Unsymmetrical cyanine dimer compounds and their application

• US 7,888,136 B2
• Filed: 04/07/2008
• Issued: 02/15/2011
• Est. Priority Date: 07/31/2003
• 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
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/099,020
• Publication Number:
  US 20080199875A1
• Time in Patent Office: 1,044 Days
• Field of Search: 436/800, 536/26.6, 435/6, 422/61
• US Class Current: 435/6.11
• 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 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

2. The reporter molecule according to claim 1, 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.

3. The reporter molecule according to claim 1, wherein the monomethine unsymmetrical cyanine monomer moiety has the general formula:

4. The reporter molecule according to claim 3, 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.

5. The reporter molecule according to claim 4, 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.

6. The reporter molecule according to claim 5, wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

7. The reporter molecule according to claim 4, 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.

8. The reporter molecule according to claim 7, 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.

9. The reporter molecule according to claim 4, 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.

10. The reporter molecule according to claim 9, 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.

11. The reporter molecule according to claim 3, wherein the linker is

12. The reporter molecule according to claim 11, wherein the reporter molecule is

13. The reporter molecule according to claim 3, wherein R⁹ is N or S and R¹² is N or S.

14. The reporter molecule according to claim 3, wherein the linker is

15. The reporter molecule according to claim 14, wherein the reporter molecule

16. The reporter molecule according to claim 3, wherein the linker is

17. The reporter molecule according to claim 16, wherein said reporter molecule

18. The reporter molecule according to claim 3, wherein the linker is

19. The reporter molecule according to claim 18, wherein the compound is

20. A nucleic acid reporter molecule that is

21. 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 and the second nucleic acid complexing monomer moiety are covalently attached to the linker, and wherein the nucleic acid reporter molecule is

22. The kit according to claim 21, wherein the kit further comprises instructions for detecting the nucleic acid.

23. The kit according to claim 21, wherein the kit further comprises instruction for detecting intracellular RNA.

24. The kit according to claim 21, 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.

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

a nucleic acid reporter molecule

wherein the molecule is

26. The kit according to claim 25, 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.

27. The kit according to claim 26, wherein the nucleic acid is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

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

a nucleic acid reporter molecule that is according to the formula

29. The kit according to claim 28, 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.

30. The kit according to claim 29, wherein E is

31. The kit according to claim 29, 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.

32. The kit according to claim 30, wherein the linker is

33. The kit according to claim 32, wherein R⁸ is S.

34. The kit according to claim 32, wherein R¹² is —

NH or —

N═

.

35. The kit according to claim 34, wherein the linker is