De-novo-designed RNA molecules are used to construct cellular computing devices that can implement complex logic functions. Toehold riboswitches are designer hairpin motifs inserted into RNA transcripts to hide ribosome binding sequences. This ensures that protein synthesis is minimal until a separate regulatory RNA species binds to and opens the hairpin. Peng Yin and colleagues combine several toehold riboswitches and antisense RNA species to implement complex Boolean regulatory logic, placing a single gene under up to 12 inputs with 5 clauses. The computation takes place in a single physical locus, making the design much more compact and efficient than existing synthetic biology approaches in which signals are encoded by DNA-binding proteins in complex regulatory networks. The work expands in vitro DNA origami concepts to in vivo RNA nanotechnology and synthetic biology. Synthetic biology aims to develop engineering-driven approaches to the programming of cellular functions that could yield transformative technologies1. Synthetic gene circuits that combine DNA, protein, and RNA components have demonstrated a range of functions such as bistability2, oscillation3,4, feedback5,6, and logic capabilities7,8,9,10,11,12,13,14,15. However, it remains challenging to scale up these circuits owing to the limited number of designable, orthogonal, high-performance parts, the empirical and often tedious composition rules, and the requirements for substantial resources for encoding and operation. Here, we report a strategy for constructing RNA-only nanodevices to evaluate complex logic in living cells. Our ‘ribocomputing’ systems are composed of de-novo-designed parts and operate through predictable and designable base-pairing rules, allowing the effective in silico design of computing devices with prescribed configurations and functions in complex cellular environments. These devices operate at the post-transcriptional level and use an extended RNA transcript to co-localize all circuit sensing, computation, signal transduction, and output elements in the same self-assembled molecular complex, which reduces diffusion-mediated signal losses, lowers metabolic cost, and improves circuit reliability. We demonstrate that ribocomputing devices in Escherichia coli can evaluate two-input logic with a dynamic range up to 900-fold and scale them to four-input AND, six-input OR, and a complex 12-input expression (A1 AND A2 AND NOT A1*) OR (B1 AND B2 AND NOT B2*) OR (C1 AND C2) OR (D1 AND D2) OR (E1 AND E2). Successful operation of ribocomputing devices based on programmable RNA interactions suggests that systems employing the same design principles could be implemented in other host organisms or in extracellular settings.

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EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.