Label-Free Fluorescence Microscopy Reveals Multiphase Organization in Biomolecular Condensates

Abstract Phase transitions of proteins and nucleic acids (NA) leading to the formation of biomolecular condensates have been linked to various biological functions. Given the growing number of proteins/NA predicted to undergo liquid-liquid phase separation (LLPS), efficient tools to investigate this behavior are critical to advancing our understanding of biomolecular condensate function. The current standard used to study LLPS involves techniques that utilize exogenous fluorophore labels. The labeling process is often costly and time-consuming and comes with associated complexity that arises from unknown interactions from the bulky fluorescent tags. These aspects limit high throughput analysis of protein/NA phase separation based on external fluorophore labeling. Here, we report the discovery that intrinsic fluorescence, well into the visible spectrum, arises as an emergent property of biomolecular condensates. Leveraging this intrinsic fluorescence, 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. Through this approach, we find that a variety of G-quadruplex DNA readily form droplets with histone H1 and display dynamic exchange. In addition, we directly demonstrate that the 3D morphology, core-shell architecture, and sub-compartmentalization of condensate droplets are tunable via the charge ratio of components in solution and NA hybridization. Our method utilizes an inherent property of condensates, thus is broadly applicable to any phase-separated systems and can advance our understanding of biological phase transition.