Method of screening for specific binding interactions

• US 20020142335A1
• Filed: 01/30/2002
• Published: 10/03/2002
• Est. Priority Date: 10/17/1997
• Status: Abandoned Application

• Current Assignee: Strittmatter, Warren J.
• Original Assignee: Strittmatter, Warren J.
• Inventors: Strittmatter, Warren J.
• Application Number:
  US10/060,639
• Publication Number:
  US 20020142335A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 530/811 Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier

Abstract

A method for detecting the binding of a test compound to a probe molecule comprises providing a test compound, the test compound having a first fluorophore bound thereto, and providing a screening substrate. The screening substrate comprises a solid support, a probe molecule bound to the solid support, and a second fluorophore bound to the solid support adjacent the probe molecule. An advantage of the invention is that this obviates the need for binding the second fluorophore directly to the probe molecule. Preferably, the second fluorophore is bound to the solid support by a flexible linker group. This enables the second fluorophore to interogate different positions on the probe molecule, which is also bound to the solid support adjacent the linker group, enhancing the ability of the method of the invention to detect positive binding events (specific binding of the test compound to the probe molecule. The first and second fluorophores together comprise the donor and acceptor fluorophores of a fluorescence resonance energy transfer (FRET) pair, or a “donor/acceptor pair.” The test compound is contacted to the screening substrate, and the screening substrate illuminated with light at a wavelength that is absorbed by the donor fluorophore. The transfer of energy from one to the other fluorophore is then detected, with the transfer of energy indicating the binding of the test compound to the probe. Substrates useful for carrying out the foregoing methods are also disclosed.

Claims

1. A method for detecting the binding of a test compound to a probe molecule, said method comprising:

providing a test compound, said test compound having a first fluorophore bound thereto;

providing a screening substrate, said screening substrate comprising a solid support, a probe molecule bound to said solid support, and a second fluorophore bound to said solid support adjacent said probe molecule;

wherein said first and second fluorophores together comprise a donor and an acceptor fluorophore of a fluorescence resonance energy transfer pair;

contacting said test compound to said screening substrate;

illuminating said screening substrate with light at a wavelength that is absorbed by said donor fluorophore; and

then

detecting the transfer of energy to said acceptor fluorophore, the transfer of energy to said acceptor fluorophore indicating the binding of said test compound to said probe.

2. A method according to claim 1, wherein said probe molecule is a protein or peptide.

3. A method according to claim 1, wherein said probe molecule is a nucleic acid.

4. A method according to claim 1, wherein said test compound is a protein or peptide.

5. A method according to claim 1, wherein said test compound is a nucleic acid.

6. A method according to claim 1, wherein said contacting step is carried out by contacting a test solution containing said test compound to said screening substrate.

7. A method according to claim 6, wherein said illuminating step is carried out with said test solution in contact with said screening substrate.

8. A method according to claim 1, wherein said first and second fluorophores absorb light at different wavelengths from one another.

9. A method according to claim 1, wherein said solid support comprises a bead.

10. A method according to claim 1, wherein said solid support comprises glass.

11. A screening substrate useful for the high throughput screening of molecular interactions, said substrate comprising:

a solid support having a surface portion, said surface portion comprising a plurality of discreet known regions;

a plurality of different probe molecules bound to said surface portion, with different probe molecules positioned on said surface portion in different ones of said discrete known regions; and

a plurality of the same fluorophores connected to said surface portion, said fluorophores positioned on said surface portion in all of said plurality of discreet known regions.

12. A screening substrate according to claim 11, wherein said probe molecules are proteins or peptides.

13. A screening substrate according to claim 11, wherein said probe molecules are nucleic acids.

14. A screening substrate according to claim 11, wherein said fluorophores are bound to said solid support by a linking group.

15. A screening substrate according to claim 14, wherein said linking group is a polymer, said polymer having a plurality of said second fluorophores conjugated thereto at separate locations along the length thereof.

