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Previously we showed that the fluorescence lifetimes of ethidium and propidium iodide were different when intercalated into cellular double-stranded DNA or RNA. Current studies of four ethidium derivatives showed that the lifetime difference of DNA and RNA bound dyes existed in all four compounds, and that the magnitude of the difference was dependent on the dye structure, the staining concentration, and the conditions in which flow cytometry (FCM) analysis of stained cells was performed. The fluorescence lifetime of both DNA and RNA bound fluorochromes was reduced with increasing dye concentration; however the level of decrease varied in different dyes. Further analysis of stained cellular DNA in dye-free solution, which favors only high affinity binding, led to elevated fluorescence lifetime values compared to lifetime values obtained from analysis in the dye solution under equilibrium binding conditions. The changes of fluorescence lifetime value reflect the difference in dye structure and distinct interaction between the dyes and nucleic acids. The staining and analysis variables used in these studies may potentially provide additional information on differences in nucleic acid conformation and function in cell populations.
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