PROJECT SUMMARY Deciphering the complex underpinning of brain structure and function requires a complete understanding of how molecular contacts between cells in the brain are regulated. Currently, there are a lack of tools to measure these intercellular protein-protein interactions (PPIs) with requisite sensitivity, precluding a complete understanding of endogenous regulatory mechanisms. Moreover, as intercellular contacts are guided by thousands of diverse PPIs, it is critical to be able to study multiple interactions simultaneously, which is presently not possible. Here, we propose to develop a completely new category of split protein-based biosensors using RNA polymerases (RNAPs). The concept is that an RNAP-based intercellular detection strategy should offer substantial improvements in sensitivity, due to the signal amplification made possible by nucleic acid amplification technologies, and multidimensionality, due to the capacity of orthogonal RNAPs to drive distinguishable RNA output signals. In preliminary data, we show an evolved split RNAP that can detect target PPIs in vivo with more than 1,200-fold dynamic range, and also show in proof-of-concept experiments that two interactions can be monitored simultaneously. We propose to develop the split RNAP platform into a new set of tools, reagents, and protocols for the study of intercellular PPIs, through the completion of three aims. First, we will transition the binary PPI analysis platform to mammalian cells for intercellular PPI detection, using the well-studied neurexin (NRX) and neuroligin (NLG) interaction as a model system. Second, we will expand the scope of detection by evolving a panel of orthogonal split RNAPs for the detection of at least four simultaneous PPIs. In addition, we will develop new imaging techniques to quantify the interactions using super-resolution imaging platforms, both for binary and multidimensional PPI detection. Finally, we will test and showcase the capabilities of the system by interrogating the “NRX interactome” using coculture experiments with primary neurons and engineered reporter cells. During the course of completing all of these steps, we will compare our technologies to the most successful split protein reporters, with metrics including sensitivity, dynamic range, and resolution as the primary attributes. Together, completion of this work will lay the foundation for a completely new approach for interrogating the intercellular interactions that guide synapse formation and brain function. We will make our new tools and protocols widely available to the broader community, accelerating and improving the work done within the BRAIN initiative. Through collaborations with other BRAIN initiative researchers, we will ultimately deploy our new technologies to uncover the complex code that guides neural coding in intact brain circuits.

PROJECT NARRATIVE Measuring the contacts between cells that control brain function with specificity, resolution, and sensitivity, requires new detection methods. In this BRAIN Initiative project, we will develop a new technology to study the intercellular protein-protein interactions that control synaptic formation and signaling. This will involve the development of new split RNAP biosensors to visualize intercellular contacts, ultimately providing a pathway toward uncovering the molecular basis of neural connectivity, brain function, and cognition.