Site-Directed Spin Labeling of RNA by Postsynthetic Modification of 2′-Amino Groups

To elucidate mechanisms that govern functions of nucleic acids, it is essential to understand their structure and dynamics. Electron paramagnetic resonance (EPR) spectroscopy is a valuable technique that is routinely used to study those aspects of nucleic acids. A prerequisite for most EPR studies of nucleic acids is incorporation of spin labels at specific sites, known as site-directed spin labeling (SDSL). There are two main strategies for SDSL through formation of covalent bonds, i.e., the phosphoramidite approach and postsynthetic spin-labeling. After describing briefly the advantages and disadvantages of these two strategies, postsynthetic labeling of 2′-amino groups in RNA is delineated. Postsynthetic labeling of 2′-amino groups in RNA using 4-isocyanato-TEMPO has long been established as a useful approach. However, this method has some drawbacks, both with regard to the spin-labeling protocol and the flexibility of the spin label itself. Recently reported isothiocyanate-substituted aromatic isoindoline-derived nitroxides can be used to quantitatively and selectively modify 2′-amino groups in RNA and do not have the drawbacks associated with 4-isocyanato-TEMPO. This chapter provides a detailed description of the postsynthetic spin-labeling methods of 2′-amino groups in RNA with a special focus on using the aromatic isothiocyanate spin labels.