exist, L 1 and L 2 It can also be directly bonded to B.

[0048] In formulas (16) and (17), E is the atomic group of (i) above, and at least one O atom in the phosphate crosslink may be replaced by a S atom.

[0049] In formula (16b) and (17b), E is the atomic group of (ii) above,

[0050] In formulae (17) and (17b), each B may be the same or different, and each E may be the same or different.

[0051] [5] The labeled single-stranded nucleic acid according to [4], wherein

[0052] The structure represented by the above formula (16) is a structure represented by the following formula (16-1) or (16-2),

[0053] The structure represented by the above formula (16b) is a structure represented by the following formula (16b-1) or (16b-2),

[0054] The structure represented by the above formula (17) is the structure represented by the following formula (17-1),

[0055] The structure represented by the above formula (17b) is a structure represented by the following formula (17b-1).

[0056] [Chemical Formula 5]

[0057]

[0058] [Chemical Formula 6]

[0059]

[0060] [Chemical Formula 7]

[0061]

[0062] [Chemical Formula 8]

[0063]

[0064] [Chemical Formula 9]

[0065]

[0066] [Chemical Formula 10]

[0067]

[0068] In formulas (16-1), (16-2), (16b-1), (16b-2), (17-1), and (17b-1),

[0069] l, m and n are any positive integers, which may be the same or different, and may or may not contain C, N, O, S, P and Si in the main chain, and may or may not contain single bonds, double bonds, triple bonds, amide bonds, ester bonds, disulfide bonds, imino groups, ether bonds, thioether bonds and thioester bonds in the main chain,

[0070] B, E, Z 11 , Z 12 and b are the same as those in the above formulas (16), (16b), (17), and (17b),

[0071] In the above formulae (16-1), (16-2), and (17-1), one or more O atoms in the phosphate crosslinks may be substituted with S atoms.

[0072] [6] The labeled single-stranded nucleic acid according to [4] or [5], wherein

[0073] The base having a pair of fluorescent atomic groups exhibiting the exciton effect has a structure represented by the above formula (16).

[0074] [7] The labeled single-stranded nucleic acid according to any one of [4] to [6], wherein

[0075] Z 11 and Z 12 Each independently represents an atomic group represented by any one of the following formulae (7) to (10).

[0076] [Chemical Formula 11]

[0077]

[0078] [Chemical Formula 12]

[0079]

[0080] [Chemical Formula 13]

[0081]

[0082] [Chemical Formula 14]

[0083]

[0084] In formulas (7) to (9),

[0085] X 1 and X 2 is S or O,

[0086] n is 0 or a positive integer,

[0087] R 1 ~R 10 、R 13 ~R 21 are independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,

[0088] R 11 and R 12 One of them is the same as L in the above formulas (16), (17), (16b), and (17b) 1 or L 2 The other of the bonded linking groups is a hydrogen atom or a lower alkyl group,

[0089] R 15 When there are multiple in formula (7), (8) or (9), they may be the same or different.

[0090] R 16 When there are multiple in formula (7), (8) or (9), they may be the same or different.

[0091] Z 11 X in 1 、X 2 and R 1 ~R 21 With Z 12 X in 1 、X 2 and R 1 ~R 21 They can be the same or different from each other.

[0092] In formula (10),

[0093] E is S or O,

[0094] R 2 ~R 12 are independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,

[0095] R 1 is the same as L in the above formulas (16), (17), (16b), and (17b) 1 or L 2 bonded linking group,

[0096] R 3 When there are multiple 's in formula (10), they may be the same or different.

[0097] R 4 When there are multiple 's in formula (10), they may be the same or different.

[0098] [8] The labeled single-stranded nucleic acid according to [7], wherein

[0099] Z 11 and Z 12 are independently an atomic group represented by the above formula (7) or (8),

[0100] Z represented by the above formula (7) or (8) 11 and Z 12 It is a group represented by the following formula (19) or (20).

[0101] [Chemical Formula 15]

[0102]

[0103] [Chemical Formula 16]

[0104]

[0105] In formulas (19) and (20),

[0106] X 1 、R 1 to R 10 、R 13 and R 14 、R 11 and R 12 Same as equations (7) to (9).

[0107] [9] The labeled single-stranded nucleic acid according to any one of [1] to [8],

[0108] It is used as a primer for amplifying a target nucleic acid or as a probe for hybridizing with a target nucleic acid.

[0109]

[10] A method for detecting a target nucleic acid, wherein:

[0110] The labeled single-stranded nucleic acid according to any one of [1] to [8] is used as a probe, and under conditions allowing hybridization with the target nucleic acid, the presence or absence of hybridization with the probe is determined by measuring fluorescence.

[0111]

[11] A method for amplifying a target nucleic acid,

[0112] The method comprises the following steps: using the labeled single-stranded nucleic acid described in any one of [1] to [8] as a primer to amplify the target nucleic acid.

[0113] Effects of the Invention

[0114] According to the present invention, it is possible to provide a labeled single-stranded nucleic acid having an excitonic oligomer as a basic skeleton, that is, a labeled single-stranded nucleic acid capable of further reducing the background of fluorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1A The following table shows the spectral measurement results of the fluorescent nucleic acid probe of the present invention, obtained in Example 1, which has two fluorescent dyes having an exciton effect. The results shown here are those using an oligonucleotide (EX16-12TOTP) having thiazole powder (TP) at the 12th base from the 3' end and thiazole orange (TO) at the 16th base from the 3' end.

[0116] Figure 1B The following table shows the spectral measurement results of the fluorescent nucleic acid probe (conventional technology) introduced with a fluorescent dye having an exciton effect, obtained in Example 1. The results shown here are obtained using an oligonucleotide (EX16.TO) having the same sequence as EX16-12TOTP and containing thiazole orange (TO) at the 16th base from the 3' end.

[0117] Figure 1C The following table shows the spectral measurement results of the fluorescent nucleic acid probe (conventional technology) introduced with a fluorescent dye exhibiting an exciton effect, obtained in Example 1. The results shown here are obtained using an oligonucleotide (EX16.TP) having the same sequence as EX16-12TOTP and containing a thiazole powder (TP) at the 12th base from the 3' end.

[0118] Figure 2AMelting curve analysis results for the fluorescent nucleic acid probe (EX8-12TOTP) obtained in Example 1, which incorporates two fluorescent dyes with an exciton effect (distance between thiazole orange (TO, 8th base from the 3' end) and thiazole powder (TP, 12th base from the 3' end): 3 bases, are shown. For comparison, melting curve analysis results for a fluorescent nucleic acid probe in which only thiazole orange (TO) was incorporated at the same position are also shown.

[0119] Figure 2B Melting curve analysis results for the fluorescent nucleic acid probe (EX10-12TOTP) obtained in Example 1, which incorporates two fluorescent dyes exhibiting an exciton effect (distance between thiazole orange (TO, 10th base from the 3' end) and thiazole powder (TP, 12th base from the 3' end): 1 base, are shown. For comparison, melting curve analysis results for a fluorescent nucleic acid probe in which only thiazole orange (TO) was incorporated at the same position are also shown.

[0120] Figure 2C Melting curve analysis results for the fluorescent nucleic acid probe (EX14-12TOTP) obtained in Example 1, which incorporates two fluorescent dyes with an exciton effect (distance between thiazole orange (TO, 14th base from the 3' end) and thiazole powder (TP, 12th base from the 3' end): 1 base, are shown. For comparison, melting curve analysis results for a fluorescent nucleic acid probe in which only thiazole orange (TO) was incorporated at the same position are also shown.

