High-resolution footprinting studies of drug-DNA complexes using chemical and enzymatic probes

Abstract

This chapter discusses high-resolution footprinting techniques of drug–DNA complexes using chemical and enzymatic probes. Footprinting is a method that has been developed for determining the sequence-specific binding of small molecules, oligonucleotides, or proteins to DNA. The technique, which was originally designed for analyzing protein–DNA interactions, is based on the ability of ligands to protect DNA from enzymatic or chemical cleavage at their binding sites. Several enzymatic and chemical agents have been developed as footprinting probes, and each has its own characteristic advantages and disadvantages. Enzymes, such as DNase I, are often easy to use but typically overestimate the size of the footprint (on account of their size) and generate uneven cleavage patterns in the absence of added ligand. Chemical agents, such as dimethyl sulfate, osmium tetroxide, or diethyl pyrocarbonate (DEPC), are of limited usefulness because they react only with specific DNA bases. Other chemical probes such as methidiumpropyl–EDTA–Fe(II) [MPE–Fe(II)] act by intercalation and so perturb the DNA structure. Arguably the best cleavage agent for accurately mapping small molecule binding sites on DNA is the hydroxyl radical.