Mechanism underlying the high regulatory performance of the doxycycline riboswitch G12.

Synthetic riboswitches provide protein-independent, modular control of gene expression, yet selecting aptamers that reliably couple ligand binding to regulatory switching remains challenging. 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.