Branched Proximity Hybridization Assay for the Quantification of Nanoscale Protein-Protein Proximity.

To better quantify the nanoscale protein-protein proximity, we developed a new branched proximity hybridization assay (bPHA). In this assay, oligo-coupled target protein-binding reagents, such as aptamer, nanobody, and antibodies, are used to convert the proximity of target proteins to the proximity of a pair of designed oligos. The closely positioned oligo pair then serve as a template for a maximum of 400-fold linear signal amplification through branched DNA (bDNA). The amplified bPHA signal is recorded and quantified by flow cytometer, providing high throughput, multiplexing, and single-cell resolution for nanoscale protein proximity studies.