A grand challenge in synthetic biology is to use our current knowledge of RNA science to perform the automatic engineering of completely synthetic sequences encoding functional RNAs in living cells. We report here a fully automated design methodology and experimental validation of synthetic RNA interaction circuits working in a cellular environment. The computational algorithm, based on a physicochemical model, produces novel RNA sequences by exploring the space of possible sequences compatible with predefined structures. We tested our methodology in Escherichia coli by designing several positive riboregulators with diverse structures and interaction models, suggesting that only the energy of formation and the activation energy (free energy barrier to overcome for initiating the hybridization reaction) are sufficient criteria to engineer RNA interaction and regulation in bacteria. The designed sequences exhibit nonsignificant similarity to any known noncoding RNA sequence. Our riboregulatory devices work independently and in combination with transcription regulation to create complex logic circuits. Our results demonstrate that a computational methodology based on first-principles can be used to engineer interacting RNAs with allosteric behavior in living cells.post-transcriptional regulation | evolutionary computation | computational RNA design | RNA synthetic biology T he understanding of RNA interactions in living cells and their subsequent exploitation as regulators is providing new synthetic biology applications (1). RNA regulation is being studied from natural systems by the analysis of the interactions of small RNAs (sRNAs) with messenger RNAs (mRNAs) (2), proteins (3) or molecules (4). However, it is also possible to follow a forward engineering approach and attempt the de novo design of RNA regulators. Rational design techniques have been applied, in both prokaryotes and eukaryotes, for repression or activation of translation (5-10), mRNA degradation (11), riboswitches and ribozymes (12)(13)(14), transcription attenuation (15-18), and scaffolding (19). On the other hand, computational methods allowed designing nucleic-acid-based logic circuits in vitro (20-23), including the redesign of allosteric ribozymes (23), hence, challenging the current knowledge of nucleic acid structure and function. Previous RNA design approaches, however, have been mostly developed to work with in vitro systems or required incorporating fragments of natural sequences.We now propose a fully automated sequence selection methodology to design general circuits based on RNA interactions to operate in living cells. Previous computational methodologies relied on the dominance of the Watson-Crick interactions (24), but they were not adapted to in vivo operations where RNA could be very unstable as it occurs in bacteria. Our approach consists of stabilizing RNA molecules by enforcing a given structure, as done in the inverse folding problem (25-27), together with targeted interactions and conformational changes. The analysis of natura...