Cholesterol‐DNA Origami Nanostructures for Synthetic Lipid Rafts Induce Early T Cell Activation Signals

ABSTRACT Lipid rafts cluster at the immunological synapse during activation and serve as signaling hubs in T cells. While cholesterol plays a crucial role in lipid raft formation, the impact of their spatial configuration remains less understood. Here, we introduce programmable DNA origami nanostructures, cholesterol nano‐patch (CNP), for nanoscale spatial control of cholesterols on live T cell membranes, enabling us to elucidate their roles in lipid raft formation, receptor activation, and intracellular signaling. We demonstrate that CNPs with high‐density cholesterol arrangements efficiently bind to the T cell plasma membrane and form a large and polarized coalescence, leading to the stabilization of ordered lipid membrane domains by increasing the local concentration of cholesterols. These synthetic lipid rafts colocalize with flotillin‐1, a raft‐associated protein, and promote the membrane reorganization and physical segregation of key signaling molecules, such as T cell receptors, Lck, LAT, and CD45. Consequently, this leads to early T cell activation in the absence of antigenic stimulation. Our DNA origami approach demonstrates the nanoscale arrangement of cholesterol plays significant roles in lipid raft formation, membrane phase separation, and protein reorganization, which are sufficient to induce early T cell activation.