Screening assay for the detection of DNA-binding molecules
First Claim
1. A method of screening for molecules capable of binding to a selected test sequence in a duplex DNA, comprising(i) adding a molecule to be screened to a test system composed of (a) a DNA binding protein that is effective to bind to a screening sequence in a duplex DNA with a binding affinity that is substantially independent of such test sequence adjacent the screening sequence, but that is sensitive to binding of molecules to such test sequence, when the test sequence is adjacent the screening sequence, and (b) a duplex DNA having said screening and test sequences adjacent one another, where the binding protein is present in an amount that saturates the screening sequence in the duplex DNA,(ii) incubating the molecule in the test system for a period sufficient to permit binding of the molecule being tested to the test sequence in the duplex DNA, and(iii) comparing the amount of binding protein bound to the duplex DNA before and after said adding.
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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. Also described herein is a method to capture DNA that has been released from the DNA:protein complex.
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17 Claims
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1. A method of screening for molecules capable of binding to a selected test sequence in a duplex DNA, comprising
(i) adding a molecule to be screened to a test system composed of (a) a DNA binding protein that is effective to bind to a screening sequence in a duplex DNA with a binding affinity that is substantially independent of such test sequence adjacent the screening sequence, but that is sensitive to binding of molecules to such test sequence, when the test sequence is adjacent the screening sequence, and (b) a duplex DNA having said screening and test sequences adjacent one another, where the binding protein is present in an amount that saturates the screening sequence in the duplex DNA, (ii) incubating the molecule in the test system for a period sufficient to permit binding of the molecule being tested to the test sequence in the duplex DNA, and (iii) comparing the amount of binding protein bound to the duplex DNA before and after said adding.
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8. A screening system for identifying molecules that are capable of binding to a test sequence in a target duplex DNA sequence, comprising
a duplex DNA having screening and test sequences adjacent one another, a DNA binding protein that is effective to bind to said screening sequence in the duplex DNA with a binding affinity that is substantially independent of a test sequence adjacent the screening sequence, but which is sensitive to binding of molecules to such test sequence, when the test sequence is adjacent the screening sequence, and where the binding protein is present in an amount that saturates the screening sequence in the duplex DNA, and means for detecting the amount of binding protein bound to the DNA.
Specification