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

Thus, by combining our previous work on pyrimidine synthesis with our current purine synthesis, we can make RNAs that provide labeling patterns that enable an important advance in NOESY assignment strategies (41, 94) . For the conventional uniformly labeled samples, the C2 and C1 resonances are both extremely crowded as discussed above. In a traditional NOESY walk all nucleotides or various permutations are fully-labeled. NOE crosspeaks between protons attached to the C1 and C2 and the C8/C6 of the same and previous nucleotides are observed for helical regions. As we have illustrated, spectral crowding can severely hinder this assignment process. However, by labeling the base of C6/C8 of each nucleotide and alternating the label on the ribose between C1 and C2 a sample is created that not only distinguishes the purines from the pyrimidines but also the A-U and the G-C pairs. We first made nucleotide specific labeled samples, and from the overlaid spectra, we could immediately tell that C/U and G/A showed more spectral overlap in their sugar resonances. Thus it was necessary to label C/G on their C1 carbons and U/A on their C2 carbons. As a proof of concept we have labeled the bacterial A-site RNA with 1 ,6-13 C-1,3-15 N CTP, 2 ,6-13 C-1,3-15 N UTP, 1 ,8-13 C GTP, and 2 ,8-13 C ATP ( Figure 5 ). By combining this alternative labeling strategy with NOESY experiments that allow for filtering/editing of 1 H crosspeaks based on the attached carbons ( 12 C versus 13 C), we can create a unique and powerful system to assign resonances without ambiguity (94) (95) (96) . For ambiguous or overlapped cross-peaks, we utilized 3D 13 C-NOESY-HSQC experiments. This alternating ribose pattern allowed us to unambiguously assign helical regions of RNA. In future work, we will streamline this methodology for use in larger RNAs.