Selective [9-¹⁵N] Guanosine for Nuclear Magnetic Resonance Studies of Large Ribonucleic Acids

Abstract

RNAs regulate various cellular processes using malleable 3D structures, and understanding the factors that control RNA structure and dynamics is critical for understanding their mechanisms of action. To mitigate factors that have limited studies of large, functionally relevant RNAs by solution NMR spectroscopy, we have extended a recently described ²H-enhanced, ¹H-¹⁵N correlation approach by developing a chemoenzymatic labeling technology that grafts selectively labeled [9-¹⁵N]-Guanine on to any labeled ribose to make [9-¹⁵N]-GTP. Our approach exploits advantageous NMR properties of the N9 nucleus which, when combined with extensive ribose deuteration and optimized NMR pulse sequences, affords sharp signals without complications that can arise using uniform [¹⁵N]-guanine labeling. The utility of the approach for NMR signal assignment and dynamics analysis is demonstrated for three large RNAs (20-78 kDa) that play critical roles in viral replication. With this approach, NMR studies of RNAs comprising 200 nt or more should now be feasible.