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Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of <jats:sup>19<\/jats:sup>F NMR methods to characterize complex exchange processes with multiple excited states.<\/jats:p>","DOI":"10.1002\/anie.202218064","type":"journal-article","created":{"date-parts":[[2023,3,27]],"date-time":"2023-03-27T07:28:55Z","timestamp":1679902135000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Multi\u2010Site Conformational Exchange in the Synthetic Neomycin\u2010Sensing Riboswitch Studied by <sup>19<\/sup>F NMR"],"prefix":"10.1002","volume":"62","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9386-7062","authenticated-orcid":false,"given":"Jan H.","family":"Overbeck","sequence":"first","affiliation":[{"name":"Department of Biophysics I Regensburg Center for Biochemistry University of Regensburg  Universit\u00e4tsstrasse 31 93051 Regensburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"V\u00f6gele","sequence":"additional","affiliation":[{"name":"Institute for Molecular Biosciences Goethe-University Frankfurt  Max-von-Laue-Str. 9 60438 Frankfurt\/M. 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