Unsymmetrical cyanine dimer compounds and their application

• US 20050074796A1
• Filed: 08/02/2004
• Published: 04/07/2005
• Est. Priority Date: 07/31/2003
• Status: Abandoned Application

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Cheung, Ching-Ying, Yue, Stephen
• Application Number:
  US10/911,423
• Publication Number:
  US 20050074796A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C09B 23/0016 the substituent being a halogen atom
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• 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 55/006 containing at least one heteroring
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Embodiments of the present invention provide methods and nucleic acid reporter molecules for the detection of nucleic acid in a sample. The nucleic acid reporter molecule comprises two unsymmetrical cyanine monomer moieties, which may be the same or different, that are covalently attached by a linker comprising at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C. The linker may be rigid, relatively flexible or some degree thereof. The unsymmetrical cyanine monomer moieties comprise a substituted or unsubstituted benzazolium moiety and a substituted or unsubstituted pyridinium or quinolinium moiety that is connected by a methine bridge that is monomethine, trimethine or pentamethine. The linkers form the cyanine dimer compounds by attaching to the pyridinium or quinolinium moiety of the monomer moieties. The present nucleic acid reporter molecules find utility in forming a nucleic acid-reporter molecule complex and detecting the nucleic acid. In particular, present nucleic acid reporter molecules with a rigid linker and monomer moieties with a monomethine bridge find utility in detecting RNA in the presence of DNA.

Claims

1. A nucleic acid reporter molecule comprising a first nucleic acid complexing monomer moiety, a second nucleic acid complexing monomer moiety and a linker that comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C, wherein the first nucleic acid complexing monomer moiety is covalently attached to the linker;

and the second nucleic acid complexing monomer moiety is covalently attached to the linker.

2. The nucleic acid reporter molecule according to claim 1, wherein the first and the second first nucleic acid complexing monomer moieties are the same.

3. The nucleic acid reporter molecule according to claim 1, wherein the first and the second first nucleic acid complexing monomer moieties are different.

4. A nucleic acid reporter molecule comprising a first unsymmetrical cyanine monomer moiety, a second unsymmetrical cyanine monomer moiety, and a linker that comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C, wherein the first unsymmetrical cyanine monomer moiety is covalently attached to the linker;

and the second unsymmetrical cyanine monomer moiety is covalently attached to the linker.

5. The nucleic acid reporter molecule according to claim 4, wherein the first and the second unsymmetrical cyanine monomer moieties are the same.

6. The nucleic acid reporter molecule according to claim 4, wherein the first and the second unsymmetrical cyanine monomer moieties are different.

7. A nucleic acid reporter molecule according to the formula:

8. The nucleic acid reporter molecule according to claim 7, wherein the molecule

11. The nucleic acid reporter molecule according to claim 7, wherein the first and the second unsymmetrical cyanine monomer moieties are different.

9. A nucleic acid reporter molecule comprising a first unsymmetrical cyanine monomer moiety, a second unsymmetrical cyanine monomer moiety, and a linker having formula (I) —

(Y)_r—

(CH₂)_m-T_q-(CH₂)_n-E-(CH₂)_n-T_q-(CH₂)_m—

(Y)_r—

;

wherein Y is a linear or branched moiety comprising 1-20 non-hydrogen atoms selected from the group consisting of C, N, O, P and S;

T is a unsubstituted heteroatom or a substituted heteroatom;

E is an aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C;

r is independently 0 or 1;

m is an integer of 0-6;

n is independently 0 or 1;

q is independently 0 or 1;

and wherein the first unsymmetrical cyanine monomer moiety is covalently attached to the linker; and

the second unsymmetrical cyanine monomer moiety is covalently attached to the linker.

10. The nucleic acid reporter molecule according to claim 9, wherein the first and the second unsymmetrical cyanine monomer moieties are the same.

12. The reporter molecule according to claim 9, wherein E is

13. The reporter molecule according to claim 9, wherein T, when present, is —

NR², —

N═

or S wherein the R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

14. The reporter molecule according to claim 9, wherein the first and the second unsymmetrical cyanine monomer moiety have the general formula:

15. The reporter molecule according to claim 14, 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.

16. The reporter molecule according to claim 15, wherein the reactive group is selected from the group consisting of 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 and a photoactivatable group.