[0121] Figure 2D Melting curve analysis results for the fluorescent nucleic acid probe (EX16-12TOTP) obtained in Example 1, which incorporates two fluorescent dyes with an exciton effect (distance between thiazole orange (TO, 16th base from the 3' end) and thiazole powder (TP, 12th base from the 3' end): 4 bases, are shown. For comparison, melting curve analysis results for a fluorescent nucleic acid probe in which only thiazole orange (TO) was incorporated at the same position are also shown.

[0122] Figure 2E Melting curve analysis results for the fluorescent nucleic acid probe (EX18-12TOTP) obtained in Example 1, which incorporates two fluorescent dyes with an exciton effect (distance between thiazole orange (TO, 18th base from the 3' end) and thiazole powder (TP, 12th base from the 3' end): 8 bases. For comparison, melting curve analysis results for a fluorescent nucleic acid probe in which only thiazole orange (TO) was incorporated at the same position are also shown. DETAILED DESCRIPTION

[0123] <Labeled Single-Stranded Nucleic Acid>

[0124] The present invention provides a labeled single-stranded nucleic acid having at least two pairs of fluorescent atomic groups that exhibit an exciton effect. Furthermore, the labeled single-stranded nucleic acid of the present invention is characterized in that:

[0125] (a) the emission peak wavelength of one of the pair of fluorescent atomic groups (the pair of fluorescent atomic groups A) is shorter than the excitation peak wavelength of the other of the pair of fluorescent atomic groups (the pair of fluorescent atomic groups B), and

[0126] (b) The pair of fluorescent atomic groups A and the pair of fluorescent atomic groups B have a Forster resonance energy transfer (FRET) effect.

[0127] A pair of fluorescent atomic groups exhibiting an exciton effect, and a labeled single-stranded nucleic acid having the pair of fluorescent atomic groups exhibiting an exciton effect, are described in Patent Documents 1 and 2, and Non-Patent Documents 2 to 5. However, a labeled single-stranded nucleic acid having at least two pairs of fluorescent atomic groups exhibiting an exciton effect, which has the characteristics of (a) and (b) described above, is not described in Patent Documents 1 and 2, and Non-Patent Documents 2 to 5.

[0128] The labeled single-stranded nucleic acid of the present invention is a single-stranded nucleic acid having at least two pairs of fluorescent atomic groups that exhibit an exciton effect.

[0129] The single-stranded nucleic acid can be DNA or RNA or a mixture thereof, or a nucleic acid partially or entirely containing non-natural nucleic acid bases. In addition, the labeled single-stranded nucleic acid of the present invention may also partially contain a double-stranded structure as long as it can hybridize with the target nucleic acid. Details will be described later.

[0130] There is no particular restriction on the base length of the labeled single-stranded nucleic acid, but since the main use is a probe or primer, and further a single-stranded nucleic acid having at least two pairs of fluorescent atomic groups that show an exciton effect, and in order to satisfy the single-stranded nucleic acid of (b) above, the base length of the single-stranded nucleic acid is, for example, in the range of 4 to 100 bases long, preferably in the range of 10 to 50 bases long, more preferably in the range of 10 to 40 bases long, and further preferably in the range of 10 to 30 bases long. The base length can be appropriately selected according to the use. For example, in the case of a capture agent for mRNA, a single-stranded nucleic acid of about 80 bases long is suitable, in the case of use as a PCR primer, a single-stranded nucleic acid of about 40 bases long is suitable, and in the case of use as a probe, a single-stranded nucleic acid of about 30 bases long is suitable.

[0131] The number of a pair of fluorescent atomic groups exhibiting an exciton effect possessed by the labeled single-stranded nucleic acid is at least two, and can be two or more. In practice, in order to exert the FRET effect, the number of a pair of fluorescent atomic groups exhibiting an exciton effect only needs to be two. However, considering the purpose of the labeled single-stranded nucleic acid, the type of fluorescent atomic group, the distance between the pair of fluorescent atomic groups, the degree of FRET effect, etc., the number can also be three, and further can be four or more.

[0132] According to the exciton effect, for example, the fluorescence intensity in the single-stranded state can be suppressed, and the double helix structure can be effectively detected. The so-called exciton effect (exciton coupling) is, for example, an effect in which fluorescence luminescence is substantially not displayed by assembling and forming an H-aggregate (H-aggregate) in parallel with multiple pigments. It is believed that this effect is caused by the excitation state of the pigment being split into two energy levels by Davydov splitting, and the excitation to the high energy level → the internal conversion to the low energy level → the luminescence is thermally forbidden. However, these explanations do not limit the present invention in any way. The so-called exciton effect can be confirmed by the absorption band of the pigment that forms the H-aggregate appearing at a wavelength shorter than the absorption band of a single pigment. As pigments that display such an effect, for example, the above-mentioned thiazole orange and its derivatives, thiazole powder and its derivatives, oxazole yellow and its derivatives, cyanine and its derivatives, semicyanine and its derivatives, methyl red and its derivatives, and the pigment group generally referred to as cyanine pigments and azo pigments can be listed.

[0133] These pigments are easily bonded by embedding double-strand, and described double-strand is by forming the DNA-DNA double-strand or DNA-RNA double-strand or thiophosphate nucleic acid or PNA (peptide nucleic acid) or locked nucleic acid (LNA) (BNA) artificial nucleic acid, with DNA or RNA formation.If a plurality of such pigments are imported in the single-stranded nucleic acid, then under common single-strand state (for example, the state of only probe or primer before hybridization), by strong exciton effect extinction, but if then aggregate is removed with target DNA or RNA hybridization, each pigment is dispersedly embedded in the double-strand.Owing to now, between pigment, there is not the interaction of electron, so do not produce exciton effect, show strong fluorescence.The absorption band of pigment now is identical with the absorption band of single pigment, is presented at and does not produce exciton effect between pigment.In addition, when pigment is embedded in the double-strand, because the distortion on the structure that pigment originally had is eliminated, so further strengthen fluorescence sometimes.

[0134] Feature (a)

[0135] The luminescence peak wavelength possessed by one of a pair of fluorescent atomic groups (a pair of fluorescent atomic groups A) is shorter than the excitation peak wavelength possessed by one of the remaining pair of fluorescent atomic groups (a pair of fluorescent atomic groups B). The luminescence peak wavelength refers to the peak wavelength of the luminescence spectrum generated when a pair of fluorescent atomic groups A is irradiated with excitation light, and changes according to the type of fluorescent atomic group A. The excitation peak wavelength refers to the peak wavelength of the excitation light spectrum that a pair of fluorescent atomic groups B can absorb, and changes according to the type of fluorescent atomic group B. There is no limitation on the luminescence peak wavelength possessed by a pair of fluorescent atomic groups A and the excitation peak wavelength possessed by a pair of fluorescent atomic groups B. However, in the case where the labeled single-stranded nucleic acid of the present invention is used as a probe or primer, and the fluorescent label is used for detection, since the fluorescent luminescence is luminescence from the fluorescent atomic group B, a fluorescent atomic group B with a luminescence intensity and wavelength suitable for detection can be selected, and the fluorescent atomic group A is selected based on the excitation peak wavelength possessed by the fluorescent atomic group B. In addition, the relationship between the emission peak wavelength possessed by the fluorescent atom group A and the excitation peak wavelength possessed by the fluorescent atom group B can be determined by taking into account the FRET effect obtained between the two. In addition, the two fluorescent atom groups possessed by a pair of fluorescent atom groups A may be the same or different, and the two fluorescent atom groups possessed by a pair of fluorescent atom groups B may be the same or different. When any one of the two fluorescent atom groups possessed by a pair of fluorescent atom groups A and the two fluorescent atom groups possessed by a pair of fluorescent atom groups B is different, with respect to each fluorescent atom group, the emission peak wavelength of at least one of the two fluorescent atom groups possessed by a pair of fluorescent atom groups A is shorter than the excitation peak wavelength of at least one of the two fluorescent atom groups possessed by a pair of fluorescent atom groups B. It is preferred that the emission peak wavelength of the two fluorescent atom groups possessed by a pair of fluorescent atom groups A is shorter than the excitation peak wavelength of the two fluorescent atom groups possessed by a pair of fluorescent atom groups B.

