Methine-substituted cyanine dye compounds

• US 20050208534A1
• Filed: 12/06/2004
• Published: 09/22/2005
• 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: Cheung, Ching-Ying, Yue, Stephen, Dallwig, Jason Alfred, Thomas, Gerald Alan, Hagen, David Carl
• Granted Patent: US 7,776,529 B2
• Time in Patent Office: Days
• 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 compound having 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 alkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered heteroaryl, or 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 thiophenyl, substituted thiophenyl, adamantyl, substituted adamantly, phenyl, substituted phenyl, alkyl, substituted alkyl, benzyl or substituted benzyl.

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 D has the formula

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

X is S;

each R¹ is H;

R³ and R⁵ are hydrogen;

R⁴ is a phenyl or substituted phenyl moiety; and

D is a substituted quinolinium, unsubstituted quinolinium, substituted benzazolium or unsubstituted benzazolium moiety.

12. The compound according to claim 1, having the formula

13. 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.

14. The compound according to claim 1, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group.

15. The compound according to claim 1, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

16. The compound according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

17. The compound according to claim 1, wherein the carrier molecule is an antibody or fragment thereof, an avidin, streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

18. The compound according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

19. The compound according to claim 1, wherein the solid support is sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

20. A composition comprising a nucleic acid reporter molecule and a sample, wherein the nucleic acid reporter molecule has the formula:

21. The composition according to claim 20, 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.

22. The composition according to claim 21, wherein the nucleic acid polymer is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

23. A complex comprising a nucleic acid reporter molecule non-covalently associated with a nucleic acid molecule, wherein the nucleic acid reporter molecule has the formula:

24. The complex according to claim 23, wherein the nucleic acid molecule is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

25. The complex according to claim 23, wherein the nucleic acid comprises mRNA, tRNA, or rRNA.

26. The complex according to claim 23, wherein the complex is present in a biological sample.

27. The complex according to claim 23, wherein the complex is present in an aqueous or aqueous miscible solution.

28. The complex according to claim 23, wherein the complex is present in an electrophoretic matrix or polymeric gel.

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

a) combining a nucleic acid reporter molecule with the sample to prepare a labeling mixture, wherein the nucleic acid reporter molecule has the formula;

each R¹ is independently hydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, aminoalkyl, substituted aminoalkyl, fused benzene, substituted fused benzene, reactive group, solid support or carrier molecule;

t is an integer from 1 to 4;

wherein R² is an alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy, substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

at least one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered aryl or a substituted 5-, 6- or 7-membered aryl; and

the remaining R³, R⁴ or R⁵ are hydrogen;

X is S, O, or Se; and

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium or unsubstituted benzazolium moiety;

b) incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule 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.

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

31. The method according to claim 29, wherein the nucleic acid is RNA.

32. The method according to claim 29, wherein the nucleic acid comprises mRNA, tRNA, or rRNA.

33. 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.

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

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

36. The method according to claim 35, further comprising quantifying the RNA present in the sample.

37. The method according to claim 35, 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.

38. The method according to claim 35, wherein the sample comprises a cell, tissue, or biological fluid.

39. The method according to claim 35, wherein the sample is present in or on a microarray or a microwell plate.

40. The method according to claim 35, wherein the fluorescence of the nucleic acid reporter molecule when non-covalently associated with RNA is distinguishable from the fluorescence of the nucleic acid reporter molecule when non-covalently associated with DNA.

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

35-1. A method for detecting RNA in the presence of DNA, wherein the method comprises the steps:

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

each R¹ is independently hydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, aminoalkyl, substituted aminoalkyl, fused benzene, substituted fused benzene, reactive group, solid support or carrier molecule;

t is an integer from 1 to 4;

wherein R² is an alkyl, substituted alkyl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkoxy, substituted alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

at least one of R³, R⁴, and R⁵ is an alkyl, substituted alkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered aryl or a substituted 5-, 6- or 7-membered aryl; and

the remaining R³, R⁴ or R⁵ are hydrogen;

X is S, O, or Se; and

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium or unsubstituted benzazolium moiety;

b) incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule 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.

42. A kit for detecting nucleic acid in a sample, wherein the kit comprises a nucleic acid reporter molecule that has the formula:

43. The kit according to claim 42, further comprising instructions for detecting RNA in the presence of DNA.

44. The kit according to claim 42, further comprising one or more of a sample preparation reagent, a buffering agent, an aqueous nucleic acid reporter molecule dilution buffer, an organic solvent, nucleic acid control, or an additional detection reagent.

45. The kit according to claim 44, wherein the nucleic acid reporter molecule dilution buffer contains a detergent.

46. The kit according to claim 45, wherein the detergent is present in a final concentration of about 0.0001% to about 0.005% (w/v).

47. The kit according to claim 45, wherein the detergent is CHAPS, Tween 20, Trinton-X or SDS.

48. The kit according to claims 44, wherein the nucleic acid reporter dilution buffer contains a low concentration of DNA.

49. The kit according to claim 42, further comprising a microarray.

50. The kit according to claim 42, further comprising a microwell plate.

51. A staining solution comprising a nucleic acid reporter molecule and a detergent, wherein the nucleic acid reporter molecule has the formula:

52. The staining solution according to claim 51, wherein the detergent present at a concentration of about 0.0001% to about 0.005%.

53. The staining solution according to claim 51, wherein the detergent is CHAPS, Triton-X, SDS or Tween 20.

54. The staining solution according to claim 51, further comprising a low concentration of DNA.

55. The staining solution according to claim 54, wherein the DNA is present at a concentration of about 0.1 μ

g/ml to about 0.5 μ

g/ml.