Exploring the Structure and Folding of RNA and RNA-Ligand-Complexes by Multifrequency EPR/ENDOR Spectroscopy

With continuous (cw) and pulsed Electron Paramagnetic Resonance/Electron Nuclear Double Resonance (EPR/ENDOR) methods performed at different microwave frequencies (3, 9 and 180 GHz) we explore structural and dynamical aspects of RNA oligonucleotides [1] as well as their structural changes upon ligand binding. The results of these studies are thought to increase the molecular understanding of specific RNA/ligand interaction and may guide a way for the design of new RNA ligands with specific capabilities.

The central EPR method in this project is the Pulsed Electron Electron Double Resonance (PELDOR) spectroscopy at 3 (S-band) and 9 GHz (X-band). The S-band PELDOR unit was build and calibrated on organic biradicals by A. Weber from our group. This PELDOR method makes is possible to measure distances between two spin centers (up to 55 Å) and their orientation from which data structural information can be gained [2].

However, to make RNA feasible for this method it has to be spin labeled. In cooperation with T. Strube from the group of Prof. J. W. Engels a selective and easy way for the spin labeling of DNA and RNA was established [3]. EPR and PELDOR measurements on mono and double labeled RNA and DNA duplexes are in progress. In the next step characteristic structural motives of RNA (hairpins, bulges, internal loops and junctions) will be studied in the same way.

With the experience from these measurements we want to explore the influence of the paramagnetic ligand Mn²⁺ (substitute for the natural occurring but diamagnetic Mg²⁺) on RNA structures and folding. We are especially interested in the manganese binding site in small ribozymes [4]. For this task multifrequency ENDOR/ESEEM (Electron Spin Echo Envelope Modulation) and measurements at 180 GHz (G-band) will provide us with information about the local surrounding of the manganese, whereas after spin labeling of the RNA with one nitroxide more global information about the structure of the ribozyme can be gained by PELDOR measurements.

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