[0136] Feature (b)

[0137] A pair of fluorescent atomic groups A and a pair of fluorescent atomic groups B show a FRET effect. The Förster resonance energy transfer (FRET) effect, also known as fluorescence resonance energy transfer, refers to a phenomenon in which the excitation energy between two adjacent chromophores is not converted into electromagnetic waves but is directly transferred through the resonance of electrons. Energy is transferred to another chromophore (acceptor) through the energy of light absorbed by one chromophore (donor), and when the acceptor is a fluorescent molecule, the acceptor emits fluorescence. In the labeled single-stranded nucleic acid of the present invention, a pair of fluorescent atomic groups A having a luminescence peak wavelength shorter than the excitation peak wavelength of a pair of fluorescent atomic groups B is configured in a manner that shows a FRET effect with a pair of fluorescent atomic groups B. The so-called configuration in which a pair of fluorescent atomic groups A and a pair of fluorescent atomic groups B show a FRET effect is, for example, a case in which a base having a pair of fluorescent atomic groups A and a base having a pair of fluorescent atomic groups B are included in the above-mentioned labeled single-stranded nucleic acid at a distance such that a pair of fluorescent atomic groups A and the above-mentioned pair of fluorescent atomic groups B have a FRET effect. The distance (base length) at which a pair of fluorescent atomic groups A and a pair of fluorescent atomic groups B have a FRET effect varies depending on the type and combination of the fluorescent atomic groups A and B, and is, for example, 1 to 11 bases, preferably 2 to 8 bases, more preferably 2 to 7 bases, more preferably 2 to 6 bases, further more preferably 2 to 5 bases, and further preferably 2 to 4 bases. In addition, the so-called distance of 1 base here means that there is a nucleic acid that does not have a fluorescent atomic group between a pair of fluorescent atomic groups A and a pair of fluorescent atomic groups B. As a combination of fluorescent atomic groups A and fluorescent atomic groups B, for example, a combination of thiazole orange (D514) and thiazole powder (D570) or D640, a combination of D436 and thiazole orange (D514), thiazole powder (D570) or D640 can be cited.

[0138] The labeled single-stranded nucleic acid of the present invention satisfies the above-mentioned characteristics (a) and (b), and either of the pair of fluorescent atomic groups A and the pair of fluorescent atomic groups B is located at a base that is two or more bases inside from the respective ends of the labeled single-stranded nucleic acid. By satisfying this characteristic, both the exciton effect and the FRET effect can be exerted.

[0139] Examples of the base having a pair of fluorescent atomic groups that exhibit an exciton effect include bases described in Patent Documents 1 and 2, and Non-Patent Documents 2 to 5. These bases are described in detail below.

[0140] The base having a pair of fluorescent atomic groups that exhibit an exciton effect may have a structure represented by the following formula (16), (16b), (17), or (17b).

[0141] [Chemical Formula 17]

[0142]

[0143] [Chemical Formula 18]

[0144]

[0145] [Chemical Formula 19]

[0146]

[0147] [Chemical Formula 20]

[0148]

[0149] In formulas (16), (16b), (17), and (17b),

[0150] B is an atomic group having a natural nucleic acid base (adenine, guanine, cytosine, thymine or uracil) skeleton or an artificial nucleic acid base skeleton,

[0151] E is (i) an atomic group having a deoxyribose backbone, a ribose backbone, or a structure derived from either of them, or (ii) an atomic group having a peptide structure or a peptoid structure,

[0152] Z 11 and Z 12 are atomic groups showing fluorescence, which may be the same or different.

[0153] L 1 , L 2 and L 3 are connecting segments (cross-linking atoms or atomic groups), the main chain length (number of main chain atoms) is arbitrary, the main chain may contain or not contain C, N, O, S, P and Si, the main chain may contain or not contain single bonds, double bonds, triple bonds, amide bonds, ester bonds, disulfide bonds, imino groups, ether bonds, thioether bonds and thioester bonds, L 1 , L 2 and L 3 They can be the same or different from each other.

[0154] D is CR, N, P, P=O, B or SiR, R is a hydrogen atom, an alkyl group or any substituent,

[0155] b is a single bond, a double bond or a triple bond,

[0156] Alternatively, in the above formulas (16) and (16b), L 1 and L 2 is the above-mentioned connecting fragment, L 3 , D and b do not exist, L1 and L 2 It can also be directly bonded to B.

[0157] In formulas (16) and (17), E is the atomic group of (i) above, and at least one O atom in the phosphate crosslink may be replaced by a S atom.

[0158] In formula (16b) and (17b), E is the atomic group of (ii) above,

[0159] In formulae (17) and (17b), each B may be the same or different, and each E may be the same or different.

[0160] In the above formulas (16), (17), (16b), and (17b), L 1 , L 2 and L 3 The main chain length (number of main chain atoms) of L is preferably an integer greater than 2. 1 , L 2 and L 3 The upper limit of the main chain length (the number of main chain atoms) is not particularly limited, but is, for example, 100 or less, more preferably 30 or less, and particularly preferably 10 or less.

[0161] It is preferred that the structure represented by the above formula (16) is the structure represented by the following formula (16-1) or (16-2), the structure represented by the above formula (16b) is the structure represented by the following formula (16b-1) or (16b-2), the structure represented by the above formula (17) is the structure represented by the following formula (17-1), and the structure represented by the above formula (17b) is the structure represented by the following formula (17b-1).

[0162] [Chemical Formula 21]

[0163]

[0164] [Chemical Formula 22]

[0165]

[0166] [Chemical Formula 23]

[0167]

[0168] [Chemical Formula 24]

[0169]

[0170] [Chemical Formula 25]

[0171]

[0172] [Chemical Formula 26]

[0173]

[0174] In formulas (16-1), (16-2), (16b-1), (16b-2), (17-1), and (17b-1),

[0175] l, m and n are any positive integers, which may be the same or different. The main chain may contain or not contain C, N, O, S, P and Si, respectively. The main chain may contain or not contain single bonds, double bonds, triple bonds, amide bonds, ester bonds, disulfide bonds, imino groups, ether bonds, thioether bonds and thioester bonds, respectively. 11 , Z 12 and b are the same as those in the above formulae (16), (16b), (17), and (17b).

[0176] In the above formulae (16-1), (16-2), and (17-1), one or more O atoms in the phosphate crosslinks may be substituted with S atoms.

[0177] Z 11 and Z 12 This is a fluorescent atomic group that exhibits an exciton effect. Therefore, for example, when a double helix structure is formed, the increase in fluorescence is large, making it possible to more effectively detect the double helix structure.

