Methine-substituted cyanine dye compounds

• US 8,470,529 B2
• Filed: 08/05/2010
• Issued: 06/25/2013
• Est. Priority Date: 12/05/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Dallwig, Jason, Hagen, David, Cheung, Ching-Ying, Thomas, Gerald, Yue, Stephen
• Primary Examiner(s): Young, Shawquia
• Application Number:
  US12/851,030
• Publication Number:
  US 20100317543A1
• Time in Patent Office: 1,055 Days
• Field of Search: 546/268.4
• US Class Current: 435/6
• CPC Class Codes: C07D 277/64 with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C09B 23/0008 substituted on the polymethine chain
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/107 four >CH- groups
• CPC Class Codes: C09B 23/164 containing one nitrogen atom
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Cyanine dye compounds having a substituted methine moiety that are nucleic acid stains, particularly for fluorescent staining of RNA, including compounds having the formula

where R¹ is a C₁-C₆ alkyl, sulfoalkyl, carboxyalkyl or C₁-C₆ alkoxy; each R² is independently selected from the group consisting of H, C₁-C₆ alkyl, C₁-C₆ alkoxy, fused benzo, trifluoromethyl, amino, sulfo, carboxy and halogen, that is optionally further substituted; at least one of R³, R⁴, and R⁵ is an alkyl, aryl, heteroaryl, cyclic, or heterocyclic moiety that is optionally substituted by alkyl, amino, aminoalkyl, carboxy, nitro, or halogen; and the remaining R³, R⁴ or R⁵ are hydrogen; X is S, O, or Se; and D is a substituted or unsubstituted pyridinium, quinolinium or benzazolium moiety.

Claims

1. A nucleic acid reporter compound of the formula:

2. The compound according to claim 1, wherein the compound exhibits a fluorescence enhancement when non-covalently associated with a nucleic acid.

3. The compound according to claim 2, wherein the fluorescence enhancement is greater when the nucleic acid is RNA than when the nucleic acid is DNA.

4. The compound according to claim 1, wherein at least one of R³, R⁴, and R⁵ is a substituted 5-, 6- or 7-membered aryl that is substituted by an alkyl, —

(CH₂)_k—

NR⁶R⁷;

—

COOR⁸, NO₂, or halogen,

wherein k is an integer from 0 to about 6,

R⁶ is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl or aminoalkyl;

R⁷ is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl or aminoalkyl; and

R⁸ is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl or aminoalkyl.

5. The compound according to claim 1, wherein at least one of R³ and R⁴ is a substituted phenyl.

6. The compound according to claim 1, wherein X is S.

7. The compound according to claim 1, wherein R¹ is alkoxy or halogen.

8. The compound according to claim 1, wherein at least one R¹ is methoxy, Br, Cl, or F.

9. The compound according to claim 1, wherein R² is methyl, ethyl, propyl, or —

(CH₂)₃SO₃₋

.

10. The compound according to claim 1, wherein R² is an alkyl;

X is S;

each R¹ is H;

R³ and R⁵ are hydrogen; and

R⁴ is a substituted phenyl moiety.

11. The compound according to claim 1, having the formula)

12. The compound according to claim 1, wherein the reactive group, solid support and carrier molecule comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

13. A composition comprising a nucleic acid reporter compound and a sample, wherein the nucleic acid reporter compound has the formula according to claim 1.

14. The composition according to claim 13, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymer, nucleosides, nucleotides, a polymeric gel or tissue sections.

15. The composition according to claim 14, wherein the nucleic acid polymer is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

16. A complex comprising a nucleic acid reporter compound non-covalently associated with a nucleic acid molecule, wherein the nucleic acid reporter compound has the formula according to claim 1.

