Enhanced Line Narrowing of Selectively Labeled [9-15N] Guanosine Enables Probing of Large RNAs

Abstract

RNA regulates various cellular processes using malleable 3D structures and characterizing their structural dynamics is critical to shedding light on their mechanism of action. To mitigate continuing limitations on studies of large RNA 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 available labeled ribose to make [9-15N]-GTP. The low CSA of the N9 nucleus (~112 ppm) in combination with extensive ribose deuteration leads to long-lived NMR signals that enable chemical shift assignment, analyze the structure of three biologically relevant large RNA constructs pivotal to viral life cycles [human hepatitis B virus ? RNA (61nt), the HIV-1 primer binding site segment RNA (103 nt), and the HIV-1 Rev response element (232 nt)], observe N9-H8 and N9-H1? correlations, and measure longitudinal and transverse relaxation rates for RNAs as large as 78 kDa. We show CSA dominates both N7 (>99%) and N9 (>90%) relaxation and enables straightforward analysis of dynamics. Taken together, application of these selective labels in conjunction with optimized NMR pulse sequences could help us push the limits of size restrictions in RNA NMR structural biology beyond 100 nt.