[0178] Z 11 and Z 12 There are no particular limitations on the fluorescent atomic groups as long as they exhibit an exciton effect. From the perspective of exhibiting an exciton effect, aromatic atomic groups are preferably used. 11 and Z 12 For example, independently and more preferably, the group derived from thiazole orange, thiazole powder, oxazole yellow, cyanine, semicyanine, other cyanine pigments, methyl red, azo pigments or their derivatives. In addition, the group derived from other known pigments can also be suitably used. There are many reports of fluorescent pigments that change fluorescence intensity by bonding with nucleic acids such as DNA. In a typical example, known ethidium bromide shows strong fluorescence by embedding in the double helix structure of DNA and is often used for DNA detection. In addition, there are also known fluorescent pigments that can control fluorescence intensity according to microscopic polarity such as pyrenecarboxamide and sodium fluorosilicate. In addition, above-mentioned thiazole orange is a fluorescent pigment formed by linking a benzothiazole ring and a quinoline ring with a methine group, which usually shows faint fluorescence, but gives strong fluorescence by embedding in the DNA with a double helix structure. In addition, for example, pigments such as fluorescein and Cy3 can also be enumerated.

[0179] Z 11 and Z 12More preferably, each independently is an atomic group represented by any one of the following formulae (7) to (10).

[0180] [Chemical Formula 27]

[0181]

[0182] [Chemical Formula 28]

[0183]

[0184] [Chemical Formula 29]

[0185]

[0186] [Chemical formula 30]

[0187]

[0188] In formulas (7) to (9),

[0189] X 1 and X 2 is S or O,

[0190] n is 0 or a positive integer,

[0191] R 1 ~R 10 、R 13 ~R 21 are independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,

[0192] R 11 and R 12 One of them is the same as L in the above formulas (16), (17), (16b), and (17b) 1 or L 2 The other of the bonded linking groups is a hydrogen atom or a lower alkyl group,

[0193] R 15 When there are multiple in formula (7), (8) or (9), they may be the same or different.

[0194] R 16 When there are multiple in formula (7), (8) or (9), they may be the same or different.

[0195] Z 11 X in 1 、X 2 and R 1 ~R 21 With Z 12 X in 1 、X 2 and R1 ~R 21 They can be the same or different from each other.

[0196] In formula (10),

[0197] E is S or O,

[0198] R 2 ~R 12 are independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,

[0199] R 1 is the same as L in the above formulas (16), (17), (16b), and (17b) 1 or L 2 bonded linking group,

[0200] R 3 When there are multiple 's in formula (10), they may be the same or different.

[0201] R 4 When there are multiple 's in formula (10), they may be the same or different.

[0202] In the above formulas (7) to (9), R 1 ~R 21 In the formula (10), the lower alkyl group is a linear or branched alkyl group having 1 to 6 carbon atoms, and the lower alkoxy group is more preferably a linear or branched alkoxy group having 1 to 6 carbon atoms. 2 ~R 12 In the above-mentioned lower alkyl group, a linear or branched alkyl group having 1 to 6 carbon atoms is preferable, and the above-mentioned lower alkoxy group is a linear or branched alkoxy group having 1 to 6 carbon atoms.

[0203] In the above formulas (7) to (9), R 11 and R 12 In addition, in the above formula (10), R 1 In the embodiment, the linking group is a polymethylene carbonyl group having 2 or more carbon atoms, and it is further preferred that the carbonyl portion and L in the above formulae (16), (16b), (17), and (17b) are connected. 1 or L 2 The upper limit of the number of carbon atoms in the polymethylene carbonyl group is not particularly limited, but is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, and particularly preferably 10 or less.

[0204] Z 11 and Z 12 When represented by the above formulae (7) to (9), for example, each independently is more preferably a group represented by formula (19) or (20).

[0205] [Chemical Formula 31]

[0206]

[0207] [Chemical Formula 32]

[0208]

[0209] In formulas (19) and (20), X 1 Indicates -S- or -O-. R 1 to R 10 、R 13 and R 14 R each independently represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group. 11 and R 12 One of them represents the same as L in the above formulas (16), (17), (16b), and (17b) 1 and L 2 Bonded linking group, R 11 and R 12 The other of represents a hydrogen atom or a lower alkyl group.

[0210] The preferred method is as follows.

[0211] (i)Z 11 and Z 12 are independently an atomic group represented by the above formula (19), wherein X 1 For S, R 1 to R 10 is a hydrogen atom, R 11 and R 12 One of them is the same as L in the above formulas (16), (17), (16b) and (17b) 1 or L 2 The other of the two bonded linking groups is a methyl group.

[0212] (ii) Z 11 and Z 12 are independently an atomic group represented by the above formula (19), wherein X 1 For S, R 1 、R 4 、R 5 、R 6 、R 7 、R 9 and R 10 is a hydrogen atom, R 2 、R 3 and R 12 is methyl, R 8is a halogen atom, R 11 is the same as L in the above formulas (16), (17), (16b) and (17b) 1 or L 2 Bonded linking group.

[0213] (iii) Z 11 and Z 12 are independently an atomic group represented by the above formula (7), wherein X 1 is S, n is 1, R 1 to R 10 、R 15 、R 16 and R 17 is a hydrogen atom, R 11 is the same as L in the above formulas (16), (17), (16b) and (17b) 1 or L 2 Bonded linking group, R 12 It is a methyl group.

[0214] Z 11 and Z 12 Each of the following chemical formulas may be independently an atomic group represented by any one of the following chemical formulas. These are, in order, thiazole orange (D514), D640, D436, D534, D543, and thiazole powder (D570). For names beginning with the atomic group D, refer to Non-Patent Document 3.

[0215] [Chemical Formula 33]

[0216]

[0217] [Chemical Formula 34]

[0218]

[0219] [Chemical Formula 35]

[0220]

[0221] [Chemical Formula 36]

[0222]

[0223] [Chemical Formula 37]

[0224]

[0225] [Chemical Formula 38]

[0226]

[0227] In the above chemical formulas, n is a positive integer.

[0228] In the above formulas (16), (17), (16b), and (17b), B may have a natural nucleic acid base backbone, but may also have an artificial nucleic acid base backbone as described above. For example, B is preferably a structure represented by Py (pyrimidine ring), Py der., Pu (purine ring), or Pu der. Here, the so-called Py is an atomic group having a covalent bond to E at the 1-position and a covalent bond to the linker segment at the 5-position in a 6-membered ring represented by the following formula (11); the so-called Py der. is an atomic group in which at least one of all the atoms in the 6-membered ring of Py is substituted with an N, C, S or O atom, and the N, C, S or O atom may appropriately have a charge, a hydrogen atom or a substituent; the so-called Pu is an atomic group in which the 9-position is covalently bonded to E and the 8-position is covalently bonded to the linker segment in a fused ring represented by the following formula (12); the so-called Pu der. is an atomic group in which at least one of all the atoms in the 5-membered ring of Pu is substituted with an N, C, S or O atom, and the N, C, S or O atom may appropriately have a charge, a hydrogen atom or a substituent.

