PROJECT SUMMARY Lipid-bilayer membranes form diverse and dynamic structures in cells to serve vital functions including nutrient uptake, waste managements, signal transduction, and so on. Understanding the molecular mechanisms by which the cell generates and changes its membrane structures has been a central task of cell biology. It is well established that the physical and chemical properties of membranes regulate their interactions with proteins, which in turn shape the membrane landscape. However, there are still major knowledge gaps regarding how proteins act upon different membrane curvature and tension, especially when the membrane structure and/or the membrane-protein interaction are transient. Cell-free systems using reconstituted membranes provide a powerful method to study such intricate molecular interplays. However, there is still a pressing need for a precisely engineered platform that (1) exquisitely controls the geometrical, biochemical and mechanical properties of membranes and (2) easily allows for biochemical and structural characterization of membrane- associating proteins. Using DNA nanotechnology, a bottom-up method that generates three-dimensional molecular assemblies with programmable shape, stiffness, chemical modification, and motion, we propose to build nanoengineered membranes to bridge the technical gap in membrane manipulation and to unravel the mechanisms of protein-mediated membrane dynamics. Specifically, we will develop tools to (1) generate accessible membranes with complex shapes for the quantitative study of curvature-dependent protein- membrane interactions, (2) build liposomes and lipid nanodiscs with dynamically tunable tension to study the role of membrane tension in regulating protein conformation and lipid transport, and (3) create transmembrane nanopores with tunable size and chemical selectivity for the reconstitution of organelle-like compartments. We expect these new tools to be enabling and potentially transformative for research in structural biology, biophysics, mechanobiology, and synthetic biology.