Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes

Abstract

This chapter describes three complementary footprinting methods and the protocols used for analysis of polyamide : DNA complexes: (1) MPE–Fe(II) footprinting, (2) affinity cleavage, and (3) quantitative DNase I footprint titration. Footprinting with methidiumpropyl–EDTA–Fe(II) [MPE–Fe(II)] is used to identify high-affinity polyamide-binding sites to near nucleotide resolution. Affinity cleavage is used to determine the orientation of the bound polyamide in the minor groove of DNA. Quantitative DNase I footprinting is used to determine equilibrium association constants (K_a) for polyamide–DNA complexes at previously identified match and mismatch sites. A pivotal step in the discovery—evaluation process of new polyamide motifs is the characterization of the affinity and specificity of next-generation molecules following the design-sythesis phase. In the design phase, it is often the case that the sequence preference, as well as the energetics of any new molecule binding at each potential site, is not perfectly understood and it is crucial to scan “libraries” of many potential DNA-binding sites in order to identify true high-affinity binding sites.