RNA Chemical Labeling with Site‐Specific, Relative Quantification by Mass Spectrometry for the Structural Study of a Neomycin‐Sensing Riboswitch Aptamer Domain

High‐resolution mass spectrometry was used for the label‐free, direct localization and relative quantification of CMC+‐modifications of a neomycin‐sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+‐modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy. High‐resolution top‐down mass spectrometry can directly localize and quantifiy nucleobase modifications from chemical probing reactions of RNA, providing important structural information. This study shows that high‐resolution mass spectrometry can be used for the localization and site‐specific, relative quantification of CMC+‐labeled RNA to within ∼5 %.