Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions

Journal article, 1997

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 presented. The simulations are based on a Markov chain analysis, assuming random distribution of the dyes along the DNA chain and energy transfer that obeys Forster kinetics. Since the investigated intercalators (ethidium bromide, YO, PO) and groove binders [4'6-diamidino-2-phenylindole (DAPI)] were found to show different depolarization dependence on binding density, the method can be used to quite sensitively characterize the binding mode. Excellent agreement between the measured and simulated anisotropy is found for all investigated intercalators. The proposed method gives an estimation of the unwinding angle for intercalators and provides information about the binding site size, and the presence or absence of sequence specificity. For the groove binder DAPI interacting with mixed sequenced DNA, the measured and computed depolarization do not agree, and this can be rationalized in terms of the high sequence specificity of this dye. However, for DAPI bound to [poly(dA-dT)](2) the measured data agree well with computed data for a groove binder that is displaced a distance 7 Angstrom from the helix axis and has a binding site size of three bases. (C) 1997 John Wiley & Sons, Inc.