Geometry-DefinedMembrane Fusion in a DNA FrameworkNanoreactor

Abstract Membrane fusion is essential yet has resisted systematic dissection because transient intermediates, heterogeneous vesicle populations, and the absence of defined reaction units obscure direct relationships among structure, dynamics, and fusion outcomes. Here, we introduce a DNA framework vesicle (DFV) nanoreactor that confines membrane interactions within nanoscale reaction windows, transforming membrane fusion into a geometry-defined and quantitatively resolvable process. By embedding lipid membranes and SNARE proteins within defined DNA apertures, DFVs convert stochastic vesicle encounters to interface-defined fusion reactions. Cryo-electron microscopy resolves a complete sequence of six fusion intermediates, while fluorescence kinetics and nanoflow cytometry provide event-resolved quantification of fusion dynamics. Fusion efficiency and multiplicity are governed by intermembrane distance, reaction interface geometry, and vesicle stoichiometry, establishing a quantitative relationship between nanoscale spatial constraints and fusion pathways. Extending this strategy to living cells, DFVs mediate SNARE-dependent surface fusion with VAMP2-expressing membranes and enable the direct cytosolic delivery of functional siRNA through fusion-driven transfer. Together, these results establish DNA framework nanoreactors as a general strategy for stabilizing, quantifying, and engineering membrane fusion, providing a chemical framework for interrogating complex membrane-associated interfacial processes.