Modular RNA-Responsive Elements for Versatile Eukaryotic Translational Control

RNA-responsive elements enable reprogramming of biological systems. Although much progress has been made in this field, eukaryotic systems still lack simple RNA responsive elements that can be placed directly in front of a target translationally controlled gene. Such a system would allow RNA therapies to be more specific based on endogenous RNA content and open up further possibilities for cellular therapies. Here, we introduce eukaryotic toehold switches (eToeholds) as a modular, ~300-500 base pair long, riboregulator that functions in eukaryotic systems. eToeholds were designed through meticulous optimization of RNA base pair mediated folding and expression methodology. eToeholds function within a range of eukaryotic organisms and may be used to sense and respond to the presence of a selected trigger RNA with up 16-fold induction of any transgene. We show a powerful application of eToeholds in the sensing of viral infection, where a Zika-targeted eToehold increases the regulated expression of luciferase 9-fold higher than controls after Zika infection. We finally demonstrate that eToeholds can be used to sense both endogenous transcript changes and cell-type specific transcript differences. The robust and specific activation and the modular design of eToeholds demonstrate their broad applicability for numerous applications in biological engineering.