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 blu e/DNA complexes over a range of NaCl and MgCl2 concentrations and a li mited range of temperature and base composition. At least four, and pr obably five, different binding sites have been identified. Component 1 has the shortest fluorescence lifetime (26 ps) and represents the mai n intercalated component under low-salt conditions. Component 2 has an intermediate fluorescence lifetime (130 ps). Component 3B has the lon gest fluorescence lifetime (620 ps), undergoes a modest amplitude (12 degrees) of local rotation, and is significantly shielded from O-2 que nching 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 intercalat ed. With increasing NaCl or MgCl2 concentration, the populations of co mponents 1 and 3B shift into 3A. With increasing temperature, the ampl itude of the longest component, ostensibly 3B, increases somewhat at t he expense of component 1, and its lifetime and residual anisotropy de crease slightly. The relative amplitudes of components 1, 2, and 3B ar e unaffected by supercoiling, which implies that they all arise from i ntercalation sites with similar unwinding angles. From a comparison of the relative photobleach amplitudes with the relative fluorescence in tensities, it can be inferred that components 3A and/or 3B dominate th e triplet yield and photobleaching amplitude under practically all con ditions. The suitability of methylene blue as the extrinsic probe in t ransient photodichroism experiments is discussed in light of these res ults.