LOCAL DYNAMICS OF DNA PROBED WITH OPTICAL-ABSORPTION SPECTROSCOPY OF BOUND ETHIDIUM-BROMIDE

We have studied the local dynamics of calf thymus double-helical DNA b y means of an ''optical labeling'' technique. The study has been perfo rmed by measuring the visible absorption band of the cationic dye ethi dium bromide, both free in solution and bound to DNA, in the temperatu re interval 360-30 K and in two different solvent conditions. The temp erature dependence of the absorption line shape has been analyzed with in the framework of the vibronic coupling theory, to extract informati on on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the pro ximity of the chromophore. For the dye free in solution, large spectra l heterogeneity and coupling to a ''bath'' of low-frequency (soft) mod es is observed; moreover, anharmonic motions become evident at suitabl y high temperatures. The average frequency of the soft modes and the a mplitude of anharmonic motions depend upon solvent composition. For th e DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motio ns are hindered. However, a new dynamic regime characterized by a larg e increase in anharmonic motions is observed at temperatures higher th an similar to 280 K. The DNA double helix therefore appears to provide , at low temperatures, a rather rigid environment for the bound chromo phore, in which conformational heterogeneity is reduced and low-freque ncy motions (both harmonic vibrations and anharmonic contributions) ar e hindered. The system becomes anharmonic at similar to 180 K; however , above similar to 280 K, anharmonicity starts to increase much more r apidly than for the dye free in solution; this can be attributed to th e onset of wobbling of the dye in its intercalation site, which is lik ely connected with the onset of (functionally relevant) DNA motions, i nvolving local opening/unwinding of the double helix. As shown by para llel measurements of the melting curves, these. motions precede the me lting of the double helix and depend upon solvent composition much mor e than does the melting itself.