PROJECT SUMMARY Cell membranes compartmentalize and modulate biochemical reactions and facilitate biomolecule transport by forming diverse and dynamic structures. For a variety of applications ranging from basic biological study of membrane trafficking to biomedical applications such as drug delivery, it is desirable to control membrane structure and dynamics precisely using in vitro methods. This has been difficult in the past due to the lack of versatile, high-precision tools to manufacture and manipulate membranes. Inspired by the protein machineries that scaffold and sculpt membranes, we propose to build DNA nanostructures with well-defined shape and motion as nanoscale mechanical tools for membrane engineering. The idea is to guide the formation and deformation of lipid bilayers using dynamic DNA nanostructures equipped with membrane-interacting molecules, hence transducing the programmable features of the DNA structures to the scaffolded membranes. This proposal builds on our recently demonstrated DNA-nanotechnology enabled membrane engineering methods, and focuses on building an arsenal of precise and versatile tools by designing DNA structures with sophisticated self-assembly and reconfiguration mechanisms. We will also incorporate membrane-remodeling protein complexes into DNA nanoscaffolds and modulate the proteins' collective behaviors. The newly developed toolkit will be tested for their ability to generate desired membrane curvatures of various geometry and dimensions in spatially and temporally controlled manner. We expect the project to (1) establish an adaptable platform for the quantitative study of membrane biophysics, (2) deliver prototype devices for sorting proteins by their membrane-curvature recognition capability, and (3) engender a unique interface between DNA nanotechnology and cell biology.