Falk Wachowius

Probing secondary structures of spin-labeled RNA by pulsed EPR spectroscopy.

The ability of RNA to interconvert between multiple conformational states is essential for the diversity of biological functions that have been discovered in the recent past. For example, the correct operation of regulatory RNA elements, such as riboswitches, is based on the precise interplay of alternative RNA conformations. Studying the molecular mechanisms of RNA function entails probing RNA-folding intermediates on the energy landscape. EPR spectroscopy, in particular, has been increasingly applied to obtain structural information on nucleic acids, including local conformational changes in RNA and the identification of metal-ion binding sites. Pulsed EPR techniques (PELDOR/ DEER) have been used to determine distances between paramagnetic centers in specifically modified RNA. PELDOR should therefore be suitable for the detection of alternative RNA conformations that involve distinct changes in base-pairing patterns. The accessibility of spin-labeled RNA still poses the major challenge for the widespread applicability of powerful EPR techniques. Nitroxide radicals are the most commonly used type of paramagnetic labels for nucleic acids. Several methods have been reported for attaching nitroxide groups at internal positions at the ribose, the phosphate backbone, or at nucleobases, often by means of multiatom linkers that provide several unwanted degrees of rotational freedom. Rigid nitroxide spin labels conjugated to the nucleobase or to nucleobase analogues have been reported for DNA. Our RNA spin-labeling approach addresses the direct attachment of nitroxide labels onto RNA nucleobases, such that conformational changes can be directly detected by PELDOR (i.e., by the change in distance between two labeled nucleotides). The nucleobase spin labels used in this study are also designed to preserve the Watson–Crick base-pairing capability of labeled nucleotides and not to interfere with alternative base-pairing patterns in different RNA conformations. Here, we describe the installation of nitroxide spin labels on exocyclic amino groups of the RNA nucleobases guanine, adenine, and cytosine (Figure 1) with unprecedented effi-

Chemical RNA Modifications for Studies of RNA Structure and Dynamics

The growing number of RNAs known to be involved in complex biological processes has increased the appreciation for the structural and functional diversity of RNA. In recent years, the scientific community has witnessed the discovery of various RNAs playing integral roles in catalysis, metabolite binding, regulation of transcription and translation events, protein recognition, and epigenetic gene regulation. A fundamental basis for many of these unanticipated biological functions is the ability of RNA to adopt complex three-dimensional structures and to interconvert rapidly between multiple functional states. However, at a molecular level, the current understanding of conformational rearrangement mechanisms and RNA folding pathways is often limited. Recent intensive biochemical and biophysical research efforts have been dedicated to the collection of much-needed information on RNA structures of different hierarchical levels and at different resolutions. An extensive set of experimental and theoretical tools is available to improve our understanding of dynamic RNA folding events and to help uncover the fundamentals of the exquisite specificity of RNA recognition processes. Many biochemical and biophysical techniques for the investigation of RNA structures and mechanisms not only benefit from, but often depend on, the availability of site-specifically modified RNA samples. Fluorescence spectroscopy is a prominent example of an effective biophysical method for monitoring global structure and conformational dynamics of RNA. Fluorescence-based assays always depend on the incorporation of fluorescent nucleoside analogues or the attachment of extrinsic chromophores to RNA residues. The applications of fluorescently labeled RNA range from measurement of fluorescence emission quenching, to fluorescence anisotropy and fluorescence lifetime, shifting of fluorescence spectra, and fluorescence resonance energy transfer. In addition to steady-state fluorescence measurements, new developments in time-resolved (e.g. , femtosecond time-resolved fluorescence spectroscopy) and advanced single-molecule techniques have shown high potential to improve our understanding of the fundamentally important dynamic aspects of RNA structures. Magnetic resonance techniques have become indispensable for the analysis of structure and dynamics of RNA. NMR spectroscopy has proven to be a powerful tool for determining the structure of small RNA motifs. Recent developments to directly detect hydrogen-bonding interactions, also in combination with time-resolved NMR spectroscopic techniques, have been used to distinguish possible secondary structures and to study mechanisms of ligand-binding events. Probing RNA dynamics in solution is based on NMR relaxation data, and information about long-range structural interactions and orientations of helical domains can be obtained by NMR residual dipolar coupling techniques. The successful application of these advanced techniques essentially relies on RNA modification strategies for the incorporation of stable isotopes, such as C, N, H, and F to alleviate the effects of signal degeneracy. Electron paramagnetic resonance (EPR) spectroscopy provides information on local and global dynamic properties and can be used to probe metal ion binding sites in RNA. Detailed information about the binding site geometry can be obtained by advanced EPR methods (e.g. , ENDOR, electron nuclear double resonance). Pulsed electron double resonance (PELDOR) yields long-range structural restraints by measuring the distance between two spin labels. The application of EPR spectroscopy is directly linked to the ability to incorporate paramagnetic centers into RNA at specific positions. Impressive high-resolution structures of small and large RNAs have recently been obtained by X-ray crystallography. De novo determination of 3D RNA structures requires sophisticated phasing techniques, which are based on derivatization with heavy atoms, by soaking crystals in salt solutions, or by chemical derivatization of nucleosides with halogen or selenium atoms. The increasing number of reported applications of modified RNAs for biophysical experiments shows that site-specific incorporation of artificial RNA modifications constitute important enabling technologies for the detailed investigation of functional RNAs. In this review, we discuss recent examples of chemical RNA modifications that were used to monitor conformational changes by NMR spectroscopy, with a focus on fluorine-modified RNA. In addition, we describe modifications to deliberately manipulate RNA secondary structure populations, and RNA analogues to observe conformational changes by fluorescence spectroscopy. Furthermore, we summarize recent developments in the areas of spin-labeled RNA for EPR spectroscopy and selenium-modified RNA for X-ray crystallography.

Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation

Pulsed electron-electron double resonance (PELDOR, also known as DEER) has become a method of choice to measure distances in biomolecules. In this work we show how the performance of the method can be improved at high EPR frequencies (94 GHz) using variable dual frequency irradiation in a dual mode cavity in order to obtain enhanced resolution toward orientation selection. Dipolar evolution traces of a representative RNA duplex and an α-helical peptide were analysed in terms of possible bi-radical structures by considering the inherent ambiguity of symmetry-related solutions.

Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label.

The nitroxide-containing nucleoside Çm is reported as the first rigid spin label for paramagnetic modification of RNA by solid-phase synthesis. The spin label is well accommodated in several RNA secondary structures as judged by its minor effect on the thermodynamic stability of hairpin and duplex RNA. Electron paramagnetic resonance (EPR) spectroscopic characterization of mono-, bi-, and trimolecular RNA structures shows that Çm will be applicable for advanced EPR studies to elucidate structural and dynamic aspects of folded RNA.