17. The reporter molecule according to claim 16, wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

18. The reporter molecule according to claim 15, wherein the carrier molecule is selected from the group consisting of 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.

19. The reporter molecule according to claim 18, wherein the carrier molecule is selected from the group consisting of an antibody or fragment thereof, an avidin or 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.

20. The reporter molecule according to claim 15, wherein the solid support is selected from the group consisting of 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, and superparamagnetic bead.

21. The reporter molecule according to claim 20, wherein the solid support is selected from the group consisting of Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose and starch.

22. The reporter molecule according to claim 9, wherein the reporter molecule has the formula:

23. A nucleic acid reporter molecule comprising a first unsymmetrical cyanine monomer moiety, a second unsymmetrical cyanine monomer moiety and a linker having formula (II) —

(CH₂)_n-T_q-(CH₂)_n-E-(CH₂)_n-T_q-(CH₂)_n—

;

wherein T is a substituted heteroatom or an unsubstituted heteroatom;

E is an aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C;

n is independently 0 or 1;

q is independently 0 or 1; and

,

the first unsymmetrical cyanine monomer moiety is covalently attached to the linker;

and the second unsymmetrical cyanine monomer moiety is covalently attached to the linker.

24. The reporter molecule according to claim 23, wherein E is

25. The reporter molecule according to claim 23, wherein T is —

NR², —

N═

or S and R² is hydrogen, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

26. The reporter molecule according to claim 23, wherein the first and the second unsymmetrical cyanine monomer moiety have the general formula:

27. The reporter molecule according to claim 23, wherein the linker is

28. The reporter molecule according to claim 23, wherein R⁸ is S.

29. The reporter molecule according to claim 27, wherein R¹² is —

NH or —

N═

.

30. The reporter molecule according to claim 27, wherein the linker is

31. A nucleic acid reporter molecule that has a RNA/DNA ratio of fluorescence enhancement greater than about one wherein the nucleic acid reporter molecule comprises a first monomethine unsymmetrical cyanine monomer moiety, a second monomethine unsymmetrical cyanine monomer moiety and a linker that is

32. The reporter molecule according to claim 31, wherein

R⁸ is —

NR², —

N═

or S wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support;

R⁹ is —

NR², —

N═

or S wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support; and

,

R¹² is —

NR², —

N═

or S wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

33. The reporter molecule according to claim 31, wherein the monomethine unsymmetrical cyanine monomer moiety has the general formula:

34. The reporter molecule according to claim 33, 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.

35. The reporter molecule according to claim 34, wherein the reactive group is selected from the group consisting of 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 and a photoactivatable group.

36. The reporter molecule according to claim 35, wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

37. The reporter molecule according to claim 34, wherein the carrier molecule is selected from the group consisting of 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.

38. The reporter molecule according to claim 37, wherein the carrier molecule is selected from the group consisting of an antibody or fragment thereof, an avidin or 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.

39. The reporter molecule according to claim 34, wherein the solid support is selected from the group consisting of 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, and superparamagnetic bead.

40. The reporter molecule according to claim 39, wherein the solid support is selected from the group consisting of Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose and starch.

41. The reporter molecule according to claim 33, wherein the linker is

42. The reporter molecule according to claim 41, wherein the reporter molecule is

43. The reporter molecule according to claim 33, wherein R⁹ is N or S and R¹² is N or S.

46. The reporter molecule according to claim 33, wherein the linker is

47. The reporter molecule according to claim 46, wherein said reporter molecule is

48. The reporter molecule according to claim 33, wherein the linker is

49. The reporter molecule according to claim 48, wherein the compound is

44. The reporter molecule according to claim 44, wherein the linker is

45. The reporter molecule according to claim 44, wherein the reporter molecule is

50. A nucleic acid reporter molecule that is

51. A complex comprising:

i. a nucleic acid reporter molecule comprising a first nucleic acid complexing monomer moiety, a second nucleic acid complexing monomer moiety and a linker that comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C, wherein the first nucleic acid complexing monomer moiety is covalently attached to the linker; and

the second nucleic acid complexing monomer moiety is covalently attached to the linker; and

,

ii. a nucleic acid molecule.

