Structure and Internal Dynamics of Short RNA Duplexes Determined by a Combination of Pulsed EPR Methods and MD Simulations.

We used EPR spectroscopy to characterize the structure of RNA duplexes and their internal twist, stretch and bending motions. We prepared eight 20-base-pair-long RNA duplexes containing the rigid spin-label Çm, a cytidine analogue, at two positions and acquired orientation-selective PELDOR/DEER data. By using different frequency bands (X-, Q-, G-band), detailed information about the distance and orientation of the labels was obtained and provided insights into the global conformational dynamics of the RNA duplex. We used 19F Mims ENDOR experiments on three singly Çm- and singly fluorine-labeled RNA duplexes to determine the exact position of the Çm spin label in the helix. In a quantitative comparison to MD simulations of RNA with and without Çm spin labels, we found that state-of-the-art force fields with explicit parameterization of the spin label were able to describe the conformational ensemble present in our experiments. The MD simulations further confirmed that the Çm spin labels are excellent mimics of cytidine inducing only small local changes in the RNA structure. Çm spin labels are thus ideally suited for high-precision EPR experiments to probe the structure and, in conjunction with MD simulations, motions of RNA.

Förster resonance energy transfer within the neomycin aptamer.

Förster resonance energy transfer (FRET) measurements between two dyes is a powerful method to interrogate both structure and dynamics of biopolymers. The intensity of a fluorescence signal in a FRET measurement is dependent on both the distance and the relative orientation of the dyes. The latter can at the same time both complicate the analysis and give more detailed information. Here we present a detailed spectroscopic study of the energy transfer between the rigid FRET labels Çmf (donor) and tCnitro (quencher/acceptor) within the neomycin aptamer N1. The energy transfer originates from multiple emitting states of the donor and occurs on a low picosecond to nanosecond time-scale. To fully characterize the energy transfer, ultrafast transient absorption measurements were performed in conjunction with static fluorescence and time-correlated single photon counting (TCSPC) measurements, showing a clear distance dependence of both signal intensity and lifetime. Using a known NMR structure of the ligand-bound neomycin aptamer, the distance between the two labels was used to estimate κ2 and, therefore, make qualitative statements about the change in orientation after ligand binding with unprecedented temporal and spatial resolution. The advantages and potential applications of absorption-based methods using rigid labels for the characterization of FRET processes are discussed.