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

The second advantage of these new labels is that the removal of the strong 13 C J-coupling leads to substantial improvements in signal intensity in the protonated base C6 and C8 positions. Additionally, these isolated spin pairs have facilitated the measurement of s-ms dynamics using CPMG and CEST pulse sequences without the complications of large carbon-carbon couplings. Finally with these isolated 'two-spin' labels, these couplings need not be explicitly taken into account in the data analysis of CEST profiles as required in previous studies using uniformly labeled RNA or protein (33, 34) , but also be able to probe more useful sites such as pyrimidine C5 and C6 sites. It is important to note that other dispersion experiments such as R 1 will also benefit from using RNAs transcribed with isolated Figure 5 . (A) NOESY walk of the bacterial A-site RNA using alternatively labeled nucleotides. Starting at the H1 of C14, the connectivity from the sugar H1 /H2 to C8/C6 of the N+1 base enables sequential assignment all the way to the H1 of G19. This allowed the consecutive assignment of 6 residues present in the helical environment. Connectivities follow the placed arrows and move sequentially from cyan to magenta to yellow to black. (B) Pymol representation of the NOESY walk using the same coloring scheme as used in the NOESY walk (PDB: 1A3M (98)). (C) Predicted versus actual chemical shift values for the assigned residues as determined by NMRViewJ (One Moon Scientific). The offset from the central line represents how far the assigned resonance is from its predicted value. Blue circles represent resonances that are less than 0.1 ppm from predicted values, red circles have predicted ppm more than 0.1 ppm away from observed chemical shift. spin systems. The improvements we see from TROSY based pulse sequences scales with the size of the RNA.