Binding of ethidium to DNA measured using a 2D diffusion-modulated gradient COSYNMR experiment

The binding of ethidium bromide to a DIVA hairpin (dU(5)-hairpin) was inves tigated using a novel 2D diffusion-modulated gradient correlation spectrosc opy (DMG-COSY) experiment to evaluate the applicability of this technique f or studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due t o molecular self-diffusion. Magnetization then evolves due to scalar coupli ng during an evolution delay, and is detected using gradient pulses for coh erence selection, The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for u ridine in DMSO solution was determined from the H5-H6 crosspeak intensities for a series of 2D DMG-COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG-COSY e xperiment. The diffusion coefficient for uridine calculated from the DMG-CO SY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24 x 10(-6) cm(2)/s at 26 degrees C). The diffusion coefficients for ethidium bromide and for the dU(5)-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromi de (4.15 x 10(-6) cm(2)/s at 26 degrees C) was considerably larger than for the dU(5)-hairpin (1.60 x 10(-6) cm(2)/s at 26 degrees C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU(5)-hairpin. consistent with complex forma tion (1.22 x 10(-6) cm(2)/s at 26 degrees C). Comples formation of 1:1 stoi chiometry between ethidium and the stem of the dU(5)-hairpin was verified i ndependently by fluorescence spectroscopy. These results demonstrate the ut ility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution, (C) 2000 Federation of European Biochemical Societ ies.