Interactions between membrane proteins are essential for cell survival and proper function, but the structural and mechanistic details of these interactions are often poorly understood. Even the biologically functional ratio of protein components within a multi-subunit membrane complex—the native stoichiometry—is difficult to establish. We have demonstrated digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors. Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence in a plate reader which in principle is generalizable to any ratio. Similar ‘single molecule in bulk’ experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

Digital nanoreactors for control over absolute stoichiometry and spatiotemporal behavior of receptors within lipid bilayers