Structural analysis of RNA and DNA by EPR spectroscopy

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Title: Structural analysis of RNA and DNA by EPR spectroscopy
Other Titles: Analyse der Struktur von RNA und DNA mittels ESR Spektroskopie
Authors: Wunnicke, Dorith
Thesis advisor: Prof. Dr. H.-J. Steinhoff
Thesis referee: Prof. Dr. B. Suess
Abstract: Natural occurring riboswitches, a class of RNA molecules, are able to control the implementation of genetic information through the regulation of gene expression. Both, natural and synthetic riboswitches, so-called aptamers, are complex folded structures, which bind specific ligands. Ligand binding in turn regulates gene expression. In the first section of this work continuous wave (cw) and pulse EPR spectroscopy in combination with site-directed spin labeling was performed to investigate the dynamics and conformational changes of the synthetic tetracycline (Tc) riboswitch. The results obtained herein indicate a thermodynamic equilibrium of two aptamer conformations in the absence of Tc, where one of these conformations is captured upon ligand binding. Aptamer structures have been modeled based on the two equilibrium conformations in the absence of the ligand, and on the captured aptamer conformation in the presence of Tc. All RNA models have been verified by MD simulations by comparing experimentally obtained interspin distances with simulated ones. The incorporation of DNA mutations such as nucleobase changes, double-strand breaks and mispairings can lead to structural and conformational changes of DNA domains which in turn are related to inheritable diseases, cancer and aging. Within this project, the copper(I)-catalyzed Huisgen-Sharpless-Meldal alkyne-azide cycloaddition (CuAAC) ‘click reaction’ was introduced as a powerful modification of existing spin labeling strategies. Interspin distances and exceptionally narrow distribution widths were determined by cw and pulse EPR experiments. The results of the MD simulations exhibit a very good agreement between simulated and experimentally obtained distances. Furthermore, the new spin labeling protocol was used to identify mismatch-induced conformational changes of spin labeled DNA. The application of cw and orientation selective pulse EPR measurements provided insights into the dynamics and structural alterations caused by mismatched base pairs. Interspin distances have been found to depend on the type and nearest neighbor environment of the mismatch. The changes are transferred to the base pairs carrying the spin labels.
Subject Keywords: EPR spectroscopy; RNA; DNA
Issue Date: 5-Sep-2011
Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB04 - E-Dissertationen

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