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Abstract

Membrane-binding and self-organization of scaffolding proteins, e.g. Bin/Amphiphysin/Rvs (BAR) domain family, mediates and regulates the shape transformations of cell membranes. The quantitative characterization of the interaction of synthetic scaffolds with lipid membranes in defined conditions will greatly add to our understanding of the fundamental physical principles driving membrane-shaping phenomena. In this work, DNA origami technology was used to create elements that bear physical features of scaffolding proteins and controllably shape lipid membranes. First, I determined the requirements for efficient membrane-binding of highly negatively charged DNA nanostructures. I show that cholesteryl-anchors positioned close to the bulky DNA nanostructures are locally hindered, and that multiple cholesteryl-anchors or DNA spacers can enhance membrane binding. Fluorescence correlation spectroscopy (FCS) data demonstrates that both the number and type of DNA spacer determine the interaction of DNA nanostructures with lipid bilayers. In addition to bilayers, we established conditions for FCS experiments investigating macromolecule-lipid monolayer interactions. Studies of the self-organization of DNA origami nanostructures on lipid membranes using high speed atomic force microscopy (HSAFM) revealed that both tip-to-tip and side-by-side interactions, and the resulting anisotropic domains, are observed on lipid membranes for per se purely repulsive DNA nanostructures. The preferred type of interaction depends on the particle surface density and thus membrane tension. I also demonstrate the use of DNA nanostructures for the study of isotropic-anisotropic phase transition of particles of different aspect-ratios in 2D. Last, we employ DNA nanostructures to shape lipid membranes, mimicking the function of scaffolding proteins. We show that curved DNA scaffolds tubulate lipid membranes, resembling not only the shape but also the action of BAR proteins. Lipid membrane deformation was correlated to the DNA scaffolds’ curvature and membrane density. To dynamically control membrane shaping, I present the design of a three-state DNA origami structure that can be switched from its passive to its active membrane-shaping conformation. A complementary approach of oxDNA molecular dynamic simulations (MD simulations) and transmission electron microscopy (TEM) imaging was used to optimize the design.