Combining computational design and molecular evolution to devise RNA aptamers directed against members of the TetR and GntR family of bacterial repressors

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Short and long non-coding RNAs are important regulators of gene expression in all kingdoms of life. Consequently, RNA molecules have become prominent in synthetic biology, and small regulatory RNAs, riboswitches and ribozymes are being applied as regulatory devices in the design of synthetic genetic circuits and networks. RNA molecules that bind their target with extraordinarily affinity and specificity can be identified de novo by in vitro selection (SELEX, Systematic Evolution of Ligands by Exponential enrichment). These so-called aptamers can adopt defined three-dimensional structures such as binding pockets or cleft-like interaction surfaces similar to those found in antibodies.The lab of the applicant Beatrix Suess has previously selected and characterized an RNA aptamer that can bind to the bacterial transcription repressor protein TetR and thus compete for its DNA binding. We demonstrated how this aptamer can be used as a synthetic device to regulate the conditional control of gene expression, for example as a splicing device. Recently, the labs of both applicants Suess and Muller joined forces to solve the crystal structure of the TetR-RNA aptamer complex and comprehensively characterize the TetR-RNA aptamer versus TetR-operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and titration calorimetry (Grau et al., 2020, NAR, PMID: 32052019).In the present proposal, the applicants seek to explore the synergistic potential of two approaches, i.e. computational design and molecular evolution, for the design of novel RNA aptamers directed against members of not only the TetR but also the GntR/HutC family of bacterial repressors. The applicants have extensively studied these repressors in the past and, at the same time, demonstrated expertise in the field of in vitro selection and computational design. The proposal also aims to expand the abilities of currently available computer software to include the design of novel RNA-protein interfaces, and hence also DNA-protein interfaces. The properties of the functional versus structural binding epitopes will be characterized in detail in these novel RNA aptamer-bacterial repressor complexes and compared to those of the natural operator DNA-repressor complexes. Initial experiments will be conducted to explore the potential of these novel aptamer-repressor pairs as tools in synthetic biology.In combination, these studies will generate new opportunities for the design of novel logic gates that could be incorporated into synthetic genetic circuits and regulatory networks. At the same time, the synergistic approach will open up new venues for the successful design of protein-RNA or even protein-DNA complexes.