Abstract
The cocaine aptamer is a DNA three-way junction that binds cocaine at its helical junction. We studied the global conformation and overall flexibility of the aptamer in the absence and presence of cocaine by pulsed electron-electron double resonance (PELDOR) spectroscopy, also called double electron-electron resonance (DEER). The rigid nitroxide spin label Ç was incorporated pairwise into two helices of the aptamer. Multi-frequency 2D PELDOR experiments allow the determination of the mutual orientation and the distances between two Çs. Since Ç is rigidly attached to double-stranded DNA, it directly reports on the aptamer dynamics. The cocaine-bound and the non-bound states could be differentiated by their conformational flexibility, which decreases upon binding to cocaine. We observed a small change in the width and mean value of the distance distribution between the two spin labels upon cocaine binding. Further structural insights were obtained by investigating the relative orientation between the two spin-labeled stems of the aptamer. We determined the bend angle between this two stems. By combining the orientation information with a priori knowledge about the secondary structure of the aptamer, we obtained a molecular model describing the global folding and flexibility of the cocaine aptamer.
Highlights
Orientation-selective pulsed electron–electron double resonance (PELDOR) experiments at X-band frequencies were performed on the aptamers in absence and presence of cocaine in order to obtain information regarding both the distances and the mutual orientations between two Ç spin labels (Fig. 2 and Fig. S10, Electronic supplementary information (ESI)†)
We studied the conformational changes of two doubly Ç-spin-labeled cocaine aptamer constructs upon cocaine binding by PELDOR spectroscopy
This study is the first example of applying PELDOR with rigid spin labels to study the changes in the structure and conformational flexibility of a nucleic acid junction upon ligand binding
Summary
Nucleic acid aptamers have been widely used in the rapidly emerging field of biochemical biosensors due to their ability to bind to specific ligands.[1,2,3] DNA and RNA aptamers are typically selected and identified in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) technology.[4,5,6] In recent years, naturally occurring RNAs, termed riboswitches, which bind to small metabolites and regulate their gene expression, have been discovered.[7,8] Studies of the structure and dynamics of aptamers provide insights into both ligand-induced conformational changes of nucleic acid architectures and principles of their molecular recognition, which in turn may offer opportunities for structure-based drug design strategies for therapeutics.[9 ]. The different variations of the modulation depth as a function of the offset frequency in the experiments on aptamers 1 and 2, with and without cocaine, (Fig. 2) reflect different conformational distributions of relative orientations between the two spin labels of the different samples, rather than experimental errors.
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