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

The binding of ethidium bromide to a DNA hairpin (dU₅-hairpin) was investigated using a novel 2D diffusion-modulated gradient correlation spectroscopy (DMG-COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self-diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for uridine 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 experiment. The diffusion coefficient for uridine calculated from the DMG-COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24x10^-6 cm²/s at 26°C). The diffusion coefficients for ethidium bromide and for the dU₅-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15x10^-6 cm²/s at 26°C) was considerably larger than for the dU₅-hairpin (1.60x10^-6 cm²/s at 26°C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU₅-hairpin, consistent with complex formation (1.22x10^-6 cm²/s at 26°C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU₅-hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution. Copyright (C) 2000 Federation of European Biochemical Societies.