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

We have transcribed a 59 nt viral RNA with 1 ,8-13 C labeling pattern as a proof of concept. The data indicate that while a majority of the nucleotides within the RNA do not experience exchange on the ms time-scale, a few residues sample a lowly populated state. Without data being fit at multiple static magnetic field strengths, the only meaningful parameter that can be extracted is a k ex value ( Figure 4A ). The exchange rates extracted from the CPMG experiments on the viral RNA match well with those from CEST experiments (unpublished). Even though similar information, and perhaps more, can be derived from R 1 data, we find that CPMG is straightforward to setup and analyse compared to R 1 experiments. Thus having labeled RNA that facilitates CPMG measurements is important for the field.