PROJECT SUMMARY Biomolecular condensates (BMCs) are emerging as a ubiquitous feature of compartmentalization within cells. These structures can form by liquid-liquid phase separation of particular proteins and RNAs from the rest of the cytoplasm or nucleoplasm and can perform important functions (e.g. enzymatic reactions, signaling regulation). BMCs of the RNA-binding protein TDP-43 are hypothesized to nucleate pathological aggregates and lead to neuron death in Amyotrophic Lateral Sclerosis (ALS). However, BMCs are difficult to characterize with traditional techniques because they lack membranes and can dissolve upon dilution and, therefore, are challenging (or impossible) to purify. Additionally, interrogating toxic effects of disease-associated BMCs has been problematic because of the difficulty of controlled induction of liquid-like or solid-like condensates within neurons. Here, we propose the development of a new technology platform, Phase-seq, that combines two approaches from different fields – biophysical in-cellulo reconstitution of protein condensation and genomic mapping of RNA organization – to address current methodological limitations for studying BMCs in neurons. This tool combines optogenetic induction of BMCs in neurons (Corelets) with a cutting-edge genomics approach (SPRITE) to interrogate the RNA compositions and functions of BMCs. First, we will lay the groundwork for Phase-seq by establishing SPRITE in primary neurons to create a genome-wide map of RNA- RNA interactions in these highly specialized cells and confirm that we can identify RNAs within individual BMCs by inducing stress granules. We will then develop models of early and late stage ALS pathology by inducing TDP-43 BMCs in neurons with Corelets, a new technology that allows for controlled optogenetic induction of condensates with tight temporal and spatial resolution. Finally, we will combine SPRITE and Corelets in neurons to create Phase-seq, a novel platform for simultaneous induction of BMCs and characterization of their functions by mapping RNA constituents. These experiments will allow us to gain valuable insight into RNA organization in neuronal BMCs, determine how TDP-43 condensates may lead to neuron death, and identify novel therapeutic avenues for ALS. We anticipate that the Phase-seq platform will be broadly applicable for interrogating the functions of endogenous BMCs in neurons and the consequences of aberrant BMCs in disease.