Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Abstract

In site-directed spin-labeling (SDSL), a stable nitroxide radical is attached to a specific location within a macromolecule and electron paramagnetic resonance (EPR) spectroscopy is used to interrogate the local environment surrounding the nitroxide. The SDSL strategy enables probing sitespecific structural and dynamic features of RNA in solution without being limited by the size of the molecule, thus serving as a unique tool in biophysical studies of RNA. This chapter describes the use of continuous-wave (cw)-EPR to study dynamic features of RNAs as well as to monitor interactions between them. Various approaches for attaching nitroxide spin labels to nucleic acids are described, followed by detailed descriptions of cw-EPR spectral acquisition and processing procedures. Specific examples are subsequently used to illustrate analysis of EPR spectra, showing how information regarding the parent RNA can be extracted.Information about RNA structure and movement is critical for our understanding of how RNA is able to carry out its multifaceted functions. One spectroscopic technique that has shown great promise to study RNA, as well as other biopolymers, is electron paramagnetic resonance (EPR) spectroscopy, also named electron spin resonance (ESR) spectroscopy. EPR is a magnetic resonance technique that monitors the behaviors of unpaired electrons, and has long been used to study structure and dynamics of biomolecules (see recent reviews by Klug and Feix, 2008;Sowa and Qin, 2008). Structural information can be obtained by distance measurements, that is, by determination of distances between two spin-centers, and is the topic of another chapter in this volume (see Chapter 16 in this volume).This chapter focuses on continuous-wave (cw)-EPR, which has been commonly used for studying dynamic features and interactions between biomolecules. The technique is capable of covering motions ranging from picosecond (ps) to millisecond (ms). Other advantages of EPR are that small amount of material (typically 0.05-1.0 nmol) is required, samples are not limited by molecular size, and the measurements can be carried out under physiological conditions. However, RNA does not contain stable unpaired electrons and, therefore, spincenters must be introduced into the RNA in order to conduct EPR studies.This chapter first summarizes methods for attaching spin labels to nucleic acids, with a special focus on RNA (Section 1). Detailed protocols for these labeling methods have been described in a series of recent publications (Edwards and Sigurdsson, 2007;Qin et al., 2007;Schiemann et al., 2007), and will not be repeated here. Acquisition and processing of cw-EPR spectrum will then be described in detail (Section 2), followed by a brief discussion of spectral analysis (Section 3). Specific examples of using cw-EPR to study dynamics and interactions in RNA will subsequently be given (Section 4). NIH Public Access Author ManuscriptMethods Enzymol. Author manuscript; available in PMC 2010 October 19. Published in final edited form as:Met...

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

“…All spectra were acquired at 5°C with the following acquisition parameters unless otherwise stated: 2 mW microwave power, 1.5 G modulation amplitude, 100 kHz modulation frequency, and 100 G scan width. The measured spectra were averaged, baseline corrected, and normalized following published procedures (Zhang et al 2009). Spectral simulations were carried out with the EPRLL program suite (Earle and Budil 2006) using the microscopic ordering macroscopic disorder (MOMD) model (Budil et al 1996;Khairy et al 2006).…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17]. The majority of biomolecules do not contain unpaired electrons from which one can obtain an EPR signal; therefore, spin-labeling approaches have been developed [15,[18][19][20][21][22][23][24][25] where site-specific persistent radicals or paramagnetic metal-probes are incorporated at specific locations within a biomolecule. Properties of the EPR spectra that originate from these probes, positioned at welldefined vantage points, provide structural and dynamic constraints.…”

Methods to Detect and Characterize Metal Ion Binding Sites in RNA

“…Unpaired electrons are commonly introduced into RNA by attaching a spin-label, usually a stable nitroxide radical, at a specific location by site-directed spin-labeling (SDSL) [94].…”

“…EPR and related methods have been used in the past to investigate global folding and the dynamics, as well as to monitor metal ion binding to RNA. To look at global folding, two spin labels need to be placed at specific locations in an oligonucleotide by chemical methods [94]. Pulsed EPR techniques like pulsed electron-electron double resonance (PELDOR or DEER) [95,96] or more specialized ones like double quantum coherence (DQC-EPR) [97] are then applied to obtain accurate distances in the range from 1.5-8…”

“…Dynamic features and interactions of biomolecules in the range from picoseconds to milliseconds can be measured by continuous-wave EPR (cw-EPR) and has been applied for example with the Hammerhead ribozyme [99]. This method has been thoroughly reviewed lately [94] and we encourage the reader to refer to this literature for more information and practical details.…”