A review on recent advances in methods for site-directed spin labeling of long RNAs.

ABSTRACT

RNAs are important biomolecules that play essential roles in various cellular processes and are crucially linked with many human diseases. The key to elucidate the mechanisms underlying their biological functions and develop RNA-based therapeutics is to investigate RNA structure and dynamics and their connections to function in detail using a variety of approaches. Magnetic resonance techniques including paramagnetic nuclear magnetic resonance (NMR) and electron magnetic resonance (EPR) spectroscopies have proved to be powerful tools to gain insights into such properties. The prerequisites for paramagnetic NMR and EPR studies on RNAs are to achieve site-specific spin labeling of the intrinsically diamagnetic RNAs, which however is not trivial, especially for long ones. In this review, we present some covalent labeling strategies that allow site-specific introduction of electron spins to long RNAs. Generally, these strategies include assembly of long RNAs via enzymatic ligation of short oligonucleotides, co- and post-transcriptional site-specific labeling empowered with the unnatural base pair system, and direct enzymatic functionalization of natural RNAs. We introduce a few case studies to discuss the advantages and limitations of each strategy, and to provide a vision for the future development.