Morphology-coupled formation and reversible gating of membrane channels in synthetic cells using reconfigurable DNA nanorafts.

Membrane reshaping and channel formation often emerge from the coordinated interplay between membranes and their associated proteins. Reconstituting such integrated behaviors in synthetic systems remains challenging, especially when seeking reversible and programmable control. Here we present a protocol for the construction and application of reconfigurable DNA nanorafts that couple membrane morphology modulation with the formation of large, gated membrane channels in synthetic cells. These nanorafts are DNA origami structures that are functionalized with cholesterol anchors and undergo reversible shape transformations. Upon membrane binding, conformational changes drive their self-arrangement into locally ordered domains that deform giant unilamellar vesicles (GUVs). During GUV shape recovery, aided by protein nanopores, the nanorafts interact with the membrane to form synthetic channels capable of transporting large biomolecules (~70 kDa). These channels can be reversibly sealed, allowing programmable control over membrane permeability. Unlike conventional DNA-based nanopores that require pre-assembly and membrane insertion, this protocol supports stepwise, membrane-coupled channel formation with reversible gating. The protocol details the preparation of DNA nanorafts, membrane-bound conformational control, GUV formation, membrane remodeling, morphology-coupled large channel formation and sealing, as well as quantitative fluorescence microscopy assays to analyze vesicle morphology changes and cargo transport. Standard DNA nanotechnology tools and fluorescence microscopy techniques are sufficient to perform the workflow, which can be completed in ~4 d. The system's modularity makes it broadly applicable for constructing artificial cellular systems with programmable structure and function.