Fluorescent nucleotides and labeling methods using the same

1. A fluorescent nucleotide represented by the formula: X—Y—Z wherein,

X represents a residue of a natural or non-natural nucleotide, an oligonucleotide, or a polynucleotide, which binds to Y at a basic moiety of said residue;

Y represents a divalent bridging group or a single bond; and

Z represents a monovalent group derived from:

a compound represented by the following general formula (II):

wherein, R 1 , R 2 , R 3 , and R 4 each independently represents a substituted or unsubstituted alkyl group, and R 3 and R 4 may bind to each other to form a saturated or unsaturated ring; V 1 , V 2 and V 3 each independently represents a hydrogen atom or a substituent, V 1 and V 2 , and/or V 2 and V 3 may bind to each other to form a saturated or unsaturated ring; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 each independently represents a substituted or unsubstituted methine group; W represents an oxygen atom, a sulfur atom, —C(R 3 )(R 4 )— or —N(R 5 )— wherein R 3 , R 4 , and R 5 each independently represents a substituted or unsubstituted alkyl group; Q represents a nitrogen atom or C(V 7 ) wherein V 7 represents a hydrogen atom or a monovalent substituent and may bind to V 6 to form a saturated or unsaturated ring; M represents a counter ion; m represents a number required to neutralize the charge of the molecule; s represents 0 or 1; t represents 0 or 1; and u represents 0 or 1; and V 8 represents a hydrogen atom or a malevolent substituent, provided that, when W represents —N(R 5 )— or —C(R 3 )(R 4 )—, V 8 may bind to a substituent on W to form a saturated or unsaturated ring; or

a compound represented by the following general formula (III):

wherein, V 1 , V 2 , V 3 , R 1 , R 2 , R 3 , R 4 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , W, M, m, s, t, and u have the same meanings as defined above; E represents a nitrogen atom or —C(R 6 )═, where R 6 represents a hydrogen atom or a monovalent substituent; V 9 represents a hydrogen atom or a monovalent substituent and V 9 may bind to R 6 to form a saturated or unsaturated ring,

and Z binds to Y at a reactive group existing in R 1 or R 2 .

2. The fluorescent nucleotide according to claim 1 , wherein at least one of R 1 and R 2 is an alkyl group substituted with an active ester group in Y, wherein said active ester group can covalently bond to an amino group, a hydroxyl group, or a thiol group.

3. The fluorescent nucleotide according to claim 1 , wherein at least one of R 1 and R 2 is an alkyl group substituted with a carboxyl group in Y.

4. The fluorescent nucleotide according to claim 1 , wherein X is a residue of a natural or non-natural nucleotide.

5. The fluorescent nucleotide according to claim 1 , wherein X is selected from the group consisting of: AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP, UTP, TMP, TDP, TTP, 2-Me-AMP, 2-Me-ADP, 2-Me-ATP, 1-Me-GMP, 1-Me-GDP, 1-Me-GTP, 5-Me-CMP, 5- Me-CDP, 5-Me-CTP, 5-MeO-CMP, 5-MeO-CDP, and 5-MeO-CTP, wherein Me represents a methyl group, MeO represents a methoxy group, and a numeral before Me or MeO represents a substituting position, and

wherein X may be a corresponding deoxynucleotide or dideoxynucleotide.

6. The fluorescent nucleotide according to claim 1 , wherein Y represents —CH 2 —, —CH═CH—, —C═C—, —CO—, —O—, —S—, —NH—, or a bridging group consisting of a combination thereof wherein a hydrogen atom on the bridging group may be replaced with other substituent.

7. The fluorescent nucleotide according to claim 1 , wherein Y represents an aminoalkyl group.

8. A method for producing a fluorescence-labeled nucleic acid which comprises a step of performing a nucleic acid synthesis reaction by using a nucleic acid synthetase, a template nucleic acid, and the fluorescent nucleotide according to claim 1 .

9. The method according to claim 8 , wherein the nucleic acid synthesis reaction comprises one or more reactions selected from the group consisting of a reverse transcription reaction, terminal transferase reaction, reactions of random prime method, PCR, and nick translation.

10. A nucleic acid probe or a primer labeled with the fluorescent nucleotide according to claim 1 .

11. An agent for detecting a nucleic acid which comprises the nucleic acid probe or the primer according to claim 10 .

12. The agent according to claim 11 , wherein said nucleic acid is indicative of a disease.

13. A kit for detecting a nucleic acid, which comprises:

(1) the fluorescent nucleotide according to claim 1 ;

(2) a nucleic acid synthetase; and

(3) a buffer.

14. An azamethine dye represented by the following general formula (II):

wherein R 1 , R 2 , R 3 , and R 4 each independently represents a substituted or unsubstituted alkyl group, and R 3 and R 4 may bind to each other to form a saturated or unsaturated ring; V 1 , V 2 and V 3 each independently represents a hydrogen atom or a substituent, V 1 and V 2 , and/or V 2 and V 3 may bind to each other to form a saturated or unsaturated ring; V 8 represents a hydrogen atom or a monovalent substituent; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 each independently represents a substituted or unsubstituted methine group; W represents an oxygen atom, a sulfur atom, —C(R 3 )(R 4 )— or —N(R 5 )— wherein R 3 , R 4 , and R 5 each independently represents a substituted or unsubstituted alkyl group, provided that, when W represents —N(R 5 )— or —C(R 3 )(R 4 )—, V 8 may bind to a substituent on W to form a saturated or unsaturated ring; M represents a counter ion; m represents a number required to neutralize the charge of the molecule; s represents 0 or 1; t represents 0 or 1; and u represents 0 or 1.

15. An azamethine dye represented by the following general formula (III):

wherein R 1 , R 2 , R 3 , and R 4 each independently represents a substituted or unsubstituted alkyl group, R 3 and R 4 may bind to each other to form a saturated or unsaturated ring; V 1 , V 2 , V 3 each independently represents a hydrogen atom or a substituent, V 9 represents hydrogen or a monovalent substituent, V 1 and V 2 , and/or V 2 and V 3 may bind to each other to form a saturated or unsaturated ring; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 each independently represents a substituted or unsubstituted methine group; W represents an oxygen atom, a sulfur atom, —C(R 3 )(R 4 )— or —N(R 5 )— wherein R 3 , R 4, and R 5 each independently represents a substituted or unsubstituted alkyl group; E represents a nitrogen atom or —C(R 6 )═, where R 6 represents a hydrogen atom or a monovalent substituent, and V 9 may bind to form a saturated or unsaturated ring; M represents a counter ion; m represents a number required to neutralize the charge of the molecule; s represents 0 or 1; t represents 0 or 1; and u represents 0 or 1.