Flexibility and conformation of the cocaine aptamer studied by PELDOR

Publisher's version (útgefin grein)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 electronelectron resonance (DEER). The rigid nitroxide spin label C 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 Cs. Since C 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.This work was supported by the Deutsche Forschungsgemeinschaft DFG (SFB 902 Molecular Principles of RNA-based Regulation, Cluster of Excellence Frankfurt Macromolecular Complexes) and the Icelandic Research Fund [120001022]. T. F. Prisner and C. M. Grytz gratefully acknowledge support by the Fond der Chemischen Industrie. We thank Dnyaneshwar B. Gophane for help in sample preparation.Peer reviewe

Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy

Post-print (lokagerð höfundar)Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron–Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology. The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached. We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR. In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created. In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously. This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center. The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif. The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.This work was supported by the SFB 902 Molecular Principles of RNA-based Regulation. C. M. G. gratefully acknowledges support by the Fonds der Chemischen Industrie. P. G. gratefully acknowledges financial support by a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS), and a Eurostars grant by the Swiss Confederation. S. Th. S. gratefully acknowledges financial support from the Icelandic Research Fund (173727). We gratefully acknowledge Friedrich A. Gollmick (Friedrich-Schiller-University, Jena, Germany) for providing the original NMR data. We thank Burkhard Endeward, Vasyl Denysenkov and Dmitry Akhmetzyanov for support in carrying out the experiments and helpful discussions.Peer reviewe

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets

Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed

Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç

The cocaine aptamer is a DNA molecule that binds cocaine at the junction of three helices. The bifunctional spectroscopic probe Ç was incorporated independently into three different positions of the aptamer and changes in structure and dynamics upon addition of the cocaine ligand were studied. Nucleoside Ç contains a rigid nitroxide spin label and can be studied directly by electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy after reduction of the nitroxide to yield the fluoroside Çf. Both the EPR and the fluorescence data for aptamer 2 indicate that helix III is formed before cocaine binding. Upon addition of cocaine, increased fluorescence of a fully base-paired Çf, placed at the three-way junction in helix III, was observed and is consistent with a helical tilt from a coaxial stack of helices II and III. EPR and fluorescence data clearly show that helix I is formed upon addition of cocaine, concomitant with the formation of the Y-shaped three-way helical junction. The EPR data indicate that nucleotides in helix I are more mobile than nucleotides in regular duplex regions and may reflect increased dynamics due to the short length of helix I

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications

Ferro- and antiferromagnetic exchange coupling constants in PELDOR spectra

Pulsed electron electron double resonance (PELDOR) is a well-established method for measuring nanometer distances between paramagnetic centres. Here, we demonstrate on three rigid and conjugated biradicals how the presence of an exchange coupling constant J and its distribtion DJ influences PELDOR data and its analysis. In principle two combinations of J and D fulfill the experimental data in each case. The correct one, including the sign of J, can be determined via simulations in case the two halves of the Pake pattern are separated enough.</p