Tuning Membrane Binding and Lipid Phase Preference of DNA Nanostructures via Synergistic Multi‐Lipidation

Lipophilic anchors are essential biomolecular modifications that mediate membrane‐dependent functions by tethering associated biomolecules, such as peripheral proteins, to the lipid bilayer. Inspired by natural multi‐lipidation systems, we introduce a DNA origami platform functionalized with lipophilic anchors spanning a wide hydrophobicity range to systematically investigate how anchor hydrophobicity governs membrane binding and lateral organization. By varying anchor type, number, and spatial arrangement, we evaluated the interactions of anchor‐modified DNA origami across homogeneous and phase‐separated giant unilamellar vesicles (GUVs). On homogeneous lipid membranes, stable attachment required a minimum per‐anchor hydrophobicity (cLogP ≈ 5) below which binding of origami to the membrane was ineffective regardless of anchor type or number. However, combining weak and strong anchors on a single nanostructure synergistically enhances binding through cooperative effects that exceed additive contributions of either anchor type. On phase‐separated GUVs, our combined‐anchor system revealed that anchor molecular identity and intrinsic hydrophobicity regulate membrane partitioning. Notably, weak anchors drive the nanostructure localization from the liquid‐ordered (Lo) to the liquid‐disordered (Ld) phase, with this shift inversely related to the hydrophobicity of the strong anchor. Collectively, our results establish design principles for multi‐anchor strategies enabling tunable membrane binding and controlled phase‐specific localization of DNA nanostructures.