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 have rationally engineered the aptamer domain of one of the largest and biotechnologically relevant flavin mononucleotide (FMN) riboswitch class, which specifically binds to synthetic ligands with high binding affinity (KD = ~54-75 nM) and regulates gene expression in vitro (EC50 = ~ 1.2 µM) and in E. Coli (EC50 = ~ 2.2 mM) while being unresponsive to FMN. To develop the orthogonal aptamers, we have rationally altered 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. Collectively, our rational approach can be extended to engineer other riboswitch classes for developing synthetic RNA devices in prokaryotes and eukaryotes for biomedical applications.