DNA branch migration across intact membranes powers an inside-out GPCR analog for lysis-free detection of intracellular RNA with on-membrane amplification

Abstract

Interrogating the sequence-specific DNA/RNA content of a living cell without destroying it, is an elusive goal in biology and diagnostics with transformative potential. A DNA/RNA-based approach, however, must relay the recognition event across the intact lipid bilayer, a step that has long been considered impossible because hydrophilic, massively charged DNA/RNA is incompatible with the hydrophobic membrane interior. Here, we show that DNA branch migration can be driven across a lipid bilayer, converting an intracellular nucleic-acid sequence into an amplified, extracellular optical signal while preserving cell viability and compartmentalization, a synthetic transmembrane signal transduction analogous to natural receptors, such as G-protein-coupled receptors (GPCRs), but operating in reverse. We demonstrate this principle with Hybridization Across Lipid for Oligonucleotide Sensing (HALOS), an amphiphilic DNA hairpin comprising a toehold for recognition, a stem for stability, a loop, and cholesterols for transmembrane anchoring. Upon binding to a sequence-specific nucleic acid target, toehold-mediated strand invasion drives DNA branch migration across the bilayer, switching HALOS from a closed to an open conformation that relays the signal to the opposite face. Molecular dynamics simulations revealed that a cholesterol belt stabilizes the DNA stem within the membrane, preserving the hairpin structure necessary for transmembrane signaling. Notably, this branch migration proceeds despite the kinetic barrier of hybridization through the hydrophobic bilayer interior. By combining HALOS with an isothermal hybridization chain reaction (HCR), we established a platform that enables intracellular nucleic acid target detection and amplified fluorescent reporting from outside synthetic vesicles and live mammalian cells, achieving nanomolar-range sensitivity. These results establish DNA branch migration across lipid membranes as a design principle for programmable lysis-free molecular sensing in intact cells.