C. Preston Linn

Spectroscopic studies of YO and YOYO fluorescent dyes in a thrombin-binding DNA ligand

The fluorescent, oxazole yellow dye YO-Pro-1 iodide (YO) and its homodimer YOYO-1 iodide (YOYO) were studied in a thrombin-binding DNA ligand, or aptamer, (tb-ligand) and in an oligomer with the same base composition in a scrambled sequence (s-ligand), both single strands of 15 bases in length. Binding constants for the dye-ligand complexes, assuming 1:1 stoichiometry, were determined to be on the order of 107M−1 for YOYO and 105M−1 for YO, which are approximately 105 smaller than estimated constants for YOYO in double-stranded DNA. In both ligands, YOYO assumes a folded conformation that promotes stability of the dye-ligand complex and excitonic coupling between the two YO groups. The folded conformation provides greater overlap of the YO groups than has been reported for YOYO in double-stranded DNA; the overlap is slightly greater in tb-ligand than in s-ligand. Both dyes exhibit bi-exponential fluorescence decay in the ligands and the lifetimes of YOYO (3–4 ns and 7–8 ns) are longer and more discrete than those of YO (1–3 ns and 4–5 ns). Fluorescence anisotropy of YOYO is a low, constant value in both ligands due to intramolecular energy transfer between the overlapping YO groups. Higher anisotropies are observed for YO, and the value is slightly higher in s-ligand than in tb-ligand. The addition of thrombin to the s-ligand affects the fluorescence intensity and anisotropy of YO but not of YOYO. The absence of intermolecular G-quartet formation of the s-ligand was demonstrated. This suggests that thrombin interacts weakly with the s-ligand but is not sensed by the fluorescence of YOYO, which is dominated by the coupling between the YO groups in the folded conformation of the bound dye. The results of these studies have implications for the application of these dyes for detection of single-stranded DNA ligands and their binding interactions.