[0229] [Chemical Formula 39]

[0230]

[0231] In the labeled single-stranded nucleic acid of the present invention, the basic skeleton of the nucleic acid is not particularly limited, and can be, for example, an oligonucleotide, a modified oligonucleotide, an oligonucleoside, a modified oligonucleoside, a polynucleotide, a modified polynucleotide, a polynucleoside, a modified polynucleoside, a DNA, a modified DNA, an RNA, a modified DNA, an LNA, a PNA (peptide nucleic acid), or any of these chimeric molecules, or other structures. In addition, the basic skeleton of the above-mentioned nucleic acid can be natural or artificially synthesized. In the case of the primer or primer pair of the present invention, the above-mentioned nucleic acid can be, for example, a nucleic acid that can form a base pair bond, and in the case of a nucleic acid sample or a target nucleic acid sequence, for example, a nucleic acid that functions as a template for synthesizing a complementary chain. Therefore, the above-mentioned nucleic acid can also be, for example, a nucleotide derivative that is partially or entirely composed of an artificial structure. As artificial bases constituting the above-mentioned nucleic acid, for example, one can select from 2-amino-6-(N,N-dimethylamino)purinepyridin-2-one, 5-methylpyridin-2-one, 2-amino-6-(2-thienyl)purine, pyrrole-2-carbaldehyde, 9-methylimidazo[(4,5)-b]pyridine, 5-iodo-2-oxo(1H)pyridine, 2-oxo-(1H)pyridine, 2-amino-6-(2-thiazolyl)purine, 7-(2-thienyl)-imidazo[4,5-b]pyridine, bromothymidine, azaadenine or azaguanine.

[0232] As the labeled single-stranded nucleic acid of the present invention, the basic skeleton is preferably, for example, an oligonucleotide, a polynucleotide, DNA, or a modified form thereof. In the present invention, the so-called "nucleotide" may be, for example, any one of a deoxynucleotide and a ribonucleotide, and the "oligonucleotide" and "polynucleotide" may be composed of, for example, any one of a deoxynucleotide and a ribonucleotide, or may contain both. In the present invention, there is no particular restriction on the number of bases constituting the nucleic acid. The term "nucleic acid" generally has the same meaning as the term "polynucleotide". The term "oligonucleotide" is generally used as a term to indicate a nucleotide with a small number of bases in a polynucleotide. Polynucleotides with a length of, for example, 2 to 100 bases, more generally 2 to 50 bases, are generally referred to as "oligonucleotides", but are not limited to these values. The term "polynucleotide" in the present invention also includes, for example, polynucleotides and oligonucleotides, as well as artificially synthesized nucleic acids such as peptide nucleic acids, morpholino nucleic acids, methylphosphonate nucleic acids, and S-oligonucleotides.

[0233] The above-mentioned peptide nucleic acid (PNA) generally has a structure in which the deoxyribose backbone of the oligonucleotide is replaced by a peptide backbone. As the above-mentioned peptide backbone, for example, repeating units of N-(2-aminoethyl)glycine bonded by amide bonds can be listed. As bases bonded to the peptide backbone of PNA, for example, naturally occurring bases such as thymine, cytosine, adenine, guanine, inosine, uracil, 5-methylcytosine, thiouracil and 2,6-diaminopurine, and artificial bases such as bromothymine, azaadenine and azaguanine can be listed, but it is not limited thereto.

[0234] LNAs are generally nucleic acids with two rings of structure, linked by a methylene crosslink between the 2'-oxygen atom and the 4'-carbon atom of the ribose sugar in the sugar-phosphate backbone. Annealing an LNA-containing oligonucleotide to DNA alters the conformation of the duplex, increasing thermal stability. Because LNAs have stronger binding forces to nucleic acids than conventional oligonucleotides, more reliable and robust hybridization can be achieved, for example, through oligonucleotide design.

[0235] The labelled single-stranded nucleic acid of the present invention is a nucleic acid comprising a labelled structure having at least two of the above-mentioned pairs of fluorescent atom groups, and as a result, compared with the unlabelled nucleic acid that for example does not comprise the above-mentioned fluorescent atom group, the specificity for the target is high, and hybridisation sometimes becomes stronger. That is, the labelled single-stranded nucleic acid of the present invention is for example compared with the unlabelled nucleic acid having the same base sequence as the basic skeleton and the same nucleic acid fragment length, and sometimes melting temperature (Tm value) improves. Therefore, compared with the above-mentioned unlabelled nucleic acid, it is sometimes possible to hybridise more firmly with the target. Therefore, in the case of the labelled single-stranded nucleic acid of the present invention with such a property, for example, efficient, specific detection can be achieved.

[0236] Since the labeled single-stranded nucleic acid of the present invention also has such characteristics, it can be used as an application technology to improve the specificity of amplification by increasing the Tm value, similar to conventional PNA or LNA. In addition, by making the basic skeleton of the labeled primer of the present invention PNA or LNA, the Tm value can sometimes be further increased compared to unlabeled PNA or LNA, so it is possible to further improve the efficiency of hybridization. In particular, as described later, when identifying mutations of one to several bases, or detecting insertions and deletions, efficient and highly specific detection can be achieved by using the labeled single-stranded nucleic acid of the present invention (e.g., also including labeled PNA, labeled LNA, etc.). If the labeled single-stranded nucleic acid of the present invention is used as a primer or probe, for example, the difference in Tm value is large depending on whether it is a perfect match or mismatch with the target sequence, and the hybridization efficiency may vary. Therefore, it is possible that the detection of mutations such as single base recognition is further facilitated. Furthermore, since the labeled single-stranded nucleic acid of the present invention has a higher Tm value than unlabeled nucleic acid, it is also possible to use it as a primer in PCR clamp methods, PNA PCR clamp methods, LNA PCR clamp methods, and PNA-LNA PCR clamp methods, for example, where it strongly bonds to a specific region and masks that region from serving as an amplification template.

[0237] Specific examples of the structure represented by formula (1) include nucleotide structures represented by the following formulas (1-3) to (1-10), or their geometric isomers, stereoisomers, or salt structures.

[0238] [Chemical Formula 40-1]

[0239]

[0240] [Chemical Formula 40-2]

[0241]

[0242] [Chemical Formula 40-3]

[0243]

[0244] [Chemical Formula 40-4]

[0245]

[0246] In the above formulas (1-3) to (1-10), n is a positive integer.

[0247] As the labeled single-stranded nucleic acid of the present invention, a labeled single-stranded nucleic acid having a pair of fluorescent atomic groups as shown in (1-1) to (1-10) above is particularly preferred.

[0248] Next, the pair of fluorescent atomic groups in the labeled single-stranded nucleic acid of the present invention is characterized in that:

[0249] (i) Two planar chemical structures within a molecule are not in the same plane and exist at a certain angle, but when the molecule is embedded in or groove-bonded with a nucleic acid, the two planar chemical structures are arranged in the same plane to produce fluorescent light, or

[0250] (ii) an atomic group consisting of two or more pigment molecules that does not emit fluorescence due to the exciton effect generated by the parallel aggregation of two or more pigment molecules, but emits fluorescence when these molecules are intercalated or groove-bonded with nucleic acids by releasing the above-mentioned aggregation state, or

[0251] (iii) A chemical structure having two or more pigment molecules within the same molecule, wherein the chemical structure does not exhibit fluorescence due to the exciton effect generated by the parallel aggregation of the two or more pigment molecules, but when these molecules are embedded in or groove-bonded to nucleic acids, the above-mentioned aggregation state is released to produce fluorescence.

[0252] In the case of (ii) or (iii) above, the pigment molecule is preferably the molecule described in (i) above.

[0253] [Synthesis of labeled single-stranded nucleic acids]

[0254] The labeled single-stranded nucleic acid in the present invention can be prepared by referring to the methods described in Patent Documents 1 and 2. For example, the compounds represented by the above formulas (1-1) to (1-10) can also be synthesized by referring to the methods described in Patent Documents 1 and 2.

