Small molecule-controlled gene expression: Design of drug-like high affinity small molecule modulators of a custom-made riboswitch

Riboswitches are regulatory RNA structures that modulate gene expression in response to a small molecule. Until now, 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 ex-pression without the need of potentially immunogenic regulatory proteins. Developing synthetic RNA switches starting from natural riboswitches will require engineering both, the ligand and the RNA sequence in order to achieve full orthogonality i.e., 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 (K _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 binding properties for our designed ligand. Based on the developed orthogonal small molecule/RNA aptamer interaction we finally constructed orthogonal 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.