4 ',6-Diamidino-2-phenylindole (DAPI) interacts with rare structures of GCpolymers

The binding of 4 ' ,6-diamidino-2-phenylindole (DAPI) to double-stranded GC polymers either in the alternating or in homopolymer sequence was investig ated using fluorescence techniques. We employed fluctuation correlation spe ctroscopy, which measures the diffusion coefficient of fluorescent particle s, to demonstrate that the fluorescence was originating from relatively slo wly diffusing entities. These entities display a very large heterogeneity o f diffusing coefficients, indicating that molecular aggregation is extensiv e in our samples. We used frequency domain fluorometry to characterize the fluorescence lifetime of the species, while varying the GC polymer-dye cove rage systematically. At very low coverage we observed a relatively bright f luorescent component with a lifetime value of approximately 4 ns. The stoic hiometry of binding of this bright species was such that it can only arise from rare molecular structures, either unusual loops or large molecular agg regates. The amount and characteristics of this bright fluorescent componen t were different between the home and the alternating polymer, indicating t hat the difference in sequence of the two polymers is responsible for the d ifferent aggregates which are then detected in the fluorescence experiment. At large GC polymer coverage we observed a relatively wide distribution of fluorescent species with short lifetime values, in the range between 0.12 and 0.2ns. Given the stoichiometry of binding of this fluorescent component , we concluded that it could arise either from intercalative and/or non-spe cific binding to the DNA double-stranded molecules. We comment on the origi n of the rare but brightly fluorescent binding sites and discuss the potent ial to detect such unusual DNA structures.