Inner Leaflet-Anchored Multiplexed DNA Toolkits for Dynamic Tracking and Programmable Regulation of Cell Membrane Functions

Biological processes at the inner membrane leaflet are fundamental to cellular function. Achieving multitargeted tracking and regulation at the cytoplasmic membrane surface critically depends on the ability to deliver multiplexed chemical tools to the inner leaflet that can engage multiple biomolecules with well-defined stoichiometric control. However, efficient incorporation of such tools across the lipid bilayer remains a major challenge. Most studies employing chemical tools such as functional DNA to study membrane-associated activities have focused on the outer leaflet, leaving the inner leaflet largely unexplored. Here, we report the precise integration of multiplexed and functionally diverse DNA toolkits onto the inner membrane leaflet via liposome fusion-based transport (LiFT), enabling dynamic monitoring and programmable manipulation of two key membrane processes: transmembrane transport and signal transduction. Distinct sets of bioorthogonal DNA sequences were incorporated with high efficiency and defined orientation, providing proximity-enhanced resolution for tracking and regulating inner leaflet-associated biological activities. Using membrane-anchored multiplexed sensors, we mapped transmembrane ion dynamics, including proton gradient fluctuations and bidirectional Na+/K+ fluxes, revealing spatiotemporal progression of chemically induced cellular events. Moreover, we report the first DNA-based programmable control of inner leaflet signaling via cytosol-to-membrane protein translocation. Using a sequence-programmable, click-type DNA-enzyme ligation, we showcased multiplexed recruitment of proteins to the inner leaflet and subsequent activation of PI3K/Akt signaling. Finally, we demonstrated that strand–displacement reaction and photolabile chemistry can modulate the inner leaflet signaling cascade in response to bioorthogonal sequence inputs or light irradiation.