New Methods to Study Biomolecular Condensates

Condensation of proteins and nucleic acids through phase separation into liquid-like droplets (biomolecular condensates) has emerged as an important phenomenon in intracellular organization and regulation of many biochemical processes. Given the growing number of proteins/nucleic acids predicted to undergo liquid-liquid phase separation (LLPS), efficient tools to investigate this behavior are critical to advancing our understanding of biomolecular condensate function. We have discovered that biomolecular condensates have intrinsic fluorescence well into the visible spectrum. Leveraging this property, we study condensate formation, directly measure their internal dynamics via Fluorescence Recovery after Photobleaching (FRAP) and examine the 3D morphology and transitions to various multiphase architectures. As this is a label-free method that utilizes an inherent property of condensates, it is efficient, cost-effective, and broadly applicable to any phase-separated system. Secondly, we have also established a new fundamental approach to quantitatively predict the formation and stability of biomolecular condensates based on single-protein thermodynamics. Applying such thermodynamics-based mechanistic analysis has the potential to guide the development of therapeutic treatments in protein aggregation-driven diseases.