Frozen DNA origami superlattice to facilitate Cryo-EM imaging of membrane proteins – REDIRECT

DNA origami nanotechnology, which uses the self-assembly of DNA molecules, has become a technique of choice to rationally design nanostructures of complex geometry. In this project, we propose to use DNA origami to develop a new method that helps to circumvent the limitations of sample preparation in Cryo-Electron Microscopy (Cryo-EM). We propose to design a two-dimensional (2D) ordered array of DNA origami which is stable in solution and with micrometer to cover holey carbon cryo-EM grids. The chemical function of the 2DNA lattice can be engineered as desired to specifically attach the target biomolecule. This strategy will create dense arrays of non-overlapping particles through site directed-conjugation. This method would thus facilitate the preparation of Cryo-EM samples. Particularly, membrane proteins by circumventing the common drawbacks associated to sample preparations (e.g., interaction at the air-water interface, aggregation, low protein concentration, etc.). We will use this method to study efflux transporters, which have become a global public health problem because they endanger the effectiveness of antibacterial therapies. The project will be carried out within the "Signaling and membrane transport" team (CiTCoM, CNRS, University of Paris Cité). The PI will benefit from the team's expertise in the study of membrane proteins and will bring her expertise in creating sophisticated DNA origami objects as a breakthrough to improve the quality of cryo-EM sample preparation and structural studies. Recently, through Cryo-EM characterization, we confirmed the formation of a large 2D origami DNA array. By combining this approach with innovative protein engineering tools, our goal is to elucidate the structural basis of efflux transporters and provide new information on their functions to develop new approaches to fight against antibiotic resistance.