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Here, we identify and mechanistically characterise G12, a doxycycline-binding aptamer with remarkably high regulatory performance in yeast and human cells. We provide evidence that RNA Capture-SELEX efficiently enriches aptamers with ligand-responsive conformational switching. We compared conventional SELEX and RNA Capture-SELEX using the same starting library followed by NGS analysis and in vivo screening, which led to the identification of G12. G12 binds doxycycline with low-nanomolar affinity and strict discrimination against close derivatives, thus enabling high-dynamic-range riboswitch control of translation in yeast and splicing in human cells. Single-molecule force spectroscopy with optical tweezers revealed that doxycycline stabilises a folding intermediate independent of the closing stem P1, which primarily acts as a scaffold for correct aptamer folding. Mutational analysis and chemical probing identified tertiary contacts between loops L2 and L3 in this intermediate state. Stopped-flow fluorescence spectroscopy further supported a two-step binding mechanism consistent with efficient regulatory switching. Together, these findings deepen our understanding of regulatory aptamer selection and function and expand the synthetic biology toolbox with a high-performance doxycycline-responsive riboswitch.<\/jats:p>","DOI":"10.1038\/s41467-026-76256-2","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T17:18:49Z","timestamp":1785777529000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mechanism underlying the high regulatory performance of the doxycycline riboswitch G12"],"prefix":"10.1038","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5858-9490","authenticated-orcid":false,"given":"J.","family":"Hoetzel","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-8275-1368","authenticated-orcid":false,"given":"A.","family":"Walbrun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Sch\u00e4fer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"T.","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A. 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