FLUORESCENCE LIFETIME ANALYSIS OF DNA INTERCALATED ETHIDIUM-BROMIDE AND QUENCHING BY FREE DYE

The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analys is for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessar y for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess Eb a binding e quilibrium is reached, and this corresponds to one Eb molecule for eve ry five nucleotides. With increasing amounts of unbound Eb, the fluore scence lifetime of the DNA-Eb complex decreases with a concomitant dro p in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This m eans that a different approach is necessary: time-resolved fluorescenc e spectroscopy directly distinguishes between a decrease in fluorescen ce intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies sug gest that techniques which measure total steady state fluorescence int ensity of bound Eb in order to infer relative amounts of double-strand ed DNA must be interpreted with caution. For such assays to be valid i t is essential that no unbound Eb be present; otherwise a variable cor rection factor is required to account for unbound Eb.