Ultrafast dynamics of 1-ethynylpyrene-modified RNA: a photophysical probe of intercalation

The photophysics of pyrene attached to an adenine base within RNA single strands and duplexes is examined with respect to the position of the pyrene within the strand and the number of pyrenes attached to one duplex. Compounds with pyrenes intercalating sequence specifically are examined, as well as a doubly modified compound, where the two pyrenes are located close enough to each other for significant excimer interaction. Femtosecond transient absorption measurements and time correlated single photon counting measurements allow a thorough examination of the local influences on the pyrene photophysics. Our results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties and lifetimes that are coupled only via the excited state as a gateway. One of them is a neutral pyrene-adenine excited state, S*, while the second one is connected to an excited charge transfer state, S*(CT). In all compounds, an ultrafast sub-ps decay from a higher excited state into the lowest excited state S* occurs, and an excited charge transfer species S*(CT) is formed within picoseconds. The fluorescence behavior of the pyrene-modified adenine, however, is strongly dependent on RNA conformation. Both S* and S*(CT) states are fluorescent, and decay within hundreds of picoseconds and approximately 2 ns, respectively. The ratio between S* and S*(CT) fluorescence depends strongly on pyrene intercalation, and it is found that the S* state is quenched selectively upon intercalation of the pyrene into RNA. The doubly modified duplex exhibits an additional fluorescent state with a lifetime of 18.7 ns, which is associated with the pyrene excimer state. This state coexists with a significant population of the pyrene monomer, since the characteristic features of the latter can still be observed. Formation of the excimer occurs on femtosecond time scales. The pyrene label thus provides a sensitive tool to monitor the local structural dynamics of RNA with the chromophore acting as a molecular beacon.