Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform

The vast datasets generated by next generation gene sequencing and expression profiling have transformed biological and translational research. However, technologies to produce large-scale functional genomics datasets, such as high-throughput detection of protein-protein interactions (PPIs), are still in early development. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform featured with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRETⁿ, which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRETⁿ in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of Hippo signaling pathway. The Hippo pathway profiling identified two unreported PPIs in addition to confirming the known ones, suggesting new mechanisms for regulation of Hippo signaling pathway. Our BRETⁿ biosensor platform with uHTS capability is expected to accelerate the systematic PPI network mapping and PPI modulator-based drug discovery. (Publication Abstract)