Method of screening for specific binding interactions

• US 6,348,322 B1
• Filed: 10/17/1997
• Issued: 02/19/2002
• Est. Priority Date: 10/17/1997
• Status: Expired due to Fees

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Strittmatter, Warren J.
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): GRUN, JAMES LESLIE
• Application Number:
  US08/953,323
• Publication Number:
  US 20020012951A1
• Time in Patent Office: 1,586 Days
• Field of Search: 435/6, 435/7.1, 435/7.8, 435/174, 435/180, 435/181, 435/287.2, 435/287.9, 435/288.7, 435/961, 435/962, 435/968, 435/973, 436/501, 436/518, 436/523, 436/524, 436/527, 436/528, 436/529, 436/531, 436/534, 436/172, 436/800, 436/805, 436/807, 530/811, 530/812, 422/50, 422/55, 422/57, 422/82.05, 422/82.08
• US Class Current: 435/7.8
• 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 mixture useful for high throughput screening of molecular interactions, said mixture comprising:

a plurality of different solid support populations, each population comprising at least one discrete solid support, each said discrete solid support having a surface portion and having probe molecules and flexible linking polymers bound to the surface portion thereof;

the probe molecules bound to each said discrete solid support of one population being different from the probe molecules bound to each said discrete solid support of another population;

each of said flexible linking polymers having opposite end portions and a length dimension, each of said linking polymers being bound by one of said end portions to said solid support surface portion adjacent at least one of said bound probe molecules and having bound along said length thereof a plurality of fluorophore molecules,

wherein each of said fluorophore molecules is a first member of a fluorescence resonance energy transfer pair of donor and acceptor fluorophore molecules, the same first pair member fluorophore molecules being bound, via said bound flexible linking polymers, onto each said discrete solid support in the mixture.

2. The mixture according to claim 1, wherein said probe molecules are proteins or peptides.

3. The mixture according to claim 1, wherein said probe molecules are nucleic acids.

4. The mixture according to claim 1, wherein said polymers are selected from the group consisting of polysaccharides, polypeptides, and polyacrylic acid.

5. The mixture according to claim 1, wherein each said discrete solid support in the mixture further comprises an additional tag component bound to the surface portion thereof, said tag component providing a detectable indication of which of the different probe molecules is bound to the discrete solid support.

6. The mixture according to claim 1, wherein each said discrete solid support is a bead having a diameter of 10 to 2000 μ

m.

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

(a) providing the mixture of claim 1;

(b) providing a test compound having a second fluorophore bound thereto, wherein the second fluorophore completes the fluorescence resonance energy transfer pair of donor and acceptor fluorophore molecules with the first pair member fluorophore molecules bound onto the discrete solid supports in the mixture;

(c) contacting said test compound with the mixture under conditions suitable for binding of test compound to any probe molecules, bound onto the discrete solid supports therein, capable of binding the test compound;

(d) illuminating at least one of said contacted discrete solid supports in the mixture with light of a wavelength that is absorbed by said donor fluorophore molecule; and

then

(e) detecting transfer of fluorescence resonance energy to said acceptor fluorophore molecule from the illuminated at least one of said contacted discrete solid supports, the transfer indicating the binding of the test compound to the probe molecules bound to said illuminated at least one of said contacted discrete solid supports.

8. The method according to claim 7, wherein said polymers are selected from the group consisting of polysaccharides, polypeptides, and polyacrylic acid.

9. The method according to claim 7, wherein said contacting step is carried out by contacting a test solution containing said test compound with said mixture.

10. The method according to claim 9, wherein said illuminating step is carried out with said test solution remaining in contact with the discrete solid supports in the mixture.

11. The method according to claim 7, wherein said illuminating and detecting steps are sequentially repeated with sequential ones of said contacted discrete solid supports.

12. The method according to claim 11, wherein said illuminating and detecting steps are sequentially repeated in a flow sorting apparatus.