DNA photofootprinting with Rh(phi)_2bpy^(3+)

Abstract

This chapter describes one method for high resolution photofootprinting of proteins or small drugs bound to DNA (1). DNA footprinting is an electrophoretic method that permits the visualization (footprint) of the binding site of a molecule site-specifically bound to DNA. The method entails the nonspecific cleavage of a radioactively end-labeled DNA containing the site-specifically bound molecule. Protection from this sequence neutral cleavage at the binding site reveals the footprint. Photofootprinting differs from other conventional footprinting techniques in that DNA cleavage occurs directly on irradiation of a photocleaving reagent, which binds to the target DNA sequence. Alternative photofootprinting methods produce DNA cleavage indirectly by creating photo-damaged DNA adducts that are labile to base treatment after irradiation of the DNA (2,3). Traditional footprinting procedures utilize either a protein like DNase I for cleavage or small molecule reagents that cleave DNA under specific chemical conditions (4–8).

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