Selective binding and local photophysics of the fluorescent cyanine dye PicoGreen in double-stranded and single-stranded DNA

Abstract

The selectivity of binding of the unsymmetric monomethine cyanine dye PicoGreen (PG) to double-stranded (ds) and single-stranded (ss) DNA has been investigated by fluorescence and by circular dichroism spectroscopy, and the photophysical properties of individual binding modes have been determined for the first time. We demonstrate that binding of PG to dsDNA preferentially occurs by intercalation between alternating GC base pairs. Intercalation is also the most important association mode for other base-pair configurations, but in many cases, binding to the exterior of DNA efficiently competes with intercalation. Intercalated PG molecules are characterized by a monoexponential fluorescence decay rate constant of (0.23 ± 0.02) ns−1, which is independent of the base pairs surrounding the dye. Dye molecules in other configurations exhibit fluorescence decay rate constants of about (0.5–0.8) ns−1, and are observed at dye∶base pair ratios larger than 0.30 in calf thymus DNA. Fluorescence yields in ssDNA homopolymers are 0.18, 0.04, 0.008 and 0.003 in poly(dG), poly(dT), poly(dC), and poly(dA), respectively, but 0.30 in random-sequence ssDNA. These differences are explained by dimeric binding to poly(dG) and poly(dT), and comparatively loose association with poly(dA) and poly(dC). Fluorescence decay is multi-exponential in all types of ssDNA, none of its components depend on base redox properties or on stacking interactions. The dye binds as a monomer to heat-denatured ss calf-thymus DNA, and the dominant mode is intercalation between two different bases, one of them being G or T.