PELDOR and hyperfine spectroscopy as complementary tools to investigate a tetracycline-binding RNA aptamer

  • Since the early 2000s, nucleic acid aptamers have gained considerable attention of life science communities. This is in particular due to the fact that aptamers are known to function as artificial riboswitches, which presents an efficient way to regulate gene expression. A promising candidate is the tetracycline-binding RNA aptamer (TC-aptamer) since the TC-aptamer is known to function in vivo and exhibits a very high affinity towards its ligand tetracycline (TC) (Kd = 800 pM at 10mM Mg2+). Although a highly resolved crystal structure exists in the ligand bound state, questions related to dynamics cannot be answered with X-ray crystallography. In this work, pulsed electron paramagnetic resonance (EPR) spectroscopy was used to study different biochemical and structural aspects of the TC-aptamer. On the one hand, pulsed hyperfine spectroscopy was used to study the binding of TC via Mn2+ to the TC-aptamer at lower and thus more physiological divalent metal ion concentrations. In a first step, a protocol for the relatively new pulsed hyperfine technique electron-electron double resonance detected NMR (ELDORdetected NMR or just EDNMR) was developed for Q-band frequencies (34 GHz). After a successful verification of the EDNMR technique at Q-band frequencies on Mn2+ model complexes ([Mn(H2O)6]2+ and Mn-DOTA), two dimensional hyperfine techniques were used to confirm the formation of a ternary RNA-Mn2+- TC complex at physiological divalent metal ion concentrations. Correlation signals between 13C (13C-labeled TC) and 31P (from the RNA backbone) to the same Mn2+ electron spin were detected with 2D-EDNMR and triple hyperfine correlation spectroscopy (THYCOS). On the other hand, pulsed electron-electron double resonance (PELDOR) spectroscopy on a doubly nitroxide-labeled TC-aptamer was used to investigate the conformational rearrangement upon ligand binding and how the conformational flexibility is affected by different Mg2+ concentrations. The Çm spin label was used as a nitroxide spin probe. Due to its rigidity and low degree of internal flexibility, the Çm spin label yields very narrow distance distributions and pronounced orientation selection (OS). As a consequence, the width of the distance distributions can be used to draw conclusions about the conformational flexibility of the spin-labeled helices. Analysis of the distance distributions showed that at high Mg2+ concentrations, the TC-aptamer is in its folded state, irrespective of the fact if TC is present or absent. Orientation selective PELDOR revealed that the orientation of the spin-labeled helices in frozen solution is the same as in the crystal structure. First Mn2+-nitroxide pulsed electron electron double resonance (PELDOR) measurements on a singly nitroxide-labeled and Mg2+/Mn2+-substituted TCaptamer at different Mn2+ concentrations in the presence and absence of TC gave insight into the affinities of the additional divalent metal ion binding sites of the TC-aptamer.

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Author:Thilo Hetzke
Place of publication:Frankfurt am Main
Referee:Thomas PrisnerORCiD, Josef WachtveitlORCiDGND
Advisor:Thomas Prisner
Document Type:Doctoral Thesis
Date of Publication (online):2020/06/02
Year of first Publication:2019
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Granting Institution:Johann Wolfgang Goethe-Universität
Date of final exam:2020/05/26
Release Date:2020/06/05
Page Number:231
Institutes:Biochemie, Chemie und Pharmazie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
Licence (German):License LogoDeutsches Urheberrecht