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The tetracycline-binding RNA aptamer (TC-aptamer) is a synthetic riboswitch that binds the antibiotic tetracycline (TC) with exceptionally high affinity. Although a crystal structure exists of the TC-bound state, little is known about the conformational dynamics and changes upon ligand binding. In this study, pulsed electron paramagnetic resonance techniques for measuring distances (PELDOR) in combination with rigid nitroxide spin labels (Çm spin label) were used to investigate the conformational flexibility of the TC-aptamer in the presence and absence of TC at different Mg2+ concentrations. TC was found to be the essential factor for stabilizing the tertiary structure at intermediate Mg2+ concentrations. At higher Mg2+ concentrations, Mg2+ alone is sufficient to stabilize the tertiary structure. In addition, the orientation of the two spin-labeled RNA helices with respect to each other was analyzed with orientation-selective PELDOR and compared to the crystal structure. These results demonstrate for the first time the unique value of the Çm spin label in combination with PELDOR to provide information about conformational flexibilities and orientations of secondary structure elements of biologically relevant RNAs.
TEMPO spin labels protected with 2-nitrobenzyloxymethyl groups were attached to the amino residues of three different nucleosides: deoxycytidine, deoxyadenosine, and adenosine. The corresponding phosphoramidites could be incorporated by unmodified standard procedures into four different self-complementary DNA and two RNA oligonucleotides. After photochemical removal of the protective group, elimination of formic aldehyde and spontaneous air oxidation, the nitroxide radicals were regenerated in high yield. The resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin–spin distances measured by PELDOR correspond well to the values obtained from molecular models.
PELDOR (pulse electron-electron double resonance) is an established method to study intramolecular distances and can give evidence for conformational changes and flexibilities. However, it can also be used to study intermolecular interactions as for example oligerimization. Here, we used PELDOR to study the ‘end-to-end’ stacking of small double stranded (ds)RNAs. For this study, the dsRNA molecules were only singly labelled with the spin label TPA to avoid multi-spin effects and to measure only the intermolecular stacking interactions. It can be shown that small dsRNAs tend to assemble to rod-like structures due to π-π-interactions between the base pairs at the end of the strands. On the one hand, these interactions can influence or complicate measurements aimed at the determining of the structure and dynamics of the dsRNA molecule itself. On the other hand, it can be interesting to study such intermolecular stacking interactions in more detail, as for example their dependence on ion concentration. We quantitatively determined the stacking probability as a function of the monovalent NaCl salt and the dsRNA concentration. From this data the dissociation constant Kd was deduced and found to depend on the ratio between the NaCl salt and dsRNA concentrations. Additionally, the distances and distance distributions obtained predict a model for the stacking geometry of dsRNAs. Introducing a nucleotide overhangs at one end of the dsRNA molecule restricts the stacking to the other end, leading only to dimer formations. Introducing such an overhang at both ends of the dsRNA molecule fully suppresses stacking, as we could demonstrate by PELDOR experiments quantitatively.