Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies

Abstract

Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems. Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.