FLUORESCENCE AND PHOTOBLEACHING STUDIES OF METHYLENE-BLUE BINDING TO DNA

Time-resolved fluorescence, fluorescence polarization anisotropy, and transient photobleaching methods are used to investigate methylene blue/DNA complexes over a range of NaCl and MgCl2 concentrations and a limited range of temperature and base composition. At least four, and probably five, different binding sites have been identified. Component 1 has the shortest fluorescence lifetime (26 ps) and represents the main intercalated component under low-salt conditions. Component 2 has an intermediate fluorescence lifetime (130 ps). Component 3B has the longest fluorescence lifetime (620 ps), undergoes a modest amplitude (12 degrees) of local rotation, and is significantly shielded from O-2 quenching of its triplet state. Its amplitude is enhanced by increasing %AT, and it appears to require AA, AT, or TA steps. Component 3A has a long fluorescence lifetime (430 ps), undergoes a very large amplitude of local rotation, and exhibits two subcomponents that are differently shielded from O-2 quenching. Component 3A is evidently not intercalated. With increasing NaCl or MgCl2 concentration, the populations of components 1 and 3B shift into 3A. With increasing temperature, the amplitude of the longest component, ostensibly 3B, increases somewhat at the expense of component 1, and its lifetime and residual anisotropy decrease slightly. The relative amplitudes of components 1, 2, and 3B are unaffected by supercoiling, which implies that they all arise from intercalation sites with similar unwinding angles. From a comparison of the relative photobleach amplitudes with the relative fluorescence intensities, it can be inferred that components 3A and/or 3B dominate the triplet yield and photobleaching amplitude under practically all conditions. The suitability of methylene blue as the extrinsic probe in transient photodichroism experiments is discussed in light of these results.