Sequence-directed DNA-binding molecules compositions and methods

• US 5,578,444 A
• Filed: 12/20/1993
• Issued: 11/26/1996
• Est. Priority Date: 06/27/1991
• Status: Expired due to Term

• Current Assignee: Genelabs Technologies, Inc. (GSK plc)
• Original Assignee: Genelabs Technologies, Inc. (GSK plc)
• Inventors: Cantor, Charles R., Fry, Kirk E., Edwards, Cynthia A., Turin, Lisa M., Andrews, Beth M.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/171,389
• Time in Patent Office: 1,072 Days
• Field of Search: 435/6, 536/23.1, 536/23.2
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 1/705 for herpetoviridae, e.g. herpes simplex, varicella zoster
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: H01L 2924/14 Integrated circuits

Abstract

The present invention defines a DNA:protein-binding assay useful for screening libraries of synthetic or biological compounds for their ability to bind DNA test sequences. The assay is versatile in that any number of test sequences can be tested by placing the test sequence adjacent to a defined protein binding screening sequence. Binding of molecules to these test sequence changes the binding characteristics of the protein molecule to its cognate binding sequence. When such a molecule binds the test sequence the equilibrium of the DNA:protein complexes is disturbed, generating changes in the concentration of free DNA probe. Numerous exemplary target test sequences (SEQ ID NO:1 to SEQ ID NO:600) are set forth. The assay of the present invention is also useful to characterize the preferred binding sequences of any selected DNA-binding molecule.

Claims

1. A method for altering the binding characteristics of a DNA-binding protein to a duplex DNA, comprising

contacting the duplex DNA with a small molecule characterized by sequence-preferential binding to a target region where, when the small molecule is bound to the target region, the small molecule is adjacent to a binding site for the DNA-binding protein and not overlapping the binding site for the DNA-binding protein by more than four basepairs, at a concentration of small molecule effective to alter the binding of the DNA-binding protein to its binding site of the duplex DNA.

2. The method of claim 1, where contacting the duplex DNA with the small molecule inhibits the binding of the DNA-binding protein to its binding site.

3. The method of claim 1, where contacting the duplex DNA with the small molecule enhances the binding of the DNA-binding protein to its binding site.

4. The method of claim 1, where the DNA binding protein is a eucaryotic general transcription factor and the target region is selected from DNA sequences adjacent the binding site for the eucaryotic transcription factor.

5. The method of claim 4, where the transcription factor is TFIID.

6. The method of claim 5, where the target region is selected from the group of DNA sequences consisting of SEQ ID NO:

1 to SEQ ID NO;

600.

7. The method of claim 1, where the DNA binding protein is a eucaryotic general transcription factor and the small molecule binds, in addition to the target region, 1 to three nucleotide pairs of the DNA-binding protein'"'"'s binding site.

8. The method of claim 7, where the eucaryotic general transcription factor is TFIID, and the small molecule binds to (i) the target region, and (ii) up to two nucleotides of the binding site for the eucaryotic transcription factor, where the nucleotides are contiguous to the target region.

9. The method of claim 1, where the DNA binding protein is a DNA replication factor.

14. The method of claim 1, wherein said small molecule binds no more than 1-3 basepairs of said binding site for said DNA-binding protein.

15. The method of claim 1, wherein said small molecule binds only to said test sequence.

10. A method for inhibiting the binding of a DNA-binding protein to duplex DNA, comprising

contacting a compound with a duplex DNA which contains a test sequence adjacent to and not overlapping by more than four basepairs a screening sequence, where the DNA-binding protein binds to the screening sequence in the absence of the compound, where the compound binds to the test sequence without binding to more than four basepairs of said screening sequence, and further where the binding of said compound to the test sequence inhibits the binding of the DNA-binding protein to the screening sequence.

11. The method of claim 10, wherein the compound is identified by the steps of

preparing a series of duplex nucleic acid fragments, each containing a test sequence composed of one of the 4N possible permutations of sequences in a sequence of base pairs having N-basepairs, where said test sequence is adjacent the screening sequence,

measuring the binding affinity of the DNA binding protein to each of the series of nucleic acid fragments in the presence of the compound, and

selecting the compound if it lowers the binding affinity of the DNA binding protein for the screening sequence.

12. The method of claim 10, wherein said compound binds no more than 1-3 basepairs of said screening sequence.

13. The method of claim 10, wherein said compound binds only to said test sequence.