Small amphiphilic DNA for programmable transmembrane signaling and amplification.

Transmembrane proteins such as G-protein coupled receptors (GPCRs) transmit molecular signals across lipid bilayers through stimulus-responsive allosteric mechanisms, where extracellular ligand binding induces G-protein dissociation to initiate downstream signaling. Mimicking such specific signal transduction pathway with DNA nanostructures has remained challenging due to the incompatibility between hydrophilic DNA and hydrophobic membranes, and the difficulty of engineering allosteric DNA reactions across lipid bilayers. Here, we overcome these limitations by exploiting DNA Hybridization Across Lipid for Optical Signaling (HALOS) using an amphiphilic DNA hairpin comprising toehold for recognition, stem for stability, loop, and cholesterols for transmembrane anchoring. Upon binding of a single-stranded nucleic acid target stimuli, strand invasion through the toehold initiates conformational switching in HALOS, enabling signal transduction across membrane. Experimentally, we demonstrate that DNA hybridization across lipid membrane can occur, contradicting the prevailing view that DNA hybridization cannot proceed through hydrophobic barriers. All-atom molecular dynamics (MD) simulations reveal that cholesterol tags stabilize the DNA stem within the bilayer, preserving the hairpin structure necessary for transmembrane signaling. By combining the HALOS with a non-enzymatic isothermal hybridization chain reaction (HCR), we establish a platform that enables intracellular nucleic acid target detection and amplified fluorescent reporting from outside synthetic vesicles and live mammalian cells achieving nanomolar sensitivity. HALOS expands the toolkit for membrane-integrated DNA nanotechnology and opens avenues for lysis-free diagnostics, synthetic cell and biology, and targeted therapeutic activation.