Multiplex selection technique for identifying protein-binding sites and DNA-binding proteins

6,066,452

08/906,691

August 06, 1997

Methods for simultaneously selecting binding site sequences for multiple DNA-binding proteins are provided. A source of DNA-binding proteins is mixed with oligonucleotide duplexes containing a randomized internal sequence. Bound oligonucleotides are isolated, amplified and analyzed, such as by DNA sequence analysis. The oligonucleotide duplexes are also used to identify and isolate DNA-binding proteins.

Weissman, Sherman M. (New Haven, CT); Nallur, Girish N. (New Haven, CT); Kulkarni, Prakash (Columbia, MD)

Yale University (New Haven, CT)

We claim

1. A method for isolating DNA-binding proteins, comprising

(a) mixing (i) a set of oligonucleotide duplexes, wherein said duplexes comprise 5' and 3' sequences that will hybridize to primers for amplification and a randomly generated internal sequence of nucleotides, (ii) an inhibitor of non-sequence specific binding of DNA-binding proteins to the oligonucleotide duplexes and (iii) a sample containing a mixture of DNA-binding proteins under conditions in which the concentration of oligonucleotide duplexes is at or below the average Kd value for specific binding between the oligonucleotide duplexes and the DNA-binding proteins;

(b) separating unbound oligonucleotide duplexes from oligonucleotide duplexes complexed with the DNA-binding proteins on the basis of differences in physical or biochemical properties;

(c) recovering the DNA-binding proteins from the complexes.

2. The method of claim 1, further comprising, subsequent to step (b)

amplifying complexed duplexes to form amplified duplexes;

mixing the amplified duplexes with the sample containing DNA-binding proteins; and

separating unbound amplified duplexes from amplified duplexes complexed with the DNA-binding proteins on the basis of differences in physical or biochemical properties;

wherein the amplifying, mixing, and separating steps are performed one or more times.

3. The method of claim 2 wherein the steps are performed three times.

4. The method of claim 1, wherein the sample is a nuclear extract or a cellular extract.

5. The method of claim 1 wherein the internal sequence contains 6 to 25 base pairs.

6. The method of claim 1 wherein the internal sequence contains 8 to 12 base pairs.

7. The method of claim 1 wherein the non-specific inhibitor is poly(dI-dC).

8. The method of claim 1 wherein the 5' and 3' sequences contain 10 to 30 bp.

9. The method of claim 1 wherein the 5' and 3' sequences are different.

10. The method of claim 1 wherein the separating step is based on differences in electrophoretic or chromatographic mobility.

11. The method of claim 10 wherein the separating step uses polyacrylamide gel electrophoresis or size-exclusion chromatography.

12. The method of claim 1 wherein the separating step is based on differences in filter-binding properties.

13. A method for identifying DNA-binding proteins in an expression library, comprising

(a) mixing (i) a set of oligonucleotide probes wherein said probes comprise 5' and 3' sequences that will hybridize to primers for amplification and a randomly generated internal sequence of nucleotides, (ii) an inhibitor of non-sequence specific binding of DNA-binding proteins to the oligonucleotide probes and (iii) a mixture of DNA binding proteins from an expression library, wherein said library comprises cells that express DNA-binding proteins, under conditions in which the concentration of oligonucleotide probes is at or below the average Kd value for specific binding between the oligonucleotide probes and the DNA-binding proteins;

(b) separating on the basis of differences in physical or biochemical properties unbound oligonucleotide probes from oligonucleotide probes complexed with the DNA-binding proteins;

(c) amplifying complexed probes to form amplified probes; and

(d) detecting the binding of amplified probes to the DNA binding proteins of the expression library;

whereby cells in the expression library that express DNA-binding proteins are identified.

14. The method of claim 13, further comprising the step of isolating cells in the expression library expressing DNA binding proteins that bind said amplified probes.

15. A method for simultaneously determining nucleotide recognition sequences for a mixture of DNA-binding proteins, comprising

(a) mixing (i) a set of oligonucleotide duplexes wherein said probes comprise 5' and 3' sequences that will hybridize to primers for amplification and a randomly generated internal sequence of nucleotides, (ii) an inhibitor of non-sequence specific binding of DNA-binding proteins to the oligonucleotide duplexes and (iii) a sample containing a mixture of DNA-binding proteins under conditions in which the concentration of oligonucleotide duplexes is at or below the average Kd value for specific binding between the oligonucleotide duplexes and the DNA-binding proteins;

(c) amplifying complexed duplexes to form amplified duplexes; and

(d) analyzing the amplified duplexes to determine nucleotide recognition sequences for the DNA-binding proteins.

16. The method of claim 15 wherein the analyzing step comprises

ligating the amplified duplexes to a vector to generate clones; and

determining the DNA sequences of the cloned duplexes.

17. The method of claim 15 wherein the analyzing step comprises

denaturing the amplified duplexes;

hybridizing the denatured duplexes to an array of single-stranded oligonucleotides having sequences; and

detecting oligonucleotides hybridized with denatured duplexes.

18. A method for simultaneously isolating nucleotide recognition sequences for a mixture of DNA-binding proteins, comprising

(a) mixing a set of oligonucleotide duplexes wherein said probes comprise 5' and 3' sequences that will hybridize to primers for amplification and a randomly generated internal sequence of nucleotides, an inhibitor of non-sequence specific binding of DNA-binding proteins to the oligonucleotide duplexes and a sample containing a mixture of DNA-binding proteins under conditions in which the concentration of oligonucleotide duplexes is at or below the average Kd value for specific binding between the oligonucleotide duplexes and the DNA-binding proteins;

(b) separating unbound oligonucleotide duplexes from oligonucleotide duplexes complexed with the DNA-binding proteins on the basis of differences in physical or biochemical properties weight;

(c) amplifying complexed duplexes to form amplified duplexes;

thereby isolating nucleotide recognition sequences for DNA-binding proteins.

19. The method of claim 18, further comprising, subsequent to step (c)

(c)(1) mixing the amplified duplexes with the sample containing DNA-binding proteins;

(c)(2) separating unbound amplified duplexes from amplified duplexes complexed with the DNA-binding proteins on the basis of differences in molecular weight; and

(c)(3) amplifying the complexed duplexes from step (c)(2) to form amplified duplexes;

wherein steps (c)(1) through (c)(3) are performed one or more times.

20. The method of claim 19 wherein steps (c)(1) to (c)(3) are performed three times.

21. The method of claim 18 wherein the 5' and 3' sequences each have a restriction site.

22. The method of claim 21 wherein the restriction sites are identical.

23. The method of claim 18 wherein the sample is a nuclear extract, cellular extract, or an extract from cells of an expression library.

24. The method of claim 18 wherein the internal sequence contains 6 to 25 base pairs.

25. The method of claim 18 wherein the non-specific inhibitor is poly(dI-dC).

26. The method of claim 18 wherein the separating step uses polyacrylamide gel electrophoresis.

27. The method of claim 18 wherein the oligonucleotide duplexes are labeled.

28. The method of claim 18 wherein the oligonucleotide duplexes comprise a tag moiety.

29. The method of claim 28 wherein said tag moiety is linked to the 5' and 3' ends of the duplexes.

30. The method of claim 29 wherein the tag moiety is biotin or a fluorescent dye.

435/6; 435/71; 435/78; 435/911; 435/912; 4353/201; 435/440

C12Q 168; C12P 1934; C12N 1563

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