Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

Abstract

AbstractBiomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, generically applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multi-point attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time FRAP imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.Significance StatementThe understanding of biomolecular condensates has significantly benefited from biochemical reconstitution with microscopy detection. Here, we present a novel surface passivation method utilizing self-assembly of Pluronic F127 on hydrophobic surfaces. This approach not only effectively minimizes non-specific binding without altering the physical properties of the condensates but also offers universal passivation across a variety of condensate systems. It demonstrates high resistance to different treatments and enables condensate immobilization through controlled anchor points. This allows for highly sensitive analytical techniques, including single-molecule imaging. The simplicity and high-performance of this method, coupled with time and cost efficiencies, could facilitate robustness and throughput of experiments, and could broaden the accessibility of biochemical phase separation studies to a wider scientific community.