A Biosensor Platform for Detecting the Dissipation of Transmembrane Gradients in Single Liposomes

Pore forming proteins are a diverse collection of polypeptides, which share little structural or amino acid sequence homology and span several mechanistic classes. Their commonality lies in their ability to create transmembrane pores in biological membranes, which places some pore forming proteins among nature's most toxic substances. Such membrane pores can dissipate chemical and electrical gradients, release cellular contents, and even deliver toxic cargo. Detecting pore forming activity commonly relies on dye release assays, which measure a change in brightness as quenched dyes are diluted. Single molecule detection provides the ultimate sensitivity, but measuring relative brightness is challenging due to intensity variation across the population. An ideal sensor could allow interrogation of the entire population of liposomes after pore formation without requiring foreknowledge of the initial intensity. To achieve this we have developed a FRET biosensor approach using ligand-responsive oligonucleotides, which are encapsulated within liposomes that sustain chemical gradients. We show that dissipation of transmembrane gradients can be measured with single liposome resolution using TIRF microscopy, which allows detection of pore forming proteins regardless of mechanistic class. Our encapsulated oligonucleotide biosensors could detect the presence of Botulinum neurotoxin down to picomolar concentrations without the need for protein-specific immunoreagents and highlighted the role of proteolytic activation in pore formation by the toxin. Adapting this approach to additional oligonucleotide sensors would provide a general platform to detect transmembrane solute movement and dissect the underlying transport mechanisms.