[0255] As the manufacturing method (synthetic method) that can be applied in the manufacture of the labeled single-stranded nucleic acid of the present invention, for example, there is the following method. That is, first, as a simple labeling method for DNA, a method of reacting the active amino group in the DNA with the activated carboxyl group in the labeling agent in a buffer solution is widely adopted. This method can be particularly applicable to the introduction of a linker fragment or a pigment. As an amino-based introduction method, there is a method utilizing the amino-modified phosphoramidite sold by GLEN RESEARCH, etc.

[0256] It is well known that there is a method for synthesizing nucleic acids with modified DNA as the basic skeleton, for example, it can be synthesized by the so-called phosphoramidite method. The phosphoramidite reagent that becomes its raw material can also be simply synthesized by a known method. When the nucleic acid of the present invention is DNA, particularly short oligo DNA, for example, it can be simply synthesized using a DNA automatic synthesizer. In addition, for example, by PCR, long-chain nucleic acids (DNA) can also be synthesized. The bonding site of DNA and the pigment molecule is not particularly limited as described above, for example, the 5th position of thymidine is particularly preferred. It is known that the triphosphate of nucleotide derivatives with various substituents extended from the 5th position of thymidine has a relatively good import efficiency using DNA polymerase. Thus, for example, not only when the nucleic acid of the present invention is short oligo DNA, but also when it is long-chain DNA, it can be simply synthesized.

[0257] For example, the fluorescent primer (labeled nucleic acid) of the present invention, which utilizes single-stranded DNA using thiazole orange, has the following advantages: (1) It can be prepared simply by contacting DNA synthesized by an automatic DNA synthesizer with the dye in a buffer solution, making synthesis easy; (2) By reacting long-stranded DNA prepared by enzymes with the dye, long-stranded fluorescent primers can also be produced. Furthermore, it can be excited with light of a relatively long wavelength, for example, around 500 nm.

[0258] The labeled single-stranded nucleic acid of the present invention is used as a probe for hybridization with a target nucleic acid or as a primer for amplifying a target nucleic acid.

[0259] The present invention includes a method for detecting a target nucleic acid, using a labeled single-stranded nucleic acid of the present invention as a probe. Under conditions permitting hybridization with the target nucleic acid, fluorescence is measured to determine whether hybridization with the probe occurs. Specifically, nucleic acid amplification methods that can be used in this method for detecting a target nucleic acid are as follows.

[0260] This nucleic acid amplification method is a method for amplifying a target nucleic acid sequence in a nucleic acid sample, and includes the following step (A) and the following step (B').

[0261] (A) a step of preparing the nucleic acid sample;

[0262] (B') a process comprising the following steps (B1') and (B2');

[0263] (B1′) a step of amplifying a target nucleic acid sequence in a nucleic acid sample using a primer or a primer pair comprising a pair of primers,

[0264] (B2') A step of hybridizing the single-stranded nucleic acid sequence amplified in the above step (B1') with a probe consisting of the labeled single-stranded nucleic acid of the present invention.

[0265] The probe composed of the labeled single-stranded nucleic acid of the present invention may be, for example, a probe comprising at least one structure represented by the above formula (16), (16b), (17), or (17b).

[0266] The primers and primer pairs used in the above-described nucleic acid amplification method are not particularly limited and can be appropriately selected depending on, for example, the target nucleic acid sequence, the type of nucleic acid amplification reaction, and the like. Furthermore, the type of nucleic acid amplification method used in the present invention is not particularly limited and includes various isothermal amplification methods such as the aforementioned SMAP method and LAMP method, as well as PCR methods, and can be performed in the same manner as in the first nucleic acid amplification method described above.

[0267] The base sequence of the labeled single-stranded nucleic acid of the present invention used as a probe can be appropriately designed according to the target nucleic acid sequence and designed in a manner that hybridizes with the above-mentioned target nucleic acid under stringent conditions. "Stringent conditions" are determined, for example, by the melting temperature Tm (°C) of the double-stranded chain of the probe of the present invention and its complementary chain, and the salt concentration of the hybridization solution. As a specific example, reference can be made to J. Sambrook, EFFrisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989) (all of which are specifically cited herein as public) and the like.

[0268] According to the above-mentioned nucleic acid amplification method, since the labeled single-stranded nucleic acid of the present invention is used as a probe, it is possible to determine, for example, whether the target nucleic acid sequence has been amplified by only detecting the fluorescence intensity of the nucleic acid amplification reaction solution. This is based on the following reasons, for example. If the probe hybridizes with the complementary nucleic acid sequence, then due to the formation of a double-stranded nucleic acid, the atomic group (pigment) of the labeled primer is embedded or groove-bonded to the double-stranded nucleic acid. At this time, since the exciton effect of the atomic group (pigment) such as the above is not generated, the atomic group produces fluorescence. On the other hand, in the absence of hybridization, the atomic group does not produce fluorescence due to the exciton effect. Therefore, for example, when the probe is not hybridized with the amplification product obtained by the nucleic acid amplification reaction, or when amplification is not caused, the atomic group that produces fluorescence is not seen, or is not increased. Therefore, if the fluorescence intensity is detected, then in the case of an increase, it can be determined that the target nucleic acid sequence has been amplified, and in the case of no increase, it can be determined that the target nucleic acid sequence has not been amplified. In particular, the labeled single-stranded nucleic acid of the present invention has the following advantages: the fluorescence background when there is no hybridization is compared with the case of using a labeled probe with an exciton effect in the past, and the detection sensitivity becomes higher.

[0269] The above-mentioned labeled single-stranded nucleic acid probe can be added to the reaction solution before the nucleic acid amplification reaction of the above-mentioned (B1') step, or it can be added to the reaction solution after the nucleic acid amplification reaction of the above-mentioned (B1'). In the former case, the detection of fluorescence intensity is carried out continuously or intermittently at the same time as the nucleic acid amplification reaction of the above-mentioned (B1'), or it can be carried out after the end of the above-mentioned (B1'). In the case where the above-mentioned (B1') step is carried out after the end of the reaction of the above-mentioned (B1'), it is preferably detected before the start of the reaction of the above-mentioned (B1') step as a background. On the other hand, in the case where the above-mentioned (B1') step and (B2') step are carried out separately, for example, the above-mentioned labeled single-stranded nucleic acid probe is added to the reaction solution after the nucleic acid amplification reaction of the above-mentioned (B1'). In this case, the detection of fluorescence intensity is carried out, for example, after the (B1') step. At this time, as a background, for example, it is preferably to detect the fluorescence intensity after the above-mentioned (B1') step and before or just after the addition of the above-mentioned labeled single-stranded nucleic acid probe. Specific examples of detection methods are as described above.

[0270] (1) The labeled single-stranded nucleic acid probe of the present invention can be used in liquid-phase homology analysis (using a 96-well microplate or capillary tube, etc.).

[0271] (2) The labeled single-stranded nucleic acid probe of the present invention can be used as a PCR probe. It can be used as a low-cost method for detecting amplification curves in DNA amplification reactions (real-time PCR) and as a replacement for TaqMan probes. It can be used as a primer label or as an internal labeling probe.

[0272] (3) The labeled single-stranded nucleic acid probe of the present invention can be used as a capture probe or a labeled probe in a DNA chip. It is a high-throughput and reagent-free system, and does not require a labeling process or a cleaning process. It can greatly avoid human errors. It can achieve simultaneous multi-item (high-throughput) analysis in glass or solid-phase carrier raw materials that replace it (substrates such as gold, ITO, copper, diamond, or plastic, etc., which can attach multiple specimens).

[0273] (4) The labeled single-stranded nucleic acid probe of the present invention can be immobilized on beads, fibers, or hydrogels. Genes can be detected in a semi-liquid / semi-solid environment. It can be transported in a solid-like manner while maintaining a liquid-like measurement environment.

