Label-free in situ approach for characterizing the macromolecular composition and water content in biomolecular condensates

Biomolecular condensates are cellular organelles that form via liquid-liquid phase separation of proteins and nucleic acids. The functional role of intracelular condensates is known to be tightly coupled to material properties such as viscosity and hydrophobicity, which act as phenomenological markers of cellular state in health and disease. In turn, the material properties of condensates essentially depend on their macromolecular composition and water content. Yet, available approaches for determining condensate composition systematically involve invasive processes that often result in the destruction of the tested sample. Here, we use Raman spectroscopy coupled with spectral phasor analysis, and develop an in situ and label-free approach that allows to resolve molecular concentrations of any chemical species within aqueous polymer solutions and biomolecular condensates. In addition to quantifying the protein-to-water volume ratio, our method allows to quantify the degree of client molecule partitioning inside condensates. We show that water hydrogen bonding resulting from protein hydration can be quantified via this approach, and find that the overwhelming proportion of water molecules within condensates largely retain their bulk-like properties. Lastly, by coupling our characterization approach with environment-sensitive fluorescent dyes we investigate the molecular origin of condensate hydrophobicity. Our results show that the hydrophobicity of condensates is by a combination of the presence of structural motifs in the backbone of polymers constituting the scaffold, and the degree of water partitioning inside condensates.