A deep dive into biomolecular condensates using single-droplet surface-enhanced Raman spectroscopy

An intense research activity reveals that living cells contain (noncanonical) membraneless organelles that are formed via phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) with nucleic acids and other biomolecules. These biomolecular condensates are involved in a myriad of critical cellular functions and neurodegenerative diseases. Unmasking the role of intrinsic disorder and conformational heterogeneity of IDPs/IDRs in promoting promiscuous and ephemeral interactions resulting in a liquid-like behavior of these condensates is crucial to understanding the molecular drivers of phase separation. While a host of existing microscopic and spectroscopic tools exist for studying phase separation, most of these methodologies are inadequate in illuminating the conformational heterogeneity and distribution within individual droplets. To overcome these limitations, we developed and adapted a novel and highly-sensitive methodology that combines the capabilities of vibrational spectroscopy and optical microscopy that can illuminate the unique molecular details of the polypeptide chains within the mesoscopic liquid condensed phase at the single-droplet resolution. Often low Raman scattering cross-section of proteins makes the recording of vibrational signatures under physiological conditions in aqueous solutions extremely challenging. In this direction, we utilize surface-engineered, plasmonic metal nanostructures giving rise to high electromagnetic/chemical enhancement of Raman signals even at extremely low analyte concentrations that can increase Raman scattering cross-section by several orders of magnitude, allowing single-molecule detection and characterization even at a much lower laser power. Our ultra-sensitive single-droplet SERS methodology captures the exquisite details involving conformational disorder, heterogeneity, and distribution within FUS condensates. Additionally, this method can capture some key structural details within the droplets formed in the presence of varying RNA-protein ratios and highlight RNA-mediated partial unwinding of the ordered domains in the C-terminal RBD that increases polypeptide chain disorder that can promote both homotypic and heterotypic interactions within the condensed phase.