Abstract
Liquid-liquid phase separation of protein and RNA complexes into biomolecular condensates has emerged as a ubiquitous phenomenon in living systems. These protein-RNA condensates are thought to be involved in many biological functions in all forms of life. One of the most sought-after properties of these condensates is their dynamical properties, as they are a major determinant of condensate physiological function and disease processes. Measurement of the diffusion dynamics of individual components in a multicomponent biomolecular condensate is therefore routinely performed. Here, we outline the experimental procedure for performing in-droplet fluorescence correlation spectroscopy (FCS) measurements to extract the diffusion coefficient of individual molecules within a biomolecular condensate in vitro. Unlike more common experiments such as fluorescence recovery after photobleaching (FRAP), where data interpretation is not straightforward and strictly model dependent, FCS offers a robust and more accurate way to quantify biomolecular diffusion rates in the dense phase. The small observation volume allows FCS experiments to report on the local diffusion coefficient within a spatial resolution of <1 μm, making it ideal for probing spatial inhomogeneities within condensates as well as variable dynamics within subcompartments of multiphasic condensates.
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Acknowledgments
The authors acknowledge the financial support from the National Institute of General Medical Sciences (R35 GM138186) and the National Institute on Aging (R21 AG064258) to P.R.B.
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Alshareedah, I., Banerjee, P.R. (2023). Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy. In: Zhou, HX., Spille, JH., Banerjee, P.R. (eds) Phase-Separated Biomolecular Condensates. Methods in Molecular Biology, vol 2563. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2663-4_9
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DOI: https://doi.org/10.1007/978-1-0716-2663-4_9
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