Probing excited conformational states of nucleic acids by nitrogen CEST NMR spectroscopy

Characterizing low-populated and short-lived excited conformational states has become increasingly important for understanding mechanisms of RNA function. Interconversion between RNA ground and excited conformational states often involves base pairing rearrangements that lead to changes in the hydrogen-bond network. Here, we present two ¹⁵N chemical exchange saturation transfer (CEST) NMR experiments that utilize protonated and non-protonated nitrogens, which are key hydrogen-bond donors and acceptors, for characterizing excited conformational states in RNA. We demonstrated these approaches on the B. Cereus fluoride riboswitch, where ¹⁵N CEST profiles complement ¹³C CEST profiles in depicting a potential pathway for ligand-dependent allosteric regulation of the excited conformational state of the fluoride riboswitch.