Biophysical characterization of biomolecular condensates through microfluidic technologies
Biomolecular condensates can form through phase separation when mixtures of pro teins and nucleic acids coalesce to form membrane-less sub compartments within the cell. These condensates have been indicated to be responsible for a range of key physiological and pathological indications in living organisms ranging from energy storage and RNA processing to driving cancer and neurodegenerative diseases. However, key biophysical questions still exist as to how condensates are able to remain stable in cells, how the transition process from individual molecules to micron-scale condensates occurs, and the exact nature of the interactions of multiple heterogeneous components within condensates. Conventional biophysical methods are often limited in their ability to describe the complexity and heterogeneity of the underlying condensate structures and their associated biophysical parameters. In my thesis, I establish and apply approaches to perform biophysical characterization of biomolecular condensates using microfluidic technology. These methods allow us to measure the stability of condensates through single-condensate zeta potential measurements, to probe the interactions between phase separating proteins and specific RNA sequences and other proteins and co polymers, as well as determine the sizes of nanoscale condensate assemblies. Beyond these characterizations I have also helped to developed high-throughput methods for assessing the phase diagram of proteins to understand how environmental factors shift the phase separation boundary. Throughout these studies, by utilizing microfluidic methods I am able to study properties of condensates under flow while allowing for the prevention of surface interactions. Overall, my findings and developments have implications for progressing our understanding of biomolecular condensates and have direct links to the design of therapeutic interventions, which will rely on detailed knowledge of the biophysical parameters defining condensate behavior.