RNA Information Processors for Complex In Vivo Logic Computation

Synthetic biology aims to create functional devices, systems, and organisms with novel and useful functions taking advantage of engineering principles applied to biology. Starting from the first demonstration of synthetic circuits in bacteria more than a decade ago, synthetic biology has substantially advanced strategies for biomedical applications. Ultimately, sophisticated therapeutic sensor-effector devices that connect diagnostic input with therapeutic output may provide platforms for future gene- and cell-based therapies. Despite great progress over the last decade, an underlying problem in synthetic biology remains the limited number of high-performance, modular, composable parts. A potential route to solve parts bottleneck problem in synthetic biology utilizes the programmability of nucleic acids inspired by molecular programming approaches that have demonstrated complex biomolecular circuits evaluating logic expressions in test tubes. Central to our current work is a novel class of in vivo RNA-based devices called toehold switches that activate gene expression in bacteria only when they detect a cognate trigger RNA. The toehold switches take trigger RNAs without any sequence constraints overcoming the limitations of conventional synthetic riboregulators. Further, these toehold switches can be forward-engineered to modulate gene expression by several hundred-fold rivaling the best protein-based regulators and provide extremely low levels of crosstalk across large library of components. Using a toolkit of orthogonal devices and modular composability, we demonstrate how toehold switches can be incorporated into decision-making RNA networks to rapidly evaluate complex logic in living cells. We have successfully demonstrated a 4-input AND gate, a 6-input OR gate, and a 10-input expression in disjunctive normal form, surpassing the complexity of previous works. The compact encoding of RNA information processing system using a library of modular parts is amenable to aggressive scale-up towards complex control of in vivo circuitry towards autonomous behaviors and biomedical applications.