Rational development of FMN-based orthogonal riboswitch that functions in response to specific non-cognate ligand

Re-engineering natural riboswitches into orthogonal RNA switches by making them functional in response to exogenous ligands but unresponsive to endogenous cognate ligands is a promising yet less explored strategy for developing gene regulatory tools. Herein, we rationally engineer the aptamer domain of one of the largest and biotechnologically relevant flavin mononucleotide (FMN) riboswitch class, which specifically binds to synthetic ligands with a high binding affinity (K_D = ~ 54-75 nM) and regulates gene expression in vitro, in prokaryotic, and eukaryotic system, while being unresponsive to FMN. To develop the orthogonal aptamers, we rationally alter key tertiary interactions, such as A/G minor motifs and base triples located in the periphery of the FMN binding pocket. The biophysical and structural probing analysis of the orthogonal aptamer and synthetic ligand complex shows binding mediated by favorable enthalpic and unfavorable entropic contributions. Our rational design approach, coupled with the adaptability to FMN aptamers derived from diverse bacterial strains, suggests the broad applicability of this strategy to numerous FMN riboswitches, each possessing a unique expression platform. This will greatly expand the current repertoire of synthetic riboswitches available for biomedical applications.