Methods for the Studies of Drug Dissociation from DNA

In addition to binding affinity, the on and off rates of drug-DNA interactions are important in determining the biological activities of a drug For example, the rate of dissociation of a drug from DNA has been shown to be related to its pharmacological activities (1 ). Various techniques have been employed to study the dissociation kinetics of drugs from DNA. These include detergent sequestration technique pioneered by Muller and Crothers (1 ), a modification of the footprinting technique for examming the dissociation of ligands from individual binding sites (2 ), relaxation methods such as T-Jump for measuring fast kinetics (3 ), and a procedure that can yield drug—DNA dissociation kinetics under conditions of active transcription of the DNA (4 ). The simplest and most widely used method IS detergent-induced dissociation rate measurement incorporating the detergent sodium dodecyl sulfate (SDS). This chapter will thus focus on the SDS-sequestration technique and include only conventional spectropliotometric and stopped-flow methods. An example of an actinomycin D dissociation measurement from the author’s laboratory will be used to illustrate the methodology