Fluorescence self-quenching of ethidium bromide intercalated in DNA

摘要

Fluorescence lifetimes of free and DNA-intercalated ethidium bromide have been established in different studies to be 1.6 - 1.8 and 20 - 25 ns, respectively. Recently, several researchers have suggested a new DNA-binding mode of ethidium based on a third fluorescence lifetime between 10 and 15 ns. We are able to reproduce the latter lifetime in ethidium:DNA complexes only when the ligand per DNA base-pair ratio (r) exceeds 0.2. Other criteria that may affect the binding properties of ethidium, such as temperature, salt concentration, length of the DNA (end effects), and base composition of the DNA only influence the appearance of this new lifetime because of their effect on the spatial distribution of intercalated ethidium. Based on single curve and global analysis of fluorescence intensity and fluorescence anisotropy decay data, we propose that the new lifetime is due to self-quenching of intercalated ethidium. Detectable quenching occurs at average intercalation site separations of less than 10 base-pairs.