PROJECT SUMMARY Membrane proteins (MPs) are molecules that can be found in membranes on the surface and the inside of all cells. They enable vital cellular functions such as transport of water, salts and nutrients across the membranes, sensing of the chemical and physical environment of the cell, communication between cells, cell adhesion and energy conversion. MPs play a role in every physiological and infectious disease and 60% of all FDA approved drug molecules target them. To understand how exactly these proteins function, what role they play in different diseases, or to simulate in a computer how new potential drugs would interact with MPs, the exact molecular structures of the MPs need to be discovered first. As MPs are naturally embedded in lipid membranes, they are not soluble in water and it is therefore much more challenging to solve their molecular structures compared water-soluble proteins. Consequently, the molecular structures of less than 100 out of ~8,000 human MPs are known. This proposal will provide new DNA-based tools that will overcome many of these challenges for MP structure determination. For this, DNA molecules without a genetic function are chemically synthesized and self-assembled into ring-shaped DNA nanostructures. These rings can then be filled with lipids and MPs, thus making MPs soluble in water. Moreover, these DNA-lipid nanodiscs provide a native cell-membrane-like environment for the MP, which is important to keep MPs in their native physiological state. By taking advantage of the programmable nature of chemical DNA synthesis, and self-assembly, the size, chemical and physical properties of these nanodiscs can be controlled with a precision and ease that alternative technologies do not provide. This will be particularly useful for solving the structures of small MPs or mechanosensitive MPs, which are actuated by molecular forces and stress in cell membranes. It is expected that the DNA-based molecular tools from this research will overcome current obstacles for MP structure determination and provide functionalities that current molecular tools cannot offer. This research will therefore enable discoveries in structural biology, pharmacology and virology, and thereby enhance the understanding and treatment of MP-associated diseases.