[0274] (5) The labeled single-stranded nucleic acid probe of the present invention can be used as a probe for blotting (Southern blot, Northern blot, dot blot, etc.). It can detect only the target gene fragment by causing it to emit light. According to the method of the present invention, washing is not required after the hybridization operation.

[0275] (6) The labeled single-stranded nucleic acid probe of the present invention can be used as a probe for detecting and tracking nucleic acids in cells. This allows for spatial and temporal analysis of DNA / RNA within cells. A fluorescence microscope or a cell classifier can be used. It can be applied to labeling DNA, tracking transfer and splicing to RNA, functional analysis of RNAi, and the like. In the method of the present invention, since washing is not required, it is suitable for functional tracking of living cells.

[0276] (7) The labeled single-stranded nucleic acid probe of the present invention can be used as a probe for fluorescence in situ hybridization (FISH). The method of the present invention can be used to stain tissues, etc. In the method of the present invention, since washing is not required, the error caused by human factors is small. That is, since the labeled single-stranded nucleic acid probe of the present invention functions as a fluorescent pigment that does not emit fluorescence when the target biomolecule is not recognized, if it is used, biological imaging that does not require a complicated washing process can be established. This is related to high reliability, low labor and real-time fluorescence observation.

[0277] (8) Since the labeled nucleic acid probes of the present invention can utilize chromophores of multiple wavelengths, they can easily be designed to minimize the presence of background light and scattered light from these excitation lights when detecting and tracking at the single-molecule level. For example, when observing biomolecules at the single-molecule level, background light and scattered light, such as leakage of excitation light, can disrupt the state, and methods to prevent this are essential. The present invention is particularly useful in such situations.

[0278] The fluorescence intensity of the labeled single-stranded nucleic acid probe of the present invention can be effectively changed, for example, by controlling the exciton interaction of the bonded pigment portion. Among the present invention, particularly, according to the proximity of utilizing the exciton interaction, as an on-off probe, a function can be achieved, so fully high extinction performance can be obtained. The design of such on-off fluorescent nucleotides is, for example, very important for establishing a bioimaging analysis that does not require cleaning. The photophysical properties displayed by the probe utilizing the exciton effect are not only very characteristic, but also suitable for the design of novel fluorescent DNA probes for monitoring and gene expression observation of DNA sequencing (sequence determination), genotyping (genotype analysis), DNA structure transitions.

[0279] In addition, if the labeled single-stranded nucleic acid of the present invention is used as a probe, then for example, by quantifying the target nucleic acid sequence, the generation of phenomena such as the amplification, decomposition, and protein bonds of the sequence can be detected immediately, and these phenomena are quantitatively measured. This detection and quantification can be carried out by the following description, which is illustrative and does not limit the present invention. That is, first, the probe (nucleic acid) of the present invention is hybridized with the above-mentioned target nucleic acid sequence at a certain amount ratio to form a double chain. Since the amount of the double chain formed is directly proportional to the amount of the target nucleic acid sequence, the target nucleic acid sequence can be detected by measuring the fluorescence intensity of the above-mentioned double chain, and its amount is quantitatively measured. In this case, the labeled single-stranded nucleic acid of the present invention is further suppressed due to the fluorescent luminescence of the background, so it will not hinder the fluorescence intensity measurement of the above-mentioned double chain, and more accurate measurement can be carried out.

[0280] A method for amplifying a target nucleic acid is provided, comprising using the labeled single-stranded nucleic acid of the present invention as a primer to amplify the target nucleic acid. The method for amplifying a target nucleic acid using the labeled single-stranded nucleic acid of the present invention as a primer can exemplify various nucleic acid amplification methods known in the past, and there is no limitation on the reaction format. As the above-mentioned nucleic acid amplification method, for example, an isothermal amplification method, a polymerase chain reaction (PCR) method, etc. can be cited. The above-mentioned isothermal amplification method is generally a method for performing a nucleic acid amplification reaction at an isothermal temperature.Examples of such methods include the strand displacement amplification (SDA) method disclosed in Japanese Patent Publication No. 7-114718 (the entire contents of which are hereby incorporated by reference herein); the improved SDA method disclosed in U.S. Patent No. 5,824,517 (the entire contents of which are hereby incorporated by reference herein); International Publication No. 99 / 09211 (the entire contents of which are hereby incorporated by reference herein); and International Publication No. 95 / 25180 (the entire contents of which are hereby incorporated by reference herein); and the nucleic acid sequence based amplification (NASBA) method disclosed in Japanese Patent No. 2,650,159 (the entire contents of which are hereby incorporated by reference herein); amplification); the loop-mediated isothermal amplification method (LAMP) disclosed in International Publication No. 00 / 28082 (the entire contents of which are expressly incorporated herein by reference); the ICAN method (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) disclosed in International Publication No. 02 / 16639 (the entire contents of which are expressly incorporated herein by reference); the self-sustained sequence replication (3SR) method; the transcription-mediated amplification, transcription-mediated amplification) method; the Qβ replicase method disclosed in Japanese Patent No. 2710159 (the entire contents of which are specifically incorporated herein as a disclosure); the method disclosed in Japanese Patent No. 389726 (the entire contents of which are specifically incorporated herein as a disclosure), Japanese Patent No. 3942627 (the entire contents of which are specifically incorporated herein as a disclosure), NATURE METHODS (Vol. 4, No. 3, March 2007, pp. 257-262) (the entire contents of which are specifically incorporated herein as a disclosure), Mitani Y., et al. 2007., Nat. Methods 4(3): 257-262. (the entire contents of which are specifically incorporated herein as a disclosure), etc. (hereinafter referred to as “SmartAmp (Smart Amplification Process, smart amplification detection) method”), the Invader method, the RCA (rolling cycle amplification) method, etc.

[0281] Example

[0282] The present invention will be further specifically described by the following examples, but the present invention is not limited by the following examples.

[0283] [Example 1]

[0284] The synthesis of the oligonucleotide DNA strand incorporating the scaffolds (Japanese: scaffold) of thiazole orange (TO) and thiazole powder (TP) was carried out by the amide method described in Patent Document 2 (for example, refer to Example 2). The incorporation of TO was carried out by reacting with TO2 diamide immediately after introducing NHS-Carboxy-dT at the target position, and the subsequent sequence was synthesized by the conventional method. Cleavage from CPG and deprotection were carried out in 28% ammonia water at 55 °C for 4 hours. Purification was carried out by HPLC equipped with a reverse phase (RP-18) column.

[0285] [Chemical formula 41]

[0286] TO2 diamide

[0287]

[0288] TO2 diamide

[0289] Thereafter, according to the method described in Patent Document 1 (for example, refer to Example 6 (synthesis of a compound incorporating two thiazole orange-derived structures in one molecule)), the purified nucleic acid was reacted with TP-ester in a sodium bicarbonate buffer solution, and purified by HPLC equipped with a reverse phase (RP-18) column to obtain the target product.

[0290] [Chemical formula 42]

[0291] TP-ester

[0292]

[0293] TP-ester

[0294] The oligonucleotide DNA strands incorporating thiazole orange (TO) and thiazole powder (TP) prepared by the above method (5 common base sequences each, SEQ ID NO: 1) are as follows.

