Nucleoside or nucleotide derivative and use thereof

• US 7,960,543 B2
• Filed: 11/08/2005
• Issued: 06/14/2011
• Est. Priority Date: 11/08/2004
• Status: Expired due to Fees

• Current Assignee: Riken
• Original Assignee: Riken
• Inventors: Hirao, Ichiro, Yokoyama, Shigeyuki
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/667,146
• Publication Number:
  US 20090275017A1
• Time in Patent Office: 2,044 Days
• Field of Search: 536/4.1, 536/23.1, 536/24.3, 536/26.6, 435/6, 435/91.1
• US Class Current: 536/26.6
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical

Abstract

The object of the present invention is to provide a nucleoside or nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base, as well as a method using the same.

In one embodiment, the nucleoside or nucleotide of the present invention has a fluorescent dye selected from the group consisting of 5-FAM, 6-FAM, 5-TAMRA, 6-TAMRA, DANSYL, 5-HEX, 6-HEX, 5-TET, 6-TET, 5-ROX and 6-ROX or a quencher dye selected from the group consisting of DABCYL, BHQ1 and BHQ2, which is attached either directly or through a linker to the 5-position of the above base.

Claims

1. A nucleic acid incorporating a nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base, and which has a fluorescent dye attached either directly or through a linker to the 5-position of the base, wherein the fluorescent dye is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), 5-(dimethylamino)naphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2′

,4,4′

,5′

,7,7′

-hexachlorofluorescein (5-HEX), 6-carboxy-2′

,4,4′

,5′

,7,7′

-hexachlorofluorescein (6-HEX), 5-carboxy-2′

,4,7,7′

-tetrachlorofluorescein (5-TET), 6-carboxy-2′

,4,7,7′

-tetrachlorofluorescein (6-TET), 5-carboxy-X-rhodamine (5-ROX), and 6-carboxy-X-rhodamine (6-ROX).

3. The nucleic acid according to claim 1 or 2, wherein the nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group forms a base pair with a nucleotide having a 6-substituted 2-aminopurin-9-yl group as a base.

4. The nucleic acid according to claim 3, wherein the 6-substituted 2-aminopurin-9-yl group is a 2-amino-6-(2-thienyl)purin-9-yl group or a 2-amino-6-(2-thiazolyl)purin-9-yl group.

5. The nucleic acid according to claim 3, wherein the nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base and the nucleotide having a 6-substituted 2-aminopurin-9-yl group as a base are present in the same single-stranded nucleic acid.

6. The nucleic acid according to claim 3 that has biological activity as antisense DNA, antisense RNA, a ribozyme or an aptamer.

7. The nucleic acid according to claim 4 that has biological activity as antisense DNA, antisense RNA, a ribozyme or an aptamer.

8. The nucleic acid according to claim 5 that has biological activity as antisense DNA, antisense RNA, a ribozyme or an aptamer.

9. A method for detecting a target protein, which comprises:

1) synthesizing a nucleic acid according to claim 1 or 2;

2) allowing the nucleic acid to bind to a target protein;

3) allowing the target protein to adhere to a support; and

4) measuring the fluorescence of the nucleic acid bound to the target protein adhered to the support.

10. A method for detecting a target protein, which comprises:

a) synthesizing a nucleic acid according to claim 1 or 2;

b) allowing the nucleic acid to bind to a target protein;

c) selectively retaining the target protein in a solution using an ultrafiltration membrane; and

d) measuring the fluorescence of the nucleic acid bound to the target protein retained in the solution.

12. A method for detecting the formation of a nucleic acid duplex, which comprises:

I) inducing hybridization between two nucleic acids according to claim 1, wherein the nucleic acids contain two mutually different fluorescent dyes which allow fluorescence resonance energy transfer (FRET) between them; and

II) measuring a change in the fluorescence spectrum.

13. A method for detecting a low-molecular weight compound, which comprises:

A) synthesizing a nucleic acid according to claim 1 or 2;

B) contacting the nucleic acid with a sample likely to contain a low-molecular weight compound; and

C) measuring a change in the fluorescence spectrum of the nucleic acid.

14. The method according to claim 13, wherein the sample is a solution and the contacting between the nucleic acid and the sample in step B) is accomplished in a solution.

15. The method according to claim 13, wherein the low-molecular weight compound is selected from the group consisting of theophylline, a base, a nucleoside or nucleotide, and an amino acid.

2. A nucleic acid incorporating a nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base, and which has a quencher dye attached either directly or through a linker to the 5-position of the base, wherein the quencher dye is selected from the group consisting of 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), N-methyl-N-[4-[2-methoxy-5-methyl-4-(2-nitro-4-methylphenylazo)phenylazo]phenyl]-4-aminobutyric acid (BHQ1) and N-methyl-N-[4-[2,5-dimethoxy-4-(4-nitrophenylazo)phenylazo]phenyl]-4-aminobutyric acid (BHQ2).

11. A method for detecting the formation of a nucleic acid duplex, which comprises:

i) inducing hybridization between a nucleic acid incorporating a nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base, and which has a fluorescent dye attached either directly or through a linker to the 5-position of the base, wherein the fluorescent dye is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), 5-(dimethylamino)naphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2′

,4,4′

,5′

,7,7′

-hexachlorofluorescein (5-HEX), 6-carboxy-2′

,4,4′

,5′

,7,7′

-hexachlorofluorescein (6-HEX), 5-carboxy-2′

,4,7,7′

-tetrachlorofluorescein (5-TET), 6-carboxy-2′

,4,7,7′

-tetrachlorofluorescein (6-TET), 5-carboxy-X-rhodamine (5-ROX), and 6-carboxy-X-rhodamine (6-ROX),

and a nucleic acid incorporating a nucleotide having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base, and which has a quencher dye attached either directly or through a linker to the 5-position of the base, wherein the quencher dye is selected from the group consisting of 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), N-methyl-N-[4-[2-methoxy-5-methyl-4-(2-nitro-4-methylphenylazo)phenylazo]phenyl]-4-aminobutyric acid (BHQ1) and N-methyl-N-[4-[2,5-dimethoxy-4-(4-nitrophenylazo)phenylazo]phenyl]-4-aminobutyric acid (BHQ2); and

ii) measuring a change in the fluorescence spectrum.