EXPERIMENTAL AND SIMULATED FLUORESCENCE DEPOLARIZATION DUE TO ENERGY-TRANSFER AS TOOLS TO STUDY DNA-DYE INTERACTIONS

A method to study DNA-dye complexes by the combination of steady state fluorescence anisotropy measurements and computer simulations of the fluorescence depolarization due to resonance energy transfer is presen ted. The simulations are based on a Markov chain analysis, assuming ra ndom distribution of the dyes along the DNA chain and energy transfer that obeys Forster kinetics. Since the investigated intercalators (eth idium bromide, YO, PO) and groove binders [4'6-diamidino-2-phenylindol e (DAPI)] were found to show different depolarization dependence on bi nding density, the method can be used to quite sensitively characteriz e the binding mode. Excellent agreement between the measured and simul ated anisotropy is found for all investigated intercalators. The propo sed method gives an estimation of the unwinding angle for intercalator s and provides information about the binding site size, and the presen ce or absence of sequence specificity. For the groove binder DAPI inte racting with mixed sequenced DNA, the measured and computed depolariza tion do not agree, and this can be rationalized in terms of the high s equence specificity of this dye. However, for DAPI bound to [poly(dA-d T)](2) the measured data agree well with computed data for a groove bi nder that is displaced a distance 7 Angstrom from the helix axis and h as a binding site size of three bases. (C) 1997 John Wiley & Sons, Inc .