[0295] 20-mer.EX16-12TOTP: 5'-TGTGZATCtTTCTCTTTCTC-3'

[0296] 20-mer.EX8-12TOTP: 5'-TGTGTATCtTTCZCTTTCTC-3'

[0297] 20-mer.EX10-12TOTP:5'-TGTGTATCtTZCTTCTTCTC-3'

[0298] 20-mer.EX14-12TOTP:5'-TGTGTAZCtTTCTCTTTCTC-3'

[0299] 20-mer.EX18-12TOTP:5'-TGZGTATCtTTCTCTTTCTC-3'

[0300] (Z represents T in TO marking, t represents T in TP marking)

[0301] [Example 2]

[0302] (Spectral comparison experiment between a fluorescent nucleic acid probe incorporating two fluorescent dyes with exciton effects and a conventional fluorescent probe with exciton effects)

[0303] The spectrum was measured by excitation with thiazole orange at an excitation wavelength of 490 nm. The spectrum was measured using a fluorescence measuring device (RF5300) from Shimadzu Corporation. Concentrations of each fluorescent probe and its complementary chain (SEQ ID NO. 2) were measured at 1 μM at a temperature of 23°C. The results are shown in Figure 1A ~C. Figure 1A In the case of using a fluorescent nucleic acid probe into which two fluorescent dyes having an exciton effect are introduced, Figure 1B and C are the spectra of conventional fluorescent probes with exciton effect. Figure 1A In the case of two exciton effects shown in , fluorescence of the wavelength (601 nm) of thiazole powder generated by the FRET effect can be confirmed. In this case, the ratio of the signal intensity (S / N ratio) when the single chain (background) is compared with the double chain (during measurement) is 4.6. When two fluorescent dyes with exciton effects are introduced, the S / N ratio of the wavelength of the measurement object is 4.6, which is different from the case of one ( Figure 1B :S / N=2.1、 Figure 1C : S / N = 1.8) is more than twice as good as that of the control.

[0304] This indicates that in the case of a single chain, the fluorescence energy of thiazole orange (533 nm) is inactivated to some extent by the exciton effect, but due to the close presence of thiazole powder, it receives energy due to the FRET effect, and this energy is also inactivated by the exciton effect.

[0305] [Example 3]

[0306] Melting curve analysis of a fluorescent nucleic acid probe containing two fluorescent dyes with exciton effects was performed using a real-time PCR device (CFX96) from BioRad. Concentrations of each fluorescent probe and its complementary strand were measured at 1 μM in a 25 μl volume. The temperature was raised from 4°C to 95°C in increments of 0.5°C. The results are shown in Figure 2A The results shown in Figure 2 are comparisons of the fluorescence of thiazole orange (excitation wavelength: 495 nm). When the distance between thiazole orange and thiazole powder is appropriate, the fluorescence of thiazole orange is greatly reduced due to the FRET effect.

[0307] Depend on Figure 2A The results of 1 to 4 suggest that, in the fluorescently labeled single-stranded nucleic acid (DNA) used in this example, the FRET effect is best achieved when the distance between the two fluorescent dyes with exciton effect is about 3 bases. It is known that when the distance is too close (one base) or too far (five bases), the FRET effect may be reduced. Figure 2A The base sequences of the fluorescent nucleic acid probes used in the melting curve analysis results shown in Figures 1 to 5E are as follows.

[0308] Industrial applicability

[0309] The present invention is useful in the field of using fluorescently labeled probes or primers.

[0310] SEQ ID NO: 1: Base sequence of the oligomeric DNA chain (20mer) synthesized in Example 1

[0311] SEQ ID NO: 2: Base sequence of the complementary chain of the oligomeric DNA chain (20mer) synthesized in Example 1

Claims

1. A labeled single-stranded nucleic acid, characterized in that It is a labeled single-stranded nucleic acid with at least two pairs of fluorescent atomic groups, wherein each pair of fluorescent atomic groups exhibits an exciton effect. The emission peak wavelength of one pair of fluorescent atomic groups A in the two pairs of fluorescent atomic groups is shorter than the excitation peak wavelength of the other pair of fluorescent atomic groups B. The pair of fluorescent atomic groups A and the pair of fluorescent atomic groups B have a Forster resonance energy transfer effect, i.e., a FRET effect. The distance between the base having the pair of fluorescent atomic groups A and the base having the pair of fluorescent atomic groups B is 2 bases to 5 bases, The pair of fluorescent atomic groups A are the same or different Z in the following formula (16): 11 and Z 12 The fluorescent atomic group showing the exciton effect is thiazole orange or its derivative represented by the following formula (7): ； In formula (7): X 1 is S or O, n=0 or 1, R 1 ~R 10 、R 17 are independently a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group, R 11 is bonded to L in the following formula (16): 1 or L 2 The connecting group, R 12 is a hydrogen atom or a lower alkyl group; The pair of fluorescent atomic groups B are the same or different Z in the following formula (16): 11 and Z 12 The fluorescent atomic group showing the exciton effect is a thiazole powder or a derivative thereof represented by formula (10). ； In formula (10): E is S or O, n=1， R 5 ~R 12 are independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group or an amino group, R 3 and R 4 is a hydrogen atom, R 2 For amino, R 1 is bonded to L in the following formula (16) 1 or L 2 a linking group; R in formula (7) 1 ~R 17 Compared with R in formula (10) 1 ~R 12 Independent of each other, in, The base having a pair of fluorescent atomic groups exhibiting the exciton effect has a structure represented by the following formula (16): ； In formula (16), B is an atomic group having a natural nucleic acid base skeleton or an artificial nucleic acid base skeleton, wherein the natural nucleic acid base is adenine, guanine, cytosine, thymine or uracil, E is an atomic group having a deoxyribose backbone, a ribose backbone, or a structure derived from either of them, At least one O atom in the phosphate bridge is replaced by a S atom, or no O atom is replaced by a S atom, Z 11 and Z 12 are respectively one of a pair of fluorescent atomic groups showing the exciton effect, and are the same as or different from each other, L 1 、L 2 and L 3 are respectively connected segments, each of which has a main chain length, i.e., the number of main chain atoms, of 10 or less, and each of which contains at least one of C, N, O, S, P, and Si in the main chain, and each of which contains at least one of a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond in the main chain, L 1 、L 2 and L 3 are the same as or different from each other, wherein the connecting fragment is a bridging atom or a group of atoms, D is CR, N, P, P=O, B or SiR, R is a hydrogen atom or an alkyl group, b is a single bond, a double bond or a triple bond.

2. The labeled single-stranded nucleic acid according to claim 1, wherein The pair of fluorescent atomic groups A is thiazole orange, and the pair of fluorescent atomic groups B is thiazole powder.

3. The labeled single-stranded nucleic acid according to claim 1, wherein The structure represented by the formula (16) has the structure represented by the following formula (16-1), ； In formula (16-1), l and m are 1 to 10, n is 1 to 10, the same or different, and the main chain contains at least one of C, N, O, S, P, and Si, and the main chain contains at least one of single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, imino group, ether bond, thioether bond, and thioester bond. B is an atomic group having a natural nucleic acid base skeleton or an artificial nucleic acid base skeleton, wherein the natural nucleic acid base is adenine, guanine, cytosine, thymine or uracil, E is an atomic group having a deoxyribose backbone, a ribose backbone, or a structure derived from either of them, Z 11 and Z 12 are respectively one of a pair of fluorescent atomic groups showing the exciton effect, b is a single bond, a double bond or a triple bond, One or more O atoms in the phosphate bridge are replaced by S atoms, or no O atoms are replaced by S atoms. The labeled single-stranded nucleic acid according to claim 1 , which is used as a primer for amplifying a target nucleic acid or a probe for hybridizing with a target nucleic acid.

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