De novo automated design of functional regulatory RNA sensors in bacteria

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Thomas Landrain, Guillermo Rodrigo, Shensi Shen, Alfonso Jaramillo

Institute for Systems and Synthetic Biology, France

A grand challenge in biology is to use our current knowledge to create innovative functionalities based on known properties of organic molecules in living cells. Based on our recent success in validating the first fully automated design methodology of synthetic RNA interaction circuits working in a cellular environment, designing several positive riboregulators with diverse structure and interaction models, we decided to extend our methodology to the design of small molecule-sensing RNAs, synthetic riboswitches. Sensing is a fundamental feature that is critical for survival and adaptation and RNA aptamers have the ability to attach specifically to almost any kind of small molecules. In order to design synthetic riboswitches for a given molecule, we take advantage of existing RNA aptamer libraries. When a RNA aptamer binds to a given ligand, its structure is altered, but RNA aptamers remain non-functional molecules as long as they can’t propagate information about their state to another molecule. In order to trigger the propagation of an allosteric change, we created a computational algorithm, based on a physicochemical model, that evolve a RNA backbone in the form of a 5’UTR around the RNA aptamer to connect it to another functional RNA domain (RBS, anti-terminator, ribozyme…). We tested our methodology in E. coli by designing several positive riboswitches from the known RNA aptamer for theophylline. The designed sequences exhibit non-significant similarity to any known non-coding RNA sequence. Our RNA 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 functional regulatory RNA sensors in living cells from non-functional RNA aptamers.