Genetically encoded RNA strand exchange circuits for programmable protein expression and computation in cells

Programmable cellular information processing could advance biomanufacturing of chemicals and medicine, and enable smart, living therapeutics and diagnostics. Nucleic acids circuits based on toehold mediated strand exchange (TMSE) show tremendous potential for cellular programming due to their scalable, composable, and biocompatible parts. However, these circuits are constrained primarily to in vitro applications because genetically encoding them is challenging and the principles of TMSE in cells remain unknown. Here we show the first demonstration of genetically encoded RNA strand exchange circuits, designed analogously to state-of-the-art TMSE circuits, in living cells. To elucidate the design principles of TMSE in cells, we develop toehold exchange riboregulators, which convert TMSE to protein expression, enabling precise control of protein translation rate. We find many of the design principles and parts used for TMSE in vitro transfer to Escherichia coli, allowing construction of multi-layer cascades and logic elements. We also identify caveats where strand exchange in cells differ substantially from cell-free systems, even in lysate from the same bacterial strain, suggesting active cellular processes are involved. Our results further highlight bounds on strand exchange circuit architectures feasible in cells. We anticipate this study will lay the groundwork for developing advanced cellular circuits, bringing nucleic acid computing from the test tube to the cell and enabling new applications by connecting TMSE to gene expression. More broadly, our results have implications for RNA:RNA interactions and gene regulation in bacteria and provide a synthetic system for exploring such phenomena.