Comparison of different footprinting methodologies for detecting binding sites for a small ligand on DNA

In order to assess the utility of different methods of footprinting applied to the study of sequence-selective small molecule-DNA interaction we have performed a homologous series of experiments on the binding of echinomycin, a bis-intercalator, to a 133 base pair DNA restriction fragment containing a small number of discrete binding sites. Two of those sites each contained a pair of closely clustered CpG steps, the cognate dinucleotide sequence which is the common denominator of sites recognised by echinomycin. DNAse I was found to be much the best enzyme for footprinting in terms of sensitivity, accuracy, and ease of handling. DNAase II and micrococcal nuclease were of limited value. Excellent results were recorded with methidiumpropyl-EDTA.FeII which picked up strong binding sites and yielded sharp footprints from which a parsimonious estimate of site size could be determined. Orthophenanthroline.CuI proved to be a very suitable, sensitive chemical nuclease but hydroxyl radical footprinting with EDTA.FeII was only partially successful. Positive footprinting with conformation-sensitive probes diethylpyrocarbonate, osmium tetroxide and potassium permanganate yielded information to complement that afforded by the enzymic and chemical nucleases. Evidence of binding to both CpG steps in the clustered pair was obtained, with indications of possible cooperativity.