Criteria for the mode of binding of DNA binding agents

A complete characterization of DNA binding agents requires that their mode of binding to DNA be established. In the absence of high resolution structural data, the mode of binding is, of necessity, usually inferred indirectly from various solution studies. The purpose of this study is to show that only certain methods can be used reliably to infer the DNA binding mode. Comparative fluorescence and hydrodynamic studies using the proven intercalator ethidium and the groove binder Hoechst 33258 are described. The results of our studies show that while fluorescence intensity, polarization, and quenching measurements can detect a binding interaction of the ligand with DNA, none are sensitive indicators of the binding mode. Fluorescence contact energy transfer studies can reliably indicate intercalation, as can viscosity measurements. Our results illustrate reliable criteria that may be used to distinguish intercalation from groove binding in the absence of high resolution structural data.