Fluorescence and Photobleaching Studies of Methylene Blue Binding to DNA

Complexes of methylene blue with DNA are characterized by time- resolved fluorescence spectroscopy and transient photobleaching methods. At least four, and probably five, spectroscopically distinct binding sites have been identified. Three of these (components 1, 2, and 3B) dominate the fluorescence decay at low ionic strength and have fluorescence lifetimes significantly different from that of the free dye. With increasing ionic strength a fourth component (3A) appears at the expense of components 1 and 3B. Component 3A exhibits two subcomponents with different degrees of shielding from O2 quenching of its triplet state. The relative amplitudes of the components at low ionic strength are strongly dependent on the composition of the DNA, and independent of superhelix density. Hence, it is inferred that components 1, 2, and 3B represent binding to different base pair steps, and that all of these components represent intercalation sites that unwind the DNA to the same degree. Component 3A is apparently not intercalated. From plots of the relative photobleach amplitudes versus the relative fluorescence intensities, we infer that the triplet yield and photobleach amplitude are dominated by components 3A and/or 3B under nearly all conditions. Our results are used to discuss the suitability of methylene blue as the extrinsic probe in transient photodichroism experiments.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.