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A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiograms produced after electrophoretic separation of digestion products allows determination of sequences that are affected by drug binding. The feasibility of the technique for locating small ligands bound to DNA and its eventual use as a quantitative thermodynamic approach to studying ligand binding to heterogeneous DNA as a function of sequence is illustrated by using actinomycin D and Netropsin.

Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.