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

• US 6,893,868 B2
• Filed: 12/22/2000
• Issued: 05/17/2005
• Est. Priority Date: 02/20/1997
• Status: Expired due to Fees

• Current Assignee: Oncolmmunin Incorporated
• Original Assignee: ONCO Immunity As
• Inventors: Komoriya, Akira, Packard, Beverly
• Primary Examiner(s): Weber, Jon
• Assistant Examiner(s): KAM, CHIH MIN
• Application Number:
  US09/747,287
• Publication Number:
  US 20030207264A1
• Time in Patent Office: 1,607 Days
• Field of Search: 435/325, 435/23, 435/7.72, 530/300, 514/2, 436/172, 536/172
• US Class Current: 435/325
• 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 mammalian cell comprising a fluorogenic composition comprising a polypeptide backbone joining two identical fluorophores whereby said fluorophores form an H-dimer resulting in the quenching of the fluorescence of said fluorophores.

2. The cell of claim 1, wherein said fluorophores are linked to the polypeptide backbone by linkers.

3. The cell of claim 1, wherein said fluorophores have an excitation wavelength between about 315 nm and about 700 nm.

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

5. The cell of claim 4, wherein said fluorophores are carboxytetramethylrhodamine.

6. The cell of claim 4, wherein said fluorophores are carboxyrhodamine-X.

7. The cell of claim 4, wherein said fluorophores are carboxyrhodamine 110.

8. The cell of claim 4, wherein said fluorophores are diethylaminocoumarin.

9. The cell of claim 4, wherein said fluorophores are carbocyanine dyes.

10. The cell of claim 1, wherein said polypeptide backbone comprises a protease cleavage site.

11. The cell of claims 4, wherein said fluorophores are xanthenes.

14. The cell of claims 1, 10, 12, 13, wherein said composition bears a hydrophobic group.

15. The cell of claim 14, wherein said hydrophobic group is selected from the group consisting of:

Fmoc (9-fluorenylmethoxycarbonyl), 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-pyridinesulphenyt (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).

16. The cell of claim 14, wherein said hydrophobic group is Fmoc (9-fluorenylmethoxycarbonyl).

17. The cell of claim 14, wherein said hydrophobic group is Fa (9-fluoreneacetyl group).

18. The cell of claim 14, wherein said hydrophobic group is attached to an amino terminus of the polypeptide.

12. A mammalian cell comprising a fluorogenic composition comprising a polypeptide backbone joining two fluorophores of the same species, wherein said fluorophores are xanthenes, whereby said fluorophores form an H-dimer resulting in the quenching of the fluorescence of said fluorophores.

13. A mammalian cell comprising a fluorogenic composition comprising a polypeptide backbone joining two fluorophores of the same species, wherein said fluorophores are carbocyanine dyes, whereby said fluorophores form an H-dimer resulting in the quenching of the fluorescence of said fluorophores.