Synthetic riboswitches: design of multi-ligand switches and novel regulatory principle

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In synthetic biology, artificial regulatory networks play a central role. Especially RNA-based regulation of gene expression is a promising application that allows a versatile control of cell behavior. Due to their modular composition and easy manipulation, regulatory RNA elements like riboswitches are of particular interest. It has been demonstrated in the recent past - in part by the applicants - that riboswitches acting both at the translational and the transcriptional level can be designed successfully on the computer. To date, such designs have been limited to the sensing of a single ligand. Here, we propose to investigate how riboswitches that react to specific concentration patterns of multiple ligands can be designed. Multiple artificial aptamer domains will be integrated in such a way that their individual structural changes in response to ligand binding are combined to an overall change in RNA structure that exerts a well-defined force on the regulatory actuator elements leading to the desired overall biological effect. The in silico predictions will be experimentally tested and qualitatively as well as quantitatively analyzed. The iterative process of computer-based predictions and wet-lab analyses will lead to a continuous improvement of the predictive power of the algorithm and also of the regulatory power of the generated riboswitches. Making use of a collection of distinct aptamer elements, we will develop a universally applicable method to design artificial riboswitches with non-trivial sensor logic and expand this also to further regulatory levels like tRNA processing reactions. In parallel, we will develop user-friendly software that can allows to solve this kind of design problems.