Using DNA Origami to Study Nanoscale Organization of Plasma Membranes

Plasma membrane (PM) lipids and proteins partition into nanodomains that regulate cellular processes by controlling local membrane organization. However, nanodomains' small size and temporal instability hinder their study in living cells. To address this, we built fluorescent DNA origami probes that insert into the PM via lipid anchors displayed on cells. Using DNA origami allows precise control over anchor number and spatial arrangement, enabling nanometer-scale sampling of the PM. Once inserted, probes diffusing across the membrane are followed by single-particle tracking to survey the PM landscape. Varying lipid anchor number and arrangement shows that origami immobilization requires simultaneous interactions with multiple nanodomains. Disrupting the actin cytoskeleton reduced immobilization, confirming its role in nanodomain stability. Moreover, acute cell stretching transiently increases origami mobility, indicating that mechanical forces can reversibly regulate PM nanodomain organization. This novel membrane-integrated DNA origami approach provides mechanistic insights into PM nanodomain architecture and dynamics in living cells.