Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques.

Publisher Summary The dye-DNA structures determined both by X-ray crystallography and solution nuclear magnetic resonance (NMR) show the molecular details of the complex formed and make the p-OH Hoechst–DNA interaction an excellent model system with which to study strong and sequence-specific binding in the minor groove of DNA. To gain insight into the binding mechanism the structural information has been complemented with the kinetics of complex formation and dissociation. This chapter presents a working example of a kinetic analysis involving a ligand that binds strongly to specific sequences of DNA. The Hoechst dye–DNA interaction is an informative model system for the study of the highly A/T-specific minor groove binding to DNA. The details of a two-exponential analysis are discussed in order to demonstrate the complexities that can be expected when dye molecules interact strongly with specific DNA sequences. Unless the data are accurate the stopped-flow curves are well described by single exponentials and the kinetic analysis suggests a simple, single-step binding mechanism. The association rate is almost diffusion controlled and the specificity of the dye comes solely from the reverse rate parameter, possibly determined by the number of dye–DNA interactions in the minor groove that must be broken for the dye molecule to escape.