Fluorogenic homogeneous binding assay methods and compositions

• US 20050239217A1
• Filed: 11/24/2004
• Published: 10/27/2005
• Est. Priority Date: 11/26/2003
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Sekar, Michael M.A., Graham, Ronald J.
• Application Number:
  US10/997,694
• Publication Number:
  US 20050239217A1
• Time in Patent Office: Days
• US Class Current: 436/528
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules

Abstract

Disclosed are binding substrate compositions, methods and kits useful for, among other things, detecting and/or characterizing binding interactions between molecules of interest.

Claims

1. A micelle comprising at least one binding substrate that comprises a hydrophobic moiety capable of integrating the binding substrate into the micelle, a fluorescent moiety and a binding moiety, wherein the fluorescence signal of the micelle is quenched in the absence of a binding partner for the binding moiety of the binding substrate.

2. The micelle of claim 1 in which the hydrophobic moiety comprises a substituted or unsubstituted, saturated or unsaturated hydrocarbon containing from 6 to 30 carbon atoms.

3. The micelle of claim 2 in which the hydrocarbon is a linear, branched or cyclic, saturated or unsaturated alkyl.

4. The micelle of claim 3 in which the alkyl is linear and contains from 10 to 26 carbon atoms.

5. The micelle of claim 4 in which the alkyl is a fully saturated n-alkyl.

6. The micelle of claim 4 in which the alkyl includes one or more carbon-carbon double bonds, each of which may, independently of the others, be in the cis or trans configuration and/or one or more carbon-carbon triple bonds.

7. The micelle of claim 1 in which the hydrophobic moiety comprises a phospholipid.

8. The micelle of claim 7 in which the phospholipid is a sphingolipid.

9. The micelle of claim 7 in which the phospholipid is a glycerophospholipid.

10. The micelle of claim 1 in which the hydrophobic moiety and the binding moiety are linked to one another through the fluorescent moiety.

11. The micelle of claim 1 in which the hydrophobic moiety and the fluorescent moiety are linked to one another through the binding moiety.

12. The micelle of claim 1 in which the hydrophobic moiety, the fluorescent moiety and the binding moiety are linked to one another via a trivalent linker.

13. The micelle of claim 12 in which the trivalent linker comprises an amino acid.

14. The micelle of claim 12 in which the trivalent linker is provided by a trivalent linker synthon illustrated in FIG. 1 G.

15. The micelle of claim 12 in which the binding substrate is selected from a binding substrate depicted in any one of FIGS. 1A-1F, wherein “

B”

comprises the binding moiety, “

D”

comprises the fluorescent moiety and R¹ comprises the hydrophobic moiety.

16. The micelle of claim 12 in which the hydrophobic moiety comprises a substituted or unsubstituted, saturated or unsaturated hydrocarbon containing from 6 to 30 carbon atoms.

17. The micelle of claim 16 in which the hydrocarbon is a linear, branched or cyclic, saturated or unsaturated alkyl.

18. The micelle of claim 17 in which the alkyl is linear and contains from 10 to 26 carbon atoms.

19. The micelle of claim 18 in which the alkyl is a fully saturated n-alkyl.

20. The micelle of claim 17 in which the alkyl includes one or more carbon-carbon double bonds, each of which may, independently of the others, be in the cis or trans configuration and/or one or more carbon-carbon triple bonds.

21. The micelle of claim 12 in which the hydrophobic moiety comprises a phospholipid.

22. The micelle of claim 21 in which the phospholipid is a sphingolipid.

23. The micelle of claim 21 in which the phospholipid is a glycerophospholipid.

24. The micelle of claim 1 in which the binding moiety comprises one member of a receptor-ligand pair, or a binding fragment thereof.

25. The micelle of claim 24 in which the binding moiety comprises the ligand.

26. The micelle of claim 24 in which the binding moiety comprises the receptor, or a binding fragment thereof.

27. The micelle of claim 1 in which the binding moiety comprises a candidate compound whose ability to bind another molecule is sought to be determined.

28. The micelle of claim 1 in which the fluorescent moiety comprises a dye having net hydrophilic character.

29. The micelle of claim 1 in which the fluorescent moiety comprises a dye selected from a xanthene dye, a rhodamine dye, a fluorescein dye, a cycanine dye, a phthalocyanine dye, a squaraine dye and a bodipy dye.

30. The micelle of claim 1 in which the fluorescent moiety comprises a xanthene dye.

31. The micelle of claim 30 in which the xanthene dye is a fluorescein dye.

32. The micelle of claim 30 in which the xanthene dye is a rhodamine dye.

33. The micelle of claim 1 in which the fluorescent moiety comprises a fluorescence donor moiety and a fluorescence acceptor moiety.

34. The micelle of claim 33 in which the fluorescence donor moiety comprises a fluorescein dye.

35. The micelle of claim 33 in which the fluorescence acceptor moiety comprises a fluorescein or a rhodamine dye.

36. The micelle of claim 35 in which the fluorescence donor moiety comprises a fluorescein dye.

37. The micelle of claim 1 in which the fluorescent moiety comprises fewer than 150 atoms.

38. A method of detecting the presence and/or quantity of a binding compound in a sample, comprising the steps of:

contacting the sample with a composition comprising a binding substrate that comprises a hydrophobic moiety capable of integrating the binding substrate into a micelle, a binding moiety and a fluorescent moiety, under conditions effective to permit binding between the binding moiety and a binding molecule therefore, if present in the sample; and

detecting a fluorescence signal, where a change in the fluorescence signal indicates the presence and/or quantity of a binding compound in the sample.

39-64. -64. (canceled)

65. A method of identifying and/or characterizing a modulator of a binding interaction, comprising the steps of:

contacting a sample comprising a candidate compound with a composition comprising;

(i) a binding substrate that comprises a hydrophobic moiety capable of integrating the binding substrate into a micelle, a fluorescent moiety and a binding moiety; and

(ii) a binding partner for the binding moiety, under conditions effective to permit binding between the binding moiety and the binding partner; and

detecting a fluorescence signal, where an increase or a decrease in the fluorescence signal as compared to a control reaction or a standard curve indicates that the candidate modulator compound modulates the binding interaction between the binding moiety and the binding partner.

66-106. -106. (canceled)

107. A method of identifying a compound that binds a receptor of interest, comprising the steps of:

contacting a sample comprising the receptor with a composition comprising a plurality of micelles, each of which comprises a binding substrate that comprises a hydrophobic moiety capable of integrating the binding substrate into a micelle, a fluorescent moiety and a binding moiety comprising a candidate binding compound, wherein the fluorescence spectra of the fluorescent moieties on the micelles are resolvable from one another and are correlated with the structure of the candidate compound comprising their binding moieties, under conditions effective to permit binding between the micelles and the receptor;

detecting a fluorescence signal, where an increase in the fluorescence signal indicates that a micelle comprises a binding compound for the receptor; and

determining the structure and/or identity of the binding compound based upon the detected fluorescence spectrum.

108-139. -139. (canceled)