ASSOCIATED ANISOTROPY DECAY OF ETHIDIUM BROMIDE INTERACTING WITH DNA

Ethidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and lifetime. In this report we studied time-resolved fluorescence properties of EB fluorophore existing simultaneously in free and bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6ns) and bound (22ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. This is because of the two existing fractions contributing to a total anisotropy change in time. The short-lived fraction associated with the free EB molecules decreases faster the than the long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecular dynamics studies. Purpose (a): To study the interaction of DNA with ethidium bromide. Methods (b): 1) Steady state fluorescence intensity measurement. 2) Time resolved measurements. 3) Steady state anisotropy. Results (c): Increase in fluorescence lifetime and fluorescence anisotropy when ethidium bromide interact with DNA. Conclusions (d): We believe that assays involving EB and DNA should be analyzed with the associated decay model. Neglecting this type of decay pattern can lead to false interpretation of results.