The binding mechanism of the molecular rotor Thioflavine-T to DNA

Thioflavin T, 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium cation (TFT), is a benzothiazolium fluorescent dye used for staining amyloid and para-amyloid tissues.Moreover, TFT is a molecular rotor, a class of molecules that have found a wide range of applications as fluorescent sensors of micro-viscosity and solvent free volume. Application examples include bulk viscosity measurement, probing dynamics of polymer formation, protein sensing and probing of protein aggregation, and micro-viscosity probing in living cells. Some data on the interaction of TFT with DNA have been reported, but the mechanistic details of the binding process are still unknown. We have undertaken a mechanistic analysis of TFT interaction with natural DNA. The results obtained all concur in indicating that the behaviour is not simple. Spectrophotometric and spectrofluorometric titrations yield different results that differently depend upon salt content and temperature. It can be inferred that two diverse binding modes are present, that, depending on the experimental conditions needed, differently contribute to the overall apparent constant measured by the two techniques. The thermodynamic parameters evaluated suggest that the two binding modes observed can be intercalation and groove binding. The kinetic analysis (T-jump technique) confirms such a model. Under conditions of polymer excess (DNA/TFT >> 1) two relaxation times are observed; under condition of dye excess (DNA/TFT < 1) biphasic relaxation curves are also found to be present, but the relevant time constant differ from those recorded for DNA/TFT >> 1. On the whole, the kinetic data, in agreement with equilibria, enable to propose the mechanistic reaction scheme shown below, where D2 is a Thioflavine-T dimer, PD is the intercalated complex and PD3 is an intercalated form containing also a groove-bound dimer and PD3* is its final, conformationally rearranged form.