52. The complex according to claim 51, wherein the first and the second first nucleic acid complexing monomer moieties are the same.

53. The complex according to claim 51, wherein the first and the second first nucleic acid complexing monomer moieties are different.

54. A complex comprising:

a) a nucleic acid reporter molecule according to the formula

wherein each R³ is independently hydrogen, unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, unsubstituted fused benzene, substituted fused benzene, unsubstituted trifluoromethyl, substituted trifluoromethyl, unsubstituted halogen, substituted halogen, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support or substituted solid support;

each R⁴ is independently a unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support or substituted solid support;

X is O, S, or CR⁶R⁷ wherein each R⁶ and R⁷ are independently a unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, substituted solid support or R⁶ and R⁷ taken together form a 5- or 6-membered saturated ring;

j is 0, 1, or 2;

linker is a series of stable covalent bonds comprising 1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S and P; and

,

K is substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium; and

,

b) a nucleic acid molecule.

55. A complex according to claim 54, wherein the nucleic acid molecule is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

56. The complex according to claim 54, wherein the molecule is

57. The complex according to claim 54, wherein the linker comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C.

58. The complex according to claim 57, wherein the linker has the formula (I) —

(Y)_r, (CH₂)_m-T_q-(CH₂)_n-E-(CH₂)_n-T_q-(CH₂)_m—

(Y)_r—

;

wherein Y is a linear or branched moiety comprising 1-20 non-hydrogen atoms selected from the group consisting of C, N, O, P and S;

T is a unsubstituted heteroatom or a substituted heteroatom;

E is an aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C;

r is independently 0 or 1;

m is an integer of 0-6;

n is independently 0 or 1;

q is independently 0 or 1;

and wherein the first unsymmetrical cyanine monomer moiety is covalently attached to the linker; and

the second unsymmetrical cyanine monomer moiety is covalently attached to the linker.

59. The complex according to claim 58, wherein E is

60. The complex according to claim 59, wherein T, when present, is —

NR², —

N═

or S wherein the R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

61. The complex according to claim 60, 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.

62. The complex according to claim 61, wherein the reactive group is selected from the group consisting of 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 and a photoactivatable group.

63. The complex according to claim 62, wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

64. The complex according to claim 61, wherein the carrier molecule is selected from the group consisting of 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.

65. The complex according to claim 64, wherein the carrier molecule is selected from the group consisting of an antibody or fragment thereof, an avidin or 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.

66. The complex according to claim 61, wherein the solid support is selected from the group consisting of 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, and superparamagnetic bead.

67. The complex according to claim 66, wherein the solid support is selected from the group consisting of Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose and starch.

68. The complex according to claim 59, wherein the linker is

69. The complex according to claim 68, wherein R⁸ is S.

70. The complex according to claim 68, wherein R¹² is —

NH or —

N═

.

71. The complex according to claim 68, wherein the linker is

72. A method for detecting nucleic acid in a sample, wherein the method comprises the steps:

a. combining a nucleic acid reporter molecule with the sample to prepare a labeling mixture, wherein the nucleic acid reporter molecule comprising a first nucleic acid complexing monomer moiety, a second nucleic acid complexing monomer moiety and a linker that comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C, wherein the first nucleic acid complexing monomer moiety is covalently attached to the linker; and

the second nucleic acid complexing monomer moiety is covalently attached to the linker;

b. incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule to associate with nucleic acid in the sample;

c. illuminating the labeling mixture with an appropriate wavelength; and

,

d. observing the illuminated labeling mixture whereby the nucleic acid is detected.

73. The method according to claim 72, wherein the first and the second first nucleic acid complexing monomer moieties are the same.

74. The method according to claim 72, wherein the first and the second first nucleic acid complexing monomer moieties are different.

