Precise Capture of Membrane Proteins Using DNA‐Origami‐Constrained Nanodiscs

Membrane proteins play a key role as cellular gatekeepers, regulating the flow of information and material across lipid membranes. Engineered, membrane‐bound biological nanosystems similarly require membrane proteins along with the molecular tools capable of placing individual copies of them with nanometer‐scale precision. To address this, we have developed DNA‐origami‐constrained nanodiscs (DOC‐NDs) by integrating nanodisc technology with DNA origami. DOC‐NDs allow for efficient capture of membrane protein‐loaded nanodiscs inside a 46 nm DNA origami ring while maintaining unobstructed physical access to the captured protein. Here, we present efficient methods for the preparation of cargo‐loaded DNA‐modified capture‐ready nanodiscs and show their capture by DOC‐NDs using atomic force microscopy (AFM) and cryogenic electron microscopy (cryo‐EM). Additionally, we show a proof‐of‐concept advanced DOC‐ND variant that provides a first step toward control of the orientation of the membrane protein relative to the DNA origami ring. We envisage that DOC‐NDs will support the development of next‐generation systems where membrane proteins can not only be precisely positioned for biochemical and structural analysis but also delivered to target lipid bilayers to tailor permeability of synthetic cells and their compartments.