测量由液-液相分离形成的隔室内功能性RNA的活性和结构。
Measuring the activity and structure of functional RNAs inside compartments formed by liquid-liquid phase separation.
作者信息
Poudyal Raghav R, Meyer McCauley O, Bevilacqua Philip C
机构信息
Department of Chemistry, Pennsylvania State University, University Park, PA, United States; Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, United States.
Department of Biochemistry, Microbiology, and Molecular Biology, Pennsylvania State University, University Park, PA, United States; Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, United States.
出版信息
Methods Enzymol. 2021;646:307-327. doi: 10.1016/bs.mie.2020.06.010. Epub 2020 Jul 10.
Liquid-liquid phase separation (LLPS) has been known to drive formation of biomolecular compartments, which can encapsulate RNA and proteins among other cosolutes. Such compartments, which lack a lipid membrane, have been implicated in origins of life scenarios as they can easily uptake and concentrate biomolecules, similar to intracellular condensates. Indeed, chemical interactions that drive LLPS in vitro have also been shown to lead to similar sub-cellular compartments in vivo. Here we describe methods to prepare compartments formed by complex coacervates, which are driven by LLPS of oppositely-charged polyions, and to probe the structures and functions of RNAs in them. These methods can be adapted to study RNA biochemistry in compartments formed by diverse artificial and biological macromolecules.
已知液-液相分离（LLPS）可驱动生物分子隔室的形成，这种隔室能够包封RNA、蛋白质及其他共溶质。此类缺乏脂质膜的隔室与生命起源假说相关，因为它们能够像细胞内凝聚物一样轻松摄取并浓缩生物分子。实际上，在体外驱动LLPS的化学相互作用在体内也已被证明会导致形成类似的亚细胞隔室。在此，我们描述了制备由复合凝聚层形成的隔室的方法，这些隔室由带相反电荷的聚离子的LLPS驱动，并对其中RNA的结构和功能进行探测。这些方法可用于研究由各种人工和生物大分子形成的隔室中的RNA生物化学。
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