Fluorescence intensity fluctuations of individual labeled DNA fragments and a DNA binding protein in solution at the single molecule level: A comparison of photobleaching, diffusion, and binding dynamics

Using an epi-illuminated confocal microscope coupled to a single photon counting avalanche photodiode, bursts of fluorescence from a: series of individual fluorophore-labeled DNA fragments were detected. The time-resolved fluorescence amplitudes were analyzed by computing the fluorescence intensity autocorrelation function. Examples of burst sizes limited by photodestruction of the dye, diffusion of the complex, and binding dynamics are presented and compared. Binding dynamics are studied using the intercalating dye thiazole orange and a conjugate of thiazole orange with a sequence-specific DNA binding peptide (a zinc finger). Unconjugated thiazole orange has a dynamic association with ds-DNA and was found to hop on and off the DNA on a time scale of a millisecond or less. The thiazole orange in the dye-protein conjugate, on the other hand, remains associated with the DNA for longer periods of time, due to the additional binding strength of the zinc finger. However, the conjugated thiazole orange is apparently conformationally hindered by its attachment to the peptide and is on the average bound to the dsDNA in a manner that is not as fluorescent as is unconjugated thiazole orange intercalated by itself.