16. A screening substrate according to claim 15, wherein said fluorophores are conjugated to said polymer linking group at separate locations along the length thereof.

17. A screening substrate according to claim 11, wherein said solid support comprises glass.

18. A method for detecting the binding of a test compound to a probe molecule, said method comprising:

providing a test compound, said test compound having a first fluorophore bound thereto;

providing a screening substrate according to claim 11, wherein said fluorophore bound to said surface portion of said solid support comprises a second fluorophore;

wherein said first and second fluorophores together comprise the donor and acceptor fluorophores of a fluorescence resonance energy transfer pair;

contacting said test compound to said screening substrate;

illuminating said screening substrate with light at a wavelength that is absorbed by said donor fluorophore; and

then

detecting the transfer of energy to said acceptor fluorophore in one of said discreet known regions, the transfer of energy to said acceptor fluorophore indicating the binding of said test compound to the probe bound to that discreet known region.

19. A method according to claim 18, wherein said contacting step is carried out by contacting a test solution containing said test compound to said screening substrate.

20. A method according to claim 19, wherein said illuminating step is carried out with said test solution in contact with said screening substrate.

21. A method according to claim 18, wherein said second fluorophores are conjugated to said solid support by a polymer linking group, said polymer linking group having a plurality of said second fluorophores conjugated thereto at separate locations along the length thereof so that energy transfer between said first and second fluorophores upon binding of the test compound to the probe molecule is enhanced.

30. A method for detecting the binding of a test compound to a probe molecule, said method comprising:

providing a test compound, said test compound having a first fluorophore bound thereto;

providing a mixture according to claim 20, wherein said fluorophore bound to said surface portion of said solid support is a second fluorophore;

wherein said first and second fluorophores together comprise the donor and acceptor fluorophores of a fluorescence resonance energy transfer pair;

contacting said test compound to at least one of said discreet solid supports;

illuminating said discreet solid support with light at a wavelength that is absorbed by said donor fluorophore; and

then

detecting the transfer of energy to said acceptor fluorophore from said discreet solid support, the transfer of energy to said acceptor fluorophore indicating the binding of said test compound to the probe bound to that discreet solid support.

31. A method according to claim 30, wherein said contacting step is carried out by contacting a test solution containing said test compound to at least one of said discreet solid supports.

32. A method according to claim 31, wherein said illuminating step is carried out with said test solution in contact with said discreet solid support.

33. A method according to claim 30, wherein said second fluorophores are conjugated to said solid support by a polymer linking group, said polymer linking group having a plurality of said second fluorophores conjugated thereto at separate locations along the length thereof so that energy transfer between said first and second fluorophores upon binding of the test compound to the probe molecule is enhanced.

34. A method according to claim 30, wherein said illuminating and detecting steps are sequentially repeated with sequential ones of said discreet solid supports.

35. A method according to claim 34, wherein said illuminating and detecting steps are sequentially repeated in a flow sorting apparatus.

22. A mixture useful for the high throughput screening of molecular interactions, said mixture comprising:

a plurality of discreet solid supports each having a surface portion, each such support having a different probe molecule bound to the surface portion thereof, and

a plurality of the same fluorophores bound to the surface portion of each of said solid supports.

23. A mixture according to claim 22, wherein said probe molecules are proteins or peptides.

24. A mixture according to claim 22, wherein said probe molecules are nucleic acids.

25. A mixture according to claim 22, wherein said fluorophores are bound to said solid support by a linking group.

26. A mixture according to claim 25, wherein said linking group is a polymer, said polymer having a plurality of said second fluorophores conjugated thereto at separate locations along the length thereof.

27. A mixture according to claim 26, wherein a plurality of said fluorophores are conjugated to said polymer linking group at separate locations along the length thereof.

28. A mixture according to claim 22, wherein each of said discreet solid supports has a different tag component bound thereto.

29. A mixture according to claim 22, wherein each of said discreet solid supports comprises a bead having a diameter of 10 to 2000 μ

m.