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

NMRViewJ was used for peak assignments. Hydrogen and carbon chemical shifts are predicted based on the secondary structure of the input RNA molecule. Expected cross peaks for different experiment types and labeling patterns were then generated using the RNA Peak Generator tool. Expected cross-peaks were generated for HSQC spectra based on the covalent structure and for NOESY spectra using inter-atomic distances typically observed in RNA helices. For bacterial A-site RNA, of which there are no deposited chemical shifts in the BMRB database, the RNA Peak Generator accurately predicted 15 of the 18 expected C1 -H1 resonances, 7 of the 9 C2 -H2 , and 20 of the 27 C6/8-H6/8 resonance within 0.1 ppm of their actual values in the HSQC spectra. Since the NOESY peak generator was used in a mode where it only predicts peaks in helical regions, peaks in bulge and tetraloop regions of the A-site RNA were not predicted. Further assignment of the NOESY spectra utilized the RNA peak slider tool within NMRViewJ. This links the predicted peaks into a network connected by atoms shared between the different peaks. Peaks are then interactively positioned in a way that utilizes the network of peaks typically connected within the NOESY 'walk'. Overall, the combined tools of NMRViewJ allowed for relatively rapid assignment of the resonance in the specifically labeled A-site RNA model system and provides a powerful tool that, when combined with selective labeling, can streamline resonance assignment for RNA than previously reported (41) .