Advanced surface passivation for high- sensitivity studies of biomolecular condensates

• 成果类型: Article
• 署名作者: Yao, Run - Wen; Rosen, Michael K.
• 署名单位: University of Texas System; University of Texas Southwestern Medical Center; Howard Hughes Medical Institute
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9789
• DOI: 10.1073/pnas.2403013121
• 发表日期: 2024-05-20
• 关键词: adsorption; chromatin

摘要

Biomolecular 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, broadly 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 multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long - time fluorescence recovery after photobleaching 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.