75. The method according to claim 72, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, a polymeric gel or tissue sections.

76. The method according to claim 72, wherein the nucleic acid is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

77. A method for detecting nucleic acid in a sample, wherein the method comprises the steps:

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

wherein each R³ is independently hydrogen, unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, unsubstituted fused benzene, substituted fused benzene, unsubstituted trifluoromethyl, substituted trifluoromethyl, unsubstituted halogen, substituted halogen, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support or substituted solid support;

each R⁴ is independently a unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support or substituted solid support;

X is O, S, or CR⁶R⁷ wherein each R⁶ and R⁷ are independently a unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, substituted solid support or R⁶ and R⁷ taken together form a 5- or 6-membered saturated ring;

j is 0, 1, or 2;

linker is a series of stable covalent bonds comprising 1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S and P; and

,

K is substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium;

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

c) illuminating the labeling mixture with an appropriate wavelength; and

,

d) observing the illuminated labeling mixture whereby the nucleic acid is detected.

78. The method according to claim 77, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, a polymeric gel or tissue sections.

79. The method according to claim 78, wherein the nucleic acid is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

80. The method according to claim 77, wherein the molecule is

81. The method according to claim 77, wherein the linker comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C.

82. The method according to claim 81, wherein the linker has the formula (I) —

(Y)_r—

(CH₂)_m-T_q-(CH₂)_n-E-(CH₂)_n-T_q-(CH₂)_m—

(Y)_r—

;

wherein Y is a linear or branched moiety comprising 1-20 non-hydrogen atoms selected from the group consisting of C, N, O, P and S;

T is a unsubstituted heteroatom or a substituted heteroatom;

E is an aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C;

r is independently 0 or 1;

m is an integer of 0-6;

n is independently 0 or 1;

q is independently 0 or 1;

and wherein the first unsymmetrical cyanine monomer moiety is covalently attached to the linker; and

the second unsymmetrical cyanine monomer moiety is covalently attached to the linker.

83. The method according to claim 82, wherein E is

84. The method according to claim 82, wherein T, when present, is —

NR², —

N═

or S wherein the R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

85. The method according to claim 84, 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.

86. The method according to claim 85, wherein the reactive group is selected from the group consisting of 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 and a photoactivatable group.

87. The method according to claim 86, wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

88. The method according to claim 85, wherein the carrier molecule is selected from the group consisting of 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.

89. The method according to claim 88, wherein the carrier molecule is selected from the group consisting of an antibody or fragment thereof, an avidin or 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.

90. The method according to claim 85, wherein the solid support is selected from the group consisting of 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, and superparamagnetic bead.

91. The method according to claim 90, wherein the solid support is selected from the group consisting of Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose and starch.

92. The method according to claim 83, wherein the linker is

93. The method according to claim 92, wherein R⁸ is S.

94. The method according to claim 92, wherein R¹² is —

NH or —

N═

.

95. The method according to claim 94, wherein the linker is

96. 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 wherein the nucleic acid reporter molecule comprises a first monomethine unsymmetrical cyanine monomer moiety, a second monomethine unsymmetrical cyanine monomer moiety and a linker that is

wherein R⁸ is a substituted heteroatom or an unsubstituted heteroatom;

R⁹ is a substituted heteroatom or an unsubstituted heteroatom;

R¹² is a substituted heteroatom or an unsubstituted heteroatom;

each Z is —

(CH₂)_g wherein g is 0 or 1; and

,

wherein the first unsymmetrical cyanine monomer moiety is covalently attached to the linker; and

the second unsymmetrical cyanine monomer moiety is covalently attached to the linker;

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

c) illuminating the labeling mixture with an appropriate wavelength; and

,

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

97. The method according to claim 96, wherein

R⁸ is —

NR², —

N═

or S and wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support;

R⁹ is —

NR², —

N═

or S and wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support; and

,

R¹² is —

NR², —

N═

or S and wherein R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

98. The method according to claim 96, wherein the monomethine unsymmetrical cyanine monomer moiety has the general formula:

99. The method according to claim 98, 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.

100. The method according to claim 99, wherein the reactive group is selected from the group consisting of 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 and a photoactivatable group.

