A synthetic lipid scramblase built from DNA

The programmable self-assembly facilitated by DNA nanotechnology provides unparalleled capabilities to construct functional devices at the nanoscale. Recently, DNA nanostructures have been developed to interact with biological membranes and serve as artificial counterparts of natural ion channels, or membrane bending, scaffolding, or fusion proteins. In this thesis, we design a synthetic DNA-built enzyme that facilitates rapid lipid mixing between the two leaflets of a lipid bilayer. It thereby mimics the function of yet another class of membrane proteins - lipid scramblases. Characterising this DNA nanostructure with gel electrophoresis, dynamic light scattering, atomic force and transmission electron microscopy, we find that the cholesterol groups required for membrane insertion, also induce clustering. Hence, we establish an easy-to-implement strategy to control hydrophobically mediated aggregation thereby introducing a solution to a common problem of amphiphilic DNA constructs. With a combination of fluorescence microscopy experiments and molecular dynamics simulations, we identify the mechanism behind the scrambling activity. The spontaneous membrane insertion of our DNA scramblase induces a toroidal pore that is lined by the lipid headgroups. This DNA-stabilised pore connects the inner and outer bilayer leaflet thereby facilitating diffusive lipid transport that rapidly equilibrates the membrane’s lipid composition. In good agreement between experiments and simulations, we find the scrambling rate catalysed by our DNA-made enzyme to exceed 10⁷ lipids per second, orders of magnitude faster than natural scramblase proteins. We thereby pioneer the use of self-assembled DNA nanostructures for controlling the lipid composition of biological membranes, opening new avenues for applications of membrane-interacting DNA systems in biophysical research and medicine.