Prying into biomolecular condensates using single-droplet surface-enhanced Raman spectroscopy

We introduce a highly sensitive methodology that captures key vibrational signatures using surface-engineered, plasmonic, metal nanoparticles spontaneously encapsulated into protein liquid droplets allowing us to unveil the inner workings of biomolecular condensates in a droplet-by-droplet manner.
Published in Chemistry

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Living cells contain a host of non-canonical membrane-less organelles that are formed via liquid-liquid phase separation (LLPS) of intrinsically disordered proteins/regions (IDPs/IDRs) along with nucleic acids and other biomolecules1,2,3. These biomolecular condensates are involved in a myriad of critical cellular functions and neurodegenerative diseases4. Unmasking the role of intrinsic disorder and conformational heterogeneity of IDPs/IDRs in promoting promiscuous and ephemeral interactions resulting in liquid-like behavior of these condensates is crucial to understanding the molecular drivers of LLPS5,6. While a host of existing microscopic and spectroscopic tools are beneficial for studying LLPS, most of these methodologies are inadequate in illuminating the conformational heterogeneity and distribution within individual droplets. For instance, microscopic tools such as confocal, super-resolution, and high-speed atomic force microscopy can directly probe the properties within individual liquid droplets7. However, these tools do not allow us to access the wealth of molecular information in a residue-specific manner. In contrast, the high-resolution structural methods such as nuclear magnetic resonance and small-angle X-ray scattering can provide atomic-resolution details of the condensed phase architecture but are inadequate in yielding molecular insights from individual droplets7.

We aimed to develop a methodology that combines the capabilities of vibrational spectroscopy and optical microscopy that can illuminate the unique molecular details of the polypeptide chains with unprecedented sensitivity within the mesoscopic liquid condensed phase at the single-droplet resolution. However, the low Raman scattering cross-section of proteins makes the recording of vibrational signatures under physiological conditions in aqueous solutions extremely challenging8. Additionally, the high laser power and magnifications required for Raman spectroscopic detection can lead to laser-induced damage, which can be detrimental to soft biological samples. In order to overcome these limitations, we have developed and adapted a novel, highly sensitive, single-droplet vibrational tool involving dispersive laser Raman spectroscopy in a microscopy format that offers a wealth of molecular information within the mesoscopic liquid condensed phase. We utilized surface-engineered, plasmonic metal nanoparticles to illuminate the inner workings of phase-separated mesoscopic liquid droplets. The near-field plasmonic enhancement by metallic nanostructured substrates gives 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 power9.

Figure. A schematic of single-droplet surface-enhanced Raman spectroscopy (SERS): We showed that the surface-modified silver nanoparticles get spontaneously encapsulated within the protein-rich condensed phase and generate plasmonic hotspots resulting in the enhancement of Raman signals.

In this direction, we engineered and prepared negatively charged iodide-modified silver nanoparticles to unveil the inner workings of mesoscopic liquid droplets of ALS-associated Fused in Sarcoma (FUS) in the absence and presence of RNA. The electrostatic interaction between the positively charged RNA-binding domain (RBD) of FUS and the negatively charged surface-coated plasmonic nanostructures leads to the spontaneous encapsulation of these SERS substrates within FUS droplets with an enhancement in the order of ≥ 104 in our SERS experiments (Figure). Adsorption of the plasmonic substrate to the structured C-terminal RBD of FUS leads to significant enhancement of arginine residues and the structured domains, primarily α-helices. Both single-droplet normal Raman and highly sensitive single-droplet SERS experiments elegantly illuminate the conformational landscape within liquid droplets of FUS. We could capture the conformational heterogeneity and intrinsic disorder of FUS within droplets with a conformationally restricted environment around several aromatic amino acid residues such as tyrosine and tryptophan residues in the condensed phase hinting at intermolecular π-π and/or cation-π interactions within the condensates.

Given the potential of single-droplet SERS, we decided to apply this method to elucidate the effect of RNA on the chain conformations within the droplets. Phase transition of FUS is well-known to be modulated by RNA-protein stoichiometry10. We showed that FUS binds stoichiometrically to RNA, which can be further used to estimate the stoichiometry of other heterotypic biomolecular condensates of proteins and nucleic acids. The negatively charged phosphate backbone of RNA competes with the plasmonic nanostructures for interaction with the C-terminal domain of FUS that modulates the polypeptide orientation on the SERS substrate surface. These changes in the orientation of the polypeptide chain cause a reduction in the enhancement of the arginine residues and changes in the intensities of several vibrational modes associated with aromatic residues. Interestingly, our SERS experiments showed that the ordered C-terminal RBD undergoes partial unwinding in the presence of RNA that can promote homotypic (protein-protein) and heterotypic (protein-RNA) interactions within the condensed phase. This unraveling of the structural domain increases the chain disorder at the expense of α-helical content within the droplets.

In summary, we have developed a unique single-droplet surface-enhanced Raman scattering (SERS) methodology that can serve as a potent tool to discern the key molecular interactions and residue-specific structural information within the condensed phase, which can potentially discern the mechanism and regulation of condensate assembly and dissolution. Thoughtful engineering of these SERS substrates using different surface functionalities and other metals can enhance a unique set of vibrational bands that can be utilized for ultra-sensitive detection, characterization, and quantification of a wide range of biomolecular condensates involved in physiology and disease.


  1. Alberti, S. & Hyman, A. A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.  Rev. Mol. Cell Biol.22, 196–213 (2021).
  2. Lyon, A. S., Peeples, W. B. & Rosen, M. K. A framework for understanding the functions of biomolecular condensates across scales.  Rev. Mol. Cell Biol.22, 215–235 (2021).
  3. Forman-Kay, J. D., Kriwacki, R. W. & Seydoux, G. Phase separation in biology and disease.  Mol. Biol.430, 4603–4606 (2018).
  4. Gomes, E. & Shorter, J. The molecular language of membraneless organelles.  Biol. Chem.294, 7115–7127 (2019).
  5. Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell174, 688–699 (2018).
  6. Vernon, R. M. et al. Pi-Pi contacts are an overlooked protein feature relevant to phase separation. eLife7, e31486 (2018).
  7. Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell176, 419–434 (2019).
  8. Rygula, A. et al. Raman spectroscopy of proteins: a review.  Raman Spectrosc.44, 1061–1076 (2013).
  9. Langer, J. et al. Present and future of surface-enhanced Raman scattering. ACS Nano.14, 28–117 (2020).
  10. Sanders, D. W. et al. Competing protein- RNA interaction networks control multiphase intracellular organization. Cell181, 306–324 (2020).

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