Split T7 RNA polymerase biosensors to study multiprotein interaction dynamics.

Protein-protein interactions (PPIs) are involved in nearly all cellular processes. PPIs are particularly crucial for mediating selectivity along signaling pathways. Thus, measuring the competitive interplay between PPIs in a cell is important for both understanding fundamental cellular regulation and developing therapeutics targeting those whose dysregulation is associated with disease. A variety of split protein reporter-based tools are available to measure if two proteins interact within a cell and thereby characterize the general determinants of their interactions. PPIs, however, occur within complex networks facilitated by dynamic biophysical nuances that determine activity and selectivity. Evolved, proximity-dependent split T7 RNA polymerase (RNAP) biosensors have recently been used to perform deep mutational scanning of PPI interfaces, and to create synthetic gene circuits. In this chapter, we present the application of proximity-dependent split RNAP biosensors as a method to measure multidimensional PPIs in live cells. Orthogonal split RNAP "tags" encode each interaction in a unique RNA signal, thereby enabling the study of multiple competitive PPIs in live cells. Each unique RNA signal can be quantified via established RNA analysis methods. Herein, we provide advice and protocols to aid other researchers in using the split RNAP biosensor, focusing primarily on how to detect multiple PPIs in mammalian cells, including their dynamic interplay in the presence of small molecule inhibitors.