Visualization of Protein-Protein Interaction In Situ Using Hybridization-Enhanced Proximity Ligation Assay.

Abstract

Understanding protein-protein interactions (PPIs) is critical for elucidating cellular functions and disease mechanisms. We present the hybridization-enhanced proximity ligation assay (HPLA), a novel approach incorporating a prehybridization step to improve the ligation efficiency of DNA probes on antibody-oligonucleotide conjugates. This step involves a splint probe hybridizing with complementary sequences on the DNA probes bound to interacting protein complexes, forming V-shaped overhangs that facilitate the circularization of two circle probes. These circularized DNA templates undergo localized rolling circle amplification, producing amplification products that can be visualized as fluorescent dots upon hybridization with dye-labeled detection probes. We demonstrated the utility of HPLA in various biological contexts, including cancer-associated protein interactions and signaling pathways, achieving clearer, more precise visualization of PPIs and enabling robust quantitative analysis.