Investigation of DNA dynamics and drug-DNA interaction by steady state fluorescence anisotropy

We have used steady-state fluorescence polarization anisotropy (FPA) of ethidium probe molecules bound to DNA to investigate DNA-DNA interactions and the effect of high densities of intercalating drugs on the internal motions of DNA responsible for depolarization of the ethidium fluorescence. To calibrate the method, we examined the effect of DNA length on (FPA) using DNA varying in size from 10-150 base pair. The association of approximately 30 base pair DNA at high concentrations was then detected by its effect on (FPA). With sample concentrations approaching those commonly used in various physical experiments (NMR, Raman) significant DNA-DNA interactions are observed. With high molecular weight DNA (greater than 500 base pair), the limiting value of the (FPA) (0.23) is due to internal motions of the DNA (and bound chromophores). The (FPA) of ethidium probe molecules (1 drug/200 base pair) is unaffected by the addition of high levels (1 drug/2 base pair) proflavine. This indicates that either the elastic properties of DNA are unaffected by high densities of intercalated drug or that the depolarization of the ethidium fluorescence is due to highly localized motions of the base pairs that are unperturbed by binding of drugs at neighboring sites.