Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.
Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.
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使用液滴内荧光相关光谱测量生物分子凝聚物内的蛋白质和核酸扩散系数。
DOI：
10.1007/978-1-0716-2663-4_9
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Banerjee,PriyaR
中科院分区：
文献类型：
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作者：
Alshareedah,Ibraheem;Banerjee,PriyaR
蛋白质和RNA复合体的液-液分离成生物分子凝聚体已成为生命系统中普遍存在的现象。这些蛋白质-RNA凝聚体被认为与所有形式的生命中的许多生物功能有关。这些凝析油最受欢迎的特性之一是它们的动力学特性，因为它们是凝析油生理功能和疾病过程的主要决定因素。因此，对多组分生物分子冷凝物中各组分的扩散动力学的测量是常规的。在这里，我们概述了执行液滴内荧光相关光谱(FCS)测量的实验程序，以提取生物分子凝聚体中单个分子的体外扩散系数。与更常见的实验，如光漂白后荧光恢复(FRAP)不同，FCS提供了一种稳健和更准确的方法来量化稠相中的生物分子扩散速率。小的观测体积使现场控制系统的实验可以报告空间分辨率为<；1μm的局部扩散系数，这使其成为探测凝析油中的空间不均质性以及多相凝析油子舱内的可变动力学的理想工具。
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.