Inserm U869 (ARNA)
CNRS UMR5248 (CBMN)
A common design of proteins, especially those involved in cellular signalling and biomolecular polymer binding, is a series of small folded protein domains connected by flexible peptide linkers. Normally these linkers provide sufficient mobility to each of the domains such they mostly behave independent of each other. This flexibility allows the domains to bind a range of elements with varying spatial separation, as required for diverse biological roles. We propose to replace these flexible linkers with rigid synthetic connectors (oligomeric quinoline foldamer helices) of several defined lengths. These rigid linked domains, or ‘protein skewers’ (brochettes de protéines), place the project within the theme of biomimetics, orthogonal systems and bionanosciences (Biomimétisme, systems orthogonaux, bionanosciences pour la biologie de synthèse). In contrast to biological proteins, the engineered protein-synthetic chemistry hybrids would contain two or more independent protein domains with a highly determined domain-domain separation and arrangement. This chemical helix rigidity linking the domains would serve biotechnological roles. First of all, the flexible linkers are areas of high protease sensitivity during recombinant production from bacteria, and their removal will increase stability in varied environments. Secondly, the defined separation of the engineered hybrids will place spatial restraints on binding targets, and will be designed to significantly reduce binding to native targets, in this case native RNA molecules. We will take advantage of this property and prepare synthetic target RNA sequences with non-native and discrete RNA elements that optimally bind only to the hybrid ‘protein skewer’ molecules within the cell. These initial steps outlined in the project will allow for feasibility studies of both the protein-quinoline molecules and RNA design, with plans toward future synthetic protein-RNA circuits within live cells, and to obtain sufficient results to apply for national funding.