Sensitive, multiplexed diagnostic assays for protein analysis

• US 20010024784A1
• Filed: 01/04/2001
• Published: 09/27/2001
• Est. Priority Date: 01/24/2000
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Phylos, Inc. (Bristol-Myers Squibb Company)
• Inventors: Wagner, Richard W.
• Granted Patent: US 6,489,116 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors

Abstract

Disclosed herein are methods for detecting multiple compounds in a sample, involving: (a) contacting the sample with a mixture of binding reagents, the binding reagents being nucleic acid-protein fusions, each having (i) a protein portion which is known to specifically bind to one of the compounds and (ii) a nucleic acid portion which encodes the protein portion and which includes a unique identification tag; (b) allowing the protein portions of the binding reagents and the compounds to form complexes; (c) capturing the binding reagent-compound complexes; (d) amplifying the nucleic acid portions of the complexed binding reagents; and (e) detecting the unique identification tag of each of the amplified nucleic acids, thereby detecting the corresponding compounds in the sample.

Also disclosed herein are kits for carrying out such methods.

Claims

1. A method for detecting multiple compounds in a sample, said method comprising:

(a) contacting said sample with a mixture of binding reagents, said binding reagents being nucleic acid-protein fusions, each having (i) a protein portion which is known to specifically bind to one of said compounds and (ii) a nucleic acid portion which encodes said protein portion and which includes a unique identification tag;

(b) allowing said protein portions of said binding reagents and said compounds to form complexes;

(c) capturing said binding reagent-compound complexes;

(d) amplifying said nucleic acid portions of said complexed binding reagents; and

(e) detecting said unique identification tag of each of said amplified nucleic acids, thereby detecting the corresponding compounds in said sample.

2. The method of

claim 1

, wherein said sample is a biological sample.

3. The method of

claim 1

, wherein said nucleic acid-protein fusion is an RNA-protein fusion.

4. The method of

claim 1

, wherein said nucleic acid-protein fusion is covalently bound.

5. The method of

claim 4

, wherein said nucleic acid-protein fusion is covalently bound through a peptide acceptor.

6. The method of

claim 5

, wherein said peptide acceptor is puromycin.

7. The method of

claim 1

, wherein said binding reagents do not bind said compounds through compound-specific antibody domains.

8. The method of

claim 1

, wherein each of said binding reagents comprises a scaffold domain.

9. The method of

claim 8

, wherein each of said binding reagents comprises a fibronectin scaffold domain.

10. The method of

claim 9

, wherein said fibronectin scaffold domain is the 10^th domain of fibronectin type III.

11. The method of

claim 8

, wherein each of said binding reagents comprises an antibody scaffold domain.

12. The method of

claim 1

, wherein said binding reagents bind said compounds with equilibrium constants of less than about 500 nM.

13. The method of

claim 1

, wherein said unique identification tags are detected using a solid support to which are immobilized nucleic acids specific for said unique identification tags and said detection is accomplished by hybridization of said unique identification tags to said immobilized nucleic acids.

14. The method of

claim 1

, wherein said amplifying step (d) is carried out using quantitative PCR.

15. The method of

claim 1

, wherein said compounds are proteins.

16. The method of

claim 1

, wherein said mixture of binding reagents comprises at least 5 different nucleic acid-protein fusions, each specifically binding to a different compound.

17. The method of

claim 16

, wherein said mixture of binding reagents comprises at least 100 different nucleic acid-protein fusions, each specifically binding to a different compound.

18. The method of

claim 17

, wherein said mixture of binding reagents comprises at least 40,000 different nucleic acid-protein fusions, each specifically binding to a different compound.

19. The method of

claim 18

, wherein said mixture of binding reagents comprises at least 500,000 different nucleic acid-protein fusions, each specifically binding to a different compound.

20. A method for detecting a compound in a sample, said method comprising:

(a) contacting said sample with a binding reagent, said binding reagent being a nucleic acid-protein fusion having (i) a protein portion which is known to specifically bind to said compound and (ii) a nucleic acid portion which encodes said protein portion and which includes a unique identification tag;

(b) allowing said protein portion of said binding reagent and said compound to form a complex;

(c) capturing said binding reagent-compound complex;

(d) amplifying said nucleic acid portion of said complexed binding reagent; and

(e) detecting said unique identification tag of said amplified nucleic acid, thereby detecting the corresponding compound in said sample.

21. A kit for carrying out compound detection, said kit comprising:

(a) a nucleic acid-protein fusion, wherein said protein portion of said fusion specifically binds said compound and said nucleic acid portion of said fusion encodes said protein portion and includes a unique identification tag;

(b) a PCR primer pair, wherein the first of said primers hybridizes to said nucleic acid portion of said fusion 5′

to said unique identification tag and the second of said primers hybridizes to said nucleic acid portion of said fusion 3′

to said unique identification tag and hybridization of said primers to said nucleic acid fusion permits amplification of said unique identification tag; and

(c) a solid support comprising a nucleic acid which can hybridize to said unique identification tag.

22. The kit of

claim 21

, wherein said kit further comprises Taq polymerase.

23. The kit of

claim 21

, wherein said nucleic acid-protein fusion is an RNA-protein fusion.

24. The kit of

claim 21

, wherein said nucleic acid-protein fusion is covalently bound.

25. The kit of

claim 24

, wherein said nucleic acid-protein fusion is covalently bound through a peptide acceptor.

26. The kit of

claim 25

, wherein said peptide acceptor is puromycin.

27. The kit of

claim 21

, wherein said nucleic acid-protein fusion does not bind said compound through a compound-specific antibody domain.

28. The kit of

claim 21

, wherein said nucleic acid-protein fusion comprises a scaffold domain.

29. The kit of

claim 28

, wherein nucleic acid-protein fusion comprises a fibronectin scaffold domain.

30. The kit of

claim 29

, wherein said fibronectin scaffold domain is the 10^th domain of fibronectin type III.

31. The kit of

claim 28

, wherein said nucleic acid-protein fusion comprises an antibody scaffold domain.

32. The kit of

claim 21

, wherein said nucleic acid-protein fusion binds said compound with an equilibrium constant of less than about 500 nM.

33. The kit of

claim 21

, wherein said solid support is a chip.

34. The kit of

claim 21

, wherein said solid support comprises an ordered array of single-stranded nucleic acids on its surface, each of said singlestranded nucleic acids being capable of hybridizing to a different said unique identification tag.

35. The kit of

claim 21

, wherein said compound is a protein.

36. The kit of

claim 21

, wherein said kit comprises at least 5 different nucleic acid-protein fusions, each specifically binding to a different compound.

37. The kit of

claim 36

, wherein said kit comprises at least 100 different nucleic acid-protein fusions, each specifically binding to a different compound.

38. The kit of

claim 37

, wherein said kit comprises at least 40,000 different nucleic acid-protein fusions, each specifically binding to a different compound.

39. The kit of

claim 38

, wherein said kit comprises at least 500,000 different nucleic acid-protein fusions, each specifically binding to a different compound.