ACRYLAMIDE AND MOLECULAR OXYGEN FLUORESCENCE QUENCHING AS A PROBE OF SOLVENT‐ACCESSIBILITY OF AROMATIC FLUOROPHORES COMPLEXED WITH DNA IN RELATION TO THEIR CONFORMATIONS: CORONENE‐DNA AND OTHER COMPLEXES

Abstract— The relationships between the accessibilities to fluorescence quenchers (O2 and acrylamide) of three different aromatic molecules and their modes of binding to double‐stranded DNA were investigated. Proflavin was selected for this study because it is a known intercalator, while Hoechst 33258 is known to bind externally in the minor groove of DNA; it is shown here that the relatively bulky polycyclic aromatic hydrocarbon coronene forms partial intercalation complexes, and thus represents a third class of fluorophore‐DNA binding. Fluorescence quenching results suggest that Hoechst 33258 bound to DNA is fully accessible to 02, that the accessibility of partially intercalated coronene is reduced to 20% of the free‐solution value, while the accessibility of intercalated proflavin to molecular oxygen is at least ten times smaller than for free polycyclic aromatic molecules in fluid solutions. In contrast, the fluorescence of all these three fluorophores bound to DNA is insensitive to acrylamide (the effective accessibilities appear to be reduced by factors greater than 50 upon binding to DNA). It is concluded that O, is useful as a qualitative probe of the type of binding and solvent exposure of fluorophore‐DNA complexes, while acrylamide appears to be of limited utility. However, acrylamide fluorescence quenching may be useful for studies of other types of fluorophore‐DNA complexes since the fluorescence of the pyrene residues in covalent adducts derived from the binding of benzo(a)pyrene diol expoxide to DNA was found to be at least partially sensitive to acrylamide.