101. The method according to claim 100, wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

102. The method according to claim 99, wherein the carrier molecule is selected from the group consisting of 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.

103. The method according to claim 102, wherein the carrier molecule is selected from the group consisting of an antibody or fragment thereof, an avidin or 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.

104. The method according to claim 99, wherein the solid support is selected from the group consisting of 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, and superparamagnetic bead.

105. The method according to claim 104, wherein the solid support is selected from the group consisting of Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose and starch.

106. The method according to claim 98, wherein the linker is

107. The method according to claim 106, wherein the reporter molecule is

108. The method according to claim 98, wherein R⁹ is N or S and R¹² is N or S.

109. The method according to claim 108, wherein the linker is

110. The method according to claim 109, wherein the reporter molecule is

111. The method according to claim 98, wherein the linker is

112. The method according to claim 111, wherein said reporter molecule is

113. The method according to claim 98, wherein the linker is

114. The method according to claim 113, wherein the compound is

115. The method according to claim 96, wherein the method further comprises combining a reporter molecule to said labeling mixture of step a) that is capable of producing a greater fluorescent intensity signal on DNA than on RNA.

116. The method according to claim 96, wherein the nucleic acid reporter molecule is

117. A kit for detecting nucleic acid in a sample, wherein the kit comprises:

a nucleic acid reporter molecule comprising a first nucleic acid complexing monomer moiety, a second nucleic acid complexing monomer moiety and a linker that comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C, wherein the first nucleic acid complexing monomer moiety is covalently attached to the linker; and

the second nucleic acid complexing monomer moiety is covalently attached to the linker.

118. The kit according to claim 117, wherein the kit further comprises instructions for detecting the nucleic acid.

119. The kit according to claim 117, wherein the kit further comprises instruction for detecting intracellular RNA.

120. The kit according to claim 117, wherein the nucleic acid reporter molecule is

121. The kit according to claim 117, wherein the kit further comprises at least one component that is a sample preparation reagent, a buffer agent, an organic solvent or an addition nucleic acid reporter molecule.

122. A kit for detecting nucleic acid in a sample, wherein the kit comprises:

a nucleic acid reporter molecule that is according to the formula

123. The kit according to claim 122, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, a polymeric gel or tissue sections.

124. The kit according to claim 123, wherein the nucleic acid is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

125. The kit according to claim 122, wherein the molecule is

126. The kit according to claim 122, wherein the linker comprises at least one aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C.

127. The kit according to claim 126, wherein the linker has the formula (I) —

(Y)_r—

(CH₂)_m-T_q-(CH₂)_n-E-(CH₂)_n-T_q-(CH₂)_m—

(Y)_r—

;

wherein Y is a linear or branched moiety comprising 1-20 non-hydrogen atoms selected from the group consisting of C, N, O, P and S;

T is a unsubstituted heteroatom or a substituted heteroatom;

E is an aromatic, heteroaromatic, cyclic or heterocyclic moiety comprising 3-20 non-hydrogen atoms selected from the group consisting of O, N, S, P and C;

r is independently 0 or 1;

m is an integer of 0-6;

n is independently 0 or 1;

q is independently 0 or 1;

and wherein the first unsymmetrical cyanine monomer moiety is covalently attached to the linker; and

the second unsymmetrical cyanine monomer moiety is covalently attached to the linker.

128. The kit according to claim 127, wherein E is

129. The kit according to claim 127, wherein T, when present, is —

NR², —

N═

or S wherein the R² is hydrogen, amine, substituted amine, substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkyl, unsubstituted reactive group, substituted reactive group, unsubstituted carrier molecule, substituted carrier molecule, unsubstituted solid support, or substituted solid support.

130. The kit according to claim 128, wherein the linker is

131. The kit according to claim 130, wherein R⁸ is S.

132. The kit according to claim 130, wherein R¹² is —

NH or —

N═

.

133. The kit according to claim 132, wherein the linker is