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

The binding of ethidium bromide to a DNA hairpin (dU 5 ‐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 (5.24×10 −6 cm 2 /s at 26°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 bromide (4.15×10 −6 cm 2 /s at 26°C) was considerably larger than for the dU 5 ‐hairpin (1.60×10 −6 cm 2 /s at 26°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 formation (1.22×10 −6 cm 2 /s at 26°C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU 5 ‐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.