Comparative studies of the binding of ethidium bromide and its photoreactive analogues to nucleic acids by fluorescence and rapid kinetics

The binding studies of the interaction of ethidium bromide with DNA have been hampered by its reversibility, which prevents direct isolation and thus characterization of the complex. The recent development of photoaffinity labeling has provided a means to circumvent this problem. However, to be useful as a probe for the parent compound, a photosensitive analogue must be shown to interact in vivo and in vitro just as the parent analogue. These studies demonstrate by steady-state and nonosecond fluorescence and stopped-flow kinetics that one of the azido analogues of ethidium, 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride, binds nucleic acids quite similarly to ethidium bromide. The interaction of the other azide, 3,8-diazido-5-ethyl-6-phenyl-phenanthridinium chloride, with DNA is qualitatively different from that of the monoazide and ethidium bromide. These results suggest that the monoazide would serve as an ideal probe for determining the actual target sites of ethidium bromide in vivo and in vitro.