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Multiparameter single-molecule fluorescence measurements of DNA intercalating fluorophores

Experiments using single-molecules of TOTO-1 intercalated into dsDNA were performed to investigate the DNA sequence dependence on the fluorescence detectable with single-molecule fluorescence spectroscopy. Previous work has shown that there is a difference in the fluorescence lifetime when TOTO-1 is intercalated in poly-AT DNA or in poly-GC DNA. The fluorescence detected from single-molecules in this work for poly-GC and poly-AT DNA showed fluorescence lifetimes of 2.1 and 1.8 nsec, respectively. Analysis of the fluorescence intensity detected from single-molecules of TOTO-1 was performed by fluorescence cross-correlation spectroscopy. TOTO-1 is shown to spend large amounts of time in dark states. These dark states reduce the detectable fluorescence intensity to approximately 10 photons per millisecond on average.