Figure 5.

Example of real‐time NMR spectroscopy: adenine‐riboswitch folding and ligand binding. For assignment a 71‐mer RNA construct was perdeuterated at H5, H3′, H4′ and H5′/5′′ positions, which substantially reduced signal overlap. A) Initial structural characterization where done with adenine and Mg²⁺ as ligands, revealing differences in H1 and H3 protons of base‐paired guanine and uridine residues, which enables following of the signal using real‐time NMR spectroscopy. B) Series of UltraSOFAST ¹H,¹⁵N HMQCs of [¹⁵N‐G]‐ or [¹⁵N‐U]‐labeled riboswitches were recorded for RT NMR experiment at rates of approximately 0.5 Hz. At sample concentration of 1 mm, four single‐scan 2D acquisitions were needed to achieve good resolution, resulting in minimal acquisition time of 1.2 s per 2D spectrum. For residues that displayed slow folding kinetics, folding could be followed at a higher resolution by extended cycles of data averaging, as for certain fast folding parts of the structure 2.4 s acquisition time is too long, while for slow‐folding part′s build‐up curve can be resolved even at 9.4 s acquisition time. C) Proposed folding model based on the RT NMR data. Reprinted with permission from ref. 30.