Homo-doubly labeled compositions for the detection of enzyme activity in biological samples

• US 20050158766A1
• Filed: 12/15/2004
• Published: 07/21/2005
• Est. Priority Date: 02/20/1997
• Status: Active Grant

• Current Assignee: Oncolmmunin Incorporated
• Original Assignee: Oncolmmunin Incorporated
• Inventors: Komoriya, Akira, Packard, Beverly
• Granted Patent: US 7,541,143 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: G01N 2333/918 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: G01N 2333/96425 from mammals
• CPC Class Codes: G01N 2333/96469 Interleukin 1-beta convertase-like enzymes
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The present invention provides for novel reagents whose fluorescence changes upon cleavage or a change in conformation of a backbone. The reagents comprise a backbone (e.g. nucleic acid, polypeptide, etc.) joining two fluorophores of the same species whereby the fluorophores form an H-dimer resulting in quenching of the fluorescence of the fluorophores. When the backbone is cleaved or changes conformation, the fluorophores are separated, no longer forming an H-type dimer, and are de-quenched thereby providing a detectable signal. The use of a single fluorophore rather than an “acceptor-donor” fluoresecence resonance energy transfer system offers synthesis and performance advantages.

Claims

1. A fluorogenic composition comprising a a nucleic acid backbone joining two fluorophores of the same species whereby said fluorophores form an H-dimer resulting in quenching of the fluorescence of said fluorophores.

2. The fluorogenic composition of claim 1, wherein said nucleic acid backbone comprises a restriction site.

3. The fluorogenic composition of claim 1, wherein said nucleic acid backbone is self-complementary and forms a hairpin.

4. The fluorogenic composition of claim 1, wherein said nucleic acid backbone ranges in length from about 10 to about 100 nucleotides.

5. The fluorogenic composition of claim 1, wherein said nucleic acid backbone ranges in length from about 15 to about 50 nucleotides.

6. The fluorogenic composition of claim 1, wherein said composition is attached to a solid support.

7. The fluorogenic composition of claim 1, wherein said composition is inside a mammalian cell.

8. The fluorogenic composition of claim 1, wherein said composition bears a hydrophobic group.

9. The fluorogenic composition of claim 8, wherein said hydrophobic group is selected from the group consisting of Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′

-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

10. The composition of claim 9, wherein said hydrophobic group is Fmoc.

11. The composition of claim 9, wherein said hydrophobic group is Fa.

12. The fluorogenic composition of claim 1, wherein said fluorophores are linked to the nucleic acid backbone by linkers.

13. The fluorogenic composition of claim 1, wherein said fluorophores have an excitation wavelength between about 310 nm and about 750 nm.

14. The fluorogenic composition of claim 1, wherein said fluorophores are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes.

15. The fluorogenic composition of claim 1, wherein said fluorophores are carboxytetramethylrhodamine.

16. The fluorogenic composition of claim 1, wherein said fluorophores are carboxyrhodamine-X.

17. The fluorogenic composition of claim 1, wherein said fluorophores are carboxyrhodamine 110.

18. The fluorogenic composition of claim 1, wherein said fluorophores are diethylaminocoumarin.

19. The fluorogenic composition of claim 1, wherein said fluorophores are carbocyanine dyes.

20. A mammalian cell comprising a fluorogenic composition comprising a nucleic acid backbone joining two identical fluorophores whereby said fluorophores form an H-dimer resulting in the quenching of the fluorescence of said fluorophores.

21. The cell of claim 20, wherein said composition comprises a nucleic acid backbone.

22. The cell of claim 20, wherein said composition bears a hydrophobic group.

23. The cell of claim 22, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′

-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

24. The cell of claim 23, wherein said hydrophobic group is Fmoc.

25. The cell of claim 20, wherein said hydrophobic group is Fa.

26. The cell of claim 20, wherein said hydrophobic group is attached to the amino terminus of the molecule.

27. The cell of claim 20, wherein said fluorophores are linked to the nucleic acid backbone or to the polypeptide backbone by linkers.

28. The cell of claim 20, wherein said fluorophores have an excitation wavelength between about 310 nm and about 750 nm.

29. The cell of claim 20, wherein said fluorophores are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes.

30. The cell of claim 29, wherein said fluorophores are carboxytetramethylrhodamine.

31. The cell of claim 29, wherein said fluorophores are carboxyrhodamine-X.

32. The cell of claim 29, wherein said fluorophores are carboxyrhodamine 110.

33. The cell of claim 29, wherein said fluorophores are diethylaminocoumarin.

34. The cell of claim 29, wherein said fluorophores are carbocyanine dyes.

35. A method of detecting the activity of a nuclease or the presence of a nucleic acid, said method comprising:

i) contacting said nuclease or said nucleic acid with a fluorogenic composition comprising a nucleic acid backbone joining two fluorophores of the same species whereby said fluorophores form an H-dimer resulting in quenching of the fluorescence of said fluorophores; and

ii) detecting a change in fluorescence or absorbance of said fluorogenic composition where an increase in fluorescence or a change in absorbance indicates that said nuclease cleaves said nucleic acid backbone or that said nucleic acid hybridizes to said backbone.

36. The method of claim 35, wherein said nucleic acid backbone comprises a restriction site.

37. The method of claim 35, wherein said nucleic acid backbone is self-complementary and forms a hairpin.

38. The method of claim 35, wherein said nucleic acid backbone ranges in length from about 10 to about 100 nucleotides.

39. The method of claim 35, wherein said nucleic acid backbone ranges in length from about 15 to about 50 nucleotides.

40. The method of claim 35, wherein said composition is attached to a solid support.

41. The method of claim 35, wherein said composition is inside a mammalian cell.

42. The method of claim 35, wherein said composition is in solution.

43. The method of claim 35, wherein said composition bears a hydrophobic group.

44. The method of claim 43, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′

-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

45. The method of claim 35, wherein said fluorophores are linked to the nucleic acid backbone or to the polypeptide backbone by linkers.

46. The method of claim 35, wherein said fluorophores have an excitation wavelength between about 310 nm and about 750 nm.

47. The method of claim 35, wherein said fluorophores are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes.

48. The method of claim 35, wherein said contacting is in a histological section.

49. The method of claim 35, wherein said contacting is in a cell culture.

50. The method of claim 35, wherein said contacting is contacting a seeded or cultured adherent cell.

51. The method of claim 35, wherein said contacting is in a cell suspension derived from a biological sample selected from the group consisting of a tissue, blood, urine, saliva, lymph, biopsy.

52. The method of claim 35, wherein said detecting is by a method selected from the group consisting of fluorescence microscopy, confocal microscopy, fluorescence microplate reader, flow cytometry, fluorometry, and absorption spectroscopy.