Quantitative footprinting analysis

This review outlines the steps for obtaining relative binding constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA site. Slopes of plots outside of drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netropsin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netropsin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.