Footprinting: A method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands

Footprinting is a simple method for assessing the sequence selectivity of DNA-binding ligands. The method is based on the ability of the ligand to protect DNA from cleavage at its binding site. This review describes the use of DNase I and hydroxyl radicals, the most commonly used footprinting probes, in footprinting experiments. The success of a footprinting experiment depends on using an appropriate DNA substrate and we describe how these can best be chosen or designed. Although footprinting was originally developed for assessing a ligand’s sequence selectivity, it can also be employed to estimate the binding strength (quantitative footprinting) and to assess the association and dissociation rate constants for slow binding reactions.