Bifunctional DNA duplexes permit efficient incorporation of pH probes into liposomes

Enzyme-mediated proton transport across biological membranes is critical for many vital cellular processes. pH-sensitive fluorescent dyes are an indispensable tool for investigating the molecular mechanism of proton-translocating enzymes. Here, we present a novel strategy to entrap pH-sensitive probes into the lumen of liposomes that has several advantages over soluble or lipid-coupled probes. In our approach, the pH sensor is linked to a DNA oligomer with a sequence complementary to a second oligomer modified with a lipophilic moiety that anchors the DNA conjugate to the inner and outer leaflet of the lipid bilayer. The use of DNA as a scaffold allows subsequent selective enzymatic removal of the probe in the outer bilayer. The method shows a high yield of insertion and is compatible with reconstitution of membrane proteins via different methods. Using two large membrane protein complexes the usefulness of the conjugate for time-resolved proton pumping measurements is demonstrated.