Ultrafast Dynamics of 1-Ethynylpyrene-Modified RNA: A Photophysical Probe of Intercalation

Abstract

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.

The Three Possible 2‐(Pyrenylethynyl) Adenosines: Rotameric Energy Barriers Govern the Photodynamics of These Structural Isomers

“…Substitution of pyrene at a single position is possible—for symmetry reasons, only the positions 1, 2 and 4 are distinct—and has frequently been utilized to generate various fluorescent nucleoside analogues for nucleic acids research . In general, both, the position of the substitution and their nature were found to affect the electronic and spectral properties of pyrene derivatives .…”

Photo-physical properties of 2-(1-ethynylpyrene)-adenosine: influence of hydrogen bonding on excited state properties

“…The photo-physics of a protected nucleoside form 60 as well as the applicability and the characteristics of PyA in modified 15-mer oligonucleotides 61 as a RNA folding probe were investigated. 62 Based on spectroscopic characteristics of CT states 12,63 and findings of other groups, which observed ICT states in similar systems 24,64 the proposed model of the photophysics involved an intramolecular charge transfer process (ICT).…”

Photophysical processes of the spectroscopic RNA probe 2-(1-ethynylpyrene)-adenosine (PyA)

“…PyA showed the existence of a locally excited (LE) state with sequence dependent lifetimes around 400 ps and presumably a relaxed S1 state with a lifetime around 2 ns. These states differ only slightly in their energy and interconvert on a timescale of 3 ps [6]. The above mentioned excimer shows an additional lifetime of approximately 19 ns and a spectral red-shift of 50 nm.…”

“…Another favorable characteristic is the ability to form excimers upon close proximity of two PyAs of complementary strands in the minor groove [4]. Experiments revealed that PyA is also able to intercalate into RNA depending on the flanking bases in 5' direction of the modified base [5,6]. A G-C base pair leads to the intercalation of PyA, whereas A-U base pairs do not show this behavior.…”

“…A G-C base pair leads to the intercalation of PyA, whereas A-U base pairs do not show this behavior. To get a further understanding of the processes, the photophysical properties of PyA were investigated with femtosecond pump-probe absorption spectroscopy and time correlated single photon counting (TCSPC) [6,7]. PyA showed the existence of a locally excited (LE) state with sequence dependent lifetimes around 400 ps and presumably a relaxed S1 state with a lifetime around 2 ns.…”