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

• US 7,541,143 B2
• Filed: 12/15/2004
• Issued: 06/02/2009
• Est. Priority Date: 02/20/1997
• Status: Expired due to Fees

• Current Assignee: OncoImmunin, Inc.
• Original Assignee: OncoImmunin, Inc.
• Inventors: Packard, Beverly, Komoriya, Akira
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US11/015,864
• Publication Number:
  US 20050158766A1
• Time in Patent Office: 1,630 Days
• Field of Search: 435/325, 435/23, 435/7.72, 435/6, 536/172, 536/23.1, 514/2, 530/300
• US Class Current: 435/6.12
• 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 nucleic acid backbone joining two fluorophores of the same species, wherein said nucleic acid backbone ranges in length from about 10 nucleotides to about 50 nucleotides, and 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 12 to about 40 nucleotides.

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

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

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

8. The fluorogenic composition of claim 7, wherein said hydrophobic group is selected from the group consisting of 9-fluorenylmethoxycarbonyl (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 (Bz10), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCI-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Born), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Burn), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

9. The composition of claim 7, wherein said hydrophobic group is Fmoc.

10. The fluorogenic composition of claim 7, wherein said hydrophobic group is a 9-fluoreneacetyl group (Fa).

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

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

13. 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.

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

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

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

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

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

19. The fluorogenic composition of claim 1, wherein said nucleic acid backbone ranges in length from about 15 to about 40 nucleotides.

20. A mammalian cell comprising a fluorogenic composition comprising a nucleic acid backbone joining two fluorophores of the same species, wherein said nucleic acid backbone ranges in length from about 10 nucleotides to about 50 nucleotides, and 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 bears a hydrophobic group.

22. The cell of claim 21, wherein said hydrophobic group is selected from the group consisting of:

9-fluorenylmethoxycarbonyl (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 (Bz10), 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 (Born), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

23. The cell of claim 21, wherein said hydrophobic group is Fmoc.

24. The cell of claim 21, wherein said hydrophobic group is attached to a terminus of the molecule.

25. The cell of claim 21, wherein said hydrophobic group is a 9-fluoreneacetyl group (Fa).

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

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

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

29. The cell of claim 28, wherein said fluorophores are carboxytetramethylrhodamine.

30. The cell of claim 28, wherein said fluorophores are carboxyrhodamine-X.

31. The cell of claim 28, wherein said fluorophores are carboxyrhodamine 110.

32. The cell of claim 28, wherein said fluorophores are diethylaminocoumarin.

33. The cell of claim 28, wherein said fluorophores are carbocyanine dyes.

34. The cell of claim 20, wherein said nucleic acid backbone ranges in length from about 12 to about 40 nucleotides.

35. The cell of claim 20, wherein said nucleic acid backbone ranges in length from about 15 to about 40 nucleotides.