SARS CoV-2 5' UTR pH-Differential Mutational Profiling

Structured elements in the SARS CoV-2 RNA-based genome are investigated using a combination of NMR and chemical probing coupled with high throughput sequencing. Case by case NMR investigations suggest some structured elements are pH responsive. To contextualize these pH-responsive elements in the context of the greater 5' UTR, we employed pH-differential mutational profiling (PD-MaP) with DMS probing to gauge the prevalence of protonation-coupled structural transitions in RNA. The RNA (which was in vitro transcribed with two flanking structured cassette helices) is chemically interrogated in a structure-dependent manner with DMS in both a low-pH and high-pH environment, reverse transcribed to encode chemical modifications as mutations, and sequenced. Reactivity profiles for high and low pH sets are calculated against a non-reacted control set and the set of the resultant reactivity profiles for the two pH settings are compared and analyzed to highlight regions that display pH-dependent reactivity changes.