Uncovering the Structural Landscape of Heat-Induced Riboswitch RNA Unfolding Using Native Variable-Temperature ESI Ion Mobility Mass Spectrometry and

RNA structures denature at elevated temperatures, leading to progressive base-pair opening and loss of secondary structure. However, the conformations adopted at solution temperatures above those at which base pairing is disrupted remain poorly understood. In this study, structural information from variable-temperature nanoelectrospray ionization ion mobility mass spectrometry, imino-proton nuclear magnetic resonance spectroscopy, and UV absorbance is combined to map the stepwise unfolding and refolding of a riboswitch aptamer. Three distinct thermal regimes were identified. In Region I (25-55 °C), the average negative charge state decreases unexpectedly and correlates with selective melting of the upper stem detected by NMR, suggesting a local reorganization that reduces the accessibility of backbone phosphate groups for deprotonation during electrospray charging. In Region II (60-75 °C), complete loss of imino resonances coincides with a sharp increase in negative charge, consistent with global loss of secondary structure and increased phosphate accessibility, enabling accommodation of higher net charge. In Region III (80-90 °C), the average charge decreases again, reflecting an increased abundance of compact conformers of the M