(12c) The Riboswitch Calculator: De Novo Design of Synthetic Cis-Acting Riboswitches From Ligand-Binding Aptamers

Riboswitches are structured 5' untranslated regions (UTRs) that regulate gene expression in response to changing ligand concentrations. As cellular sensors, riboswitches have the ability to detect toxic chemicals, trigger the in vivo production of therapeutics, or report the cell's internal metabolic state for metabolic engineering efforts. Currently, the majority of engineered riboswitches respond to theophylline or similarly structured ligands; however, thousands of ligand-binding mRNAs, called aptamers, have been generated through in vitro selection methods. The great challenge of riboswitch engineering has been to convert these aptamers into riboswitches capable of regulating gene expression with practically useful dynamic ranges.

We have developed and experimentally validated a computational design method, the Riboswitch Calculator, that designs synthetic riboswitches capable of binding to diverse chemical ligands (3+) and activating translation rates by 10- to 100-fold.  We combine a predictive statistical thermodynamic model of translation initiation -- the RBS Calculator v1.2 -- with known aptamer sequences to identify synthetic 5' UTRs that will undergo targeted RNA shape changes to maximally regulate translation initiation in response to ligand binding events.   Importantly, no additional parameterization was necessary to predict translation rates in response to ligand binding events.

We have employed both in vitro and in vivo expression assays to measure changes in translation rates and to identify the key rate-limiting steps in ligand-dependent riboswitch activation. We have identified the key reasons why some riboswitches fail to regulate gene expression, and developed quantitative criteria to discard such sequences during computational optimization. We also identify instrinsic limitations to utilizing certain aptamer sequences.  The Riboswitch Calculator provides a quantitative and falsifiable approach to engineering synthetic riboswitches with predictable behaviors. It has also provided an fruitful avenue for further advancing our understanding of RNA biophysics inside cells.