The fluorescence enhancement mechanisms of a series of DNA stains of the oxazole yellow (YO) family have been investigated in detail using steady-state and ultrafast time-resolved fluorescence spectroscopy. The strong increase in the fluorescence quantum yield of these dyes upon DNA binding is shown to originate from the inhibition of two distinct processes: 1) isomerisation through large-amplitude motion that non-radiatively deactivates the excited state within a few picoseconds and 2) formation of weakly emitting H-dimers. As the H-dimers are not totally non-fluorescent, their formation is less efficient than isomerisation as a fluorescent contrast mechanism. The propensity of the dyes to form H-dimers and thus to reduce their fluorescence contrast upon DNA binding is shown to depend on several of their structural parameters, such as their monomeric (YO) or homodimeric (YOYO) nature, their substitution and their electric charge. Moreover, these parameters also have a substantial influence on the affinity of the dyes for DNA and on the ensuing sensitivity for DNA detection. The results give new insight into the development and optimisation of fluorescent DNA probes with the highest contrast.

The excited-state dynamics of the DNA bisintercalator YOYO-1 and of two derivatives has been investigated using ultrafast fluorescence up-conversion and time-correlated single photon counting. The free dyes in water exist in two forms: nonaggregated dyes and intramolecular H-type aggregates, the latter form being only very weakly fluorescent because of excitonic interaction. The excited-state dynamics of the nonaggregated dyes is dominated by a nonradiative decay with a time constant of the order of 5 ps associated with large amplitude motion around the monomethine bridge of the cyanine chromophores. The strong fluorescence enhancement observed upon binding of the dyes to DNA is due to both the inhibition of this nonradiative deactivation of the nonaggregated dyes and the dissociation of the aggregates and thus to the disruption of the excitonic interaction. However, the interaction between the two chromophoric moieties in DNA is sufficient to enable ultrafast hopping of the excitation energy as revealed by the decay of the fluorescence anisotropy. Finally, these dyes act as solvation probes since a dynamic fluorescence Stokes shift was observed both in bulk water and in DNA. Very similar time scales were found in bulk water and in DNA.