Small Molecule-Controlled Gene Expression: Design of Drug-like High-Affinity Modulators of a Custom-Made Riboswitch

Riboswitches are regulatory RNA structures that modulate gene expression in response to a small molecule. Until now, most efforts to design ligand analogs were motivated by their potential antibiotic activity. However, riboswitches are ideally suited as tools for gene therapy, enabling precise control of gene expression without the need for potentially immunogenic regulatory proteins. Developing synthetic RNA switches starting from natural riboswitches will require to engineer both the ligand and the RNA sequence to achieve sensitivity to the designed small molecule modulator but not to the natural ligand. We present the structure-based design of a drug-like small molecule ligand of the thiamine pyrophosphate (TPP) aptamer, BI-5232. BI-5232 is structurally highly diverse from the natural ligand TPP but rivals its binding affinity (<i>K</i>_D = 1.0 nM). Importantly, in our design, the pyrophosphate of TPP was replaced by an uncharged heterocycle that interacts with the PP-helix in an unprecedented way, as revealed by Molecular Dynamics simulations. Subsequently, we altered the aptamer sequence to drastically reduce its affinity to TPP while retaining the binding properties of our designed ligand. Based on the developed small molecule/RNA aptamer interaction, we finally constructed ribozyme-based ON- and OFF-switches of gene expression in human cell lines. Such systems are valuable additions to the synthetic toolbox for conditionally controlling gene expression, with potential applications in next-generation gene therapies.