17. The complex according to claim 16, wherein the nucleic acid molecule is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

18. The complex according to claim 16, wherein the nucleic acid comprises mRNA, tRNA, or rRNA.

19. The complex according to claim 16, wherein the complex is present in a biological sample.

20. The complex according to claim 16, wherein the complex is present in an aqueous or aqueous miscible solution.

21. The complex according to claim 16, wherein the complex is present in an electrophoretic matrix or polymeric gel.

22. A method for detecting the presence or absence of nucleic acid in a sample, wherein the method comprises:

a) combining a nucleic acid reporter compound with the sample to prepare a labeling mixture, wherein the nucleic acid reporter compound has the formula according to claim 1;

b) incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter compound to associate with the nucleic acid in the sample to form an incubated mixture;

c) illuminating the incubated mixture with an appropriate wavelength to form an illuminated mixture; and

d) observing the illuminated mixture whereby the presence or absence of the nucleic acid in the sample is detected.

23. The method according to claim 22, further comprising quantifying the nucleic acid present in the sample.

24. The method according to claim 22, wherein the nucleic acid is RNA.

25. The method according to claim 22, wherein the nucleic acid comprises mRNA, tRNA, or rRNA.

26. The method according to claim 22, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, nucleosides, a polymeric gel or tissue sections.

27. The method according to claim 22, wherein the sample comprises a cell, tissue, or biological fluid.

28. The method according to claim 22, wherein the sample is present in or on a microarray or a microwell plate.

29. A method for detecting RNA in the presence of DNA, wherein the method comprises the steps:

a) combining a nucleic acid reporter compound with a sample to prepare a labeling mixture, wherein the nucleic acid reporter compound has a RNA/DNA ratio of fluorescence enhancement greater than about one and wherein the nucleic acid reporter compound has the formula according to claim 1;

b) incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter compound to associate with RNA in the sample to form an incubated mixture;

c) illuminating the incubated mixture with an appropriate wavelength to form an illuminated mixture; and

d) observing the illuminated mixture whereby the RNA is detected in the presence of DNA.

30. The method according to claim 29, further comprising quantifying the RNA present in the sample.

31. The method according to claim 29, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, nucleosides, a polymeric gel or tissue sections.

32. The method according to claim 29, wherein the sample comprises a cell, tissue, or biological fluid.

33. The method according to claim 29, wherein the sample is present in or on a microarray or a microwell plate.

34. The method according to claim 29, wherein the fluorescence of the nucleic acid reporter compound when non-covalently associated with RNA is distinguishable from the fluorescence of the nucleic acid reporter compound when non-covalently associated with DNA.

35. The method according to claim 29, wherein the RNA comprises mRNA, tRNA, or rRNA.

36. A kit for detecting nucleic acid in a sample, wherein the kit comprises a nucleic acid reporter compound that has the formula according to claim 1.

37. The kit according to claim 36, further comprising instructions for detecting RNA in the presence of DNA.

38. The kit according to claim 36, further comprising one or more of a sample preparation reagent, a buffering agent, an aqueous nucleic acid reporter compound dilution buffer, an organic solvent, nucleic acid control, or an additional detection reagent.

39. The kit according to claim 38, wherein the nucleic acid reporter compound dilution buffer contains a detergent.

40. The kit according to claim 39, wherein the detergent is present in a final concentration of about 0.0001% to about 0.005% (w/v).

41. The kit according to claim 39, wherein the detergent is CHAPS, Tween 20, Trinton-X or SDS.

42. The kit according to claim 36, wherein the nucleic acid reporter compound dilution buffer contains a low concentration of DNA.

43. The kit according to claim 36, further comprising a microarray.

44. The kit according to claim 36, further comprising a microwell plate.

45. A staining solution comprising a nucleic acid reporter compound and a detergent, wherein the nucleic acid reporter compound has the formula according to claim 1.

46. The staining solution according to claim 45, wherein the detergent present at a concentration of about 0.0001% to about 0.005%.

47. The staining solution according to claim 45, wherein the detergent is CHAPS, Triton-X, SDS or Tween 20.

48. The staining solution according to claim 45, further comprising a low concentration of DNA.

49. The staining solution according to claim 48, wherein the DNA is present at a concentration of about 0.1 μ

g/ml to about 0.5 μ

g/ml.