Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

When studying large RNAs (>40 nts) by NMR, slow molecular tumbling leads to broadened linewidths and losses in signal intensity. Careful selection of appropriate NMR experiments to address these losses are necessary for successful measurement of many NMR parameters. TROSY experiments take advantage of the interference between the dipolar coupling and chemical shift anisotropy (CSA) components of T 2 relaxation (66) . For the base C8 position of adenine and guanine, these contributions effectively cancel at ∼800 MHz field strength leading to reduction in the R 2 relaxation rate (80, 81) . Thus, RNAs synthesized with our selective site-specifically labeled NTPs should benefit from TROSY based NMR experiments that reduce the problems of crowding, fast signal decay, low resolution, and decreased S/N ratios (12, 34, 31, (66) (67) (80) (81) .