Quantitative Analysis of RNA Chaperone Activity by Native Gel Electrophoresis and Fluorescence Spectroscopy.
Quantitative Analysis of RNA Chaperone Activity by Native Gel Electrophoresis and Fluorescence Spectroscopy.
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DOI：
10.1007/978-1-0716-0231-7_2
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Woodson SA
中科院分区：
文献类型：
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作者：
Panja S;Małecka EM;Santiago-Frangos A;Woodson SA
不同类型的RNA结合蛋白通过增加RNA碱基配对的速率和通过稳定最终的RNA双链体来陪伴非编码RNA的相互作用。急诊coli蛋白Hfq促进小的非编码RNA与其靶mRNA之间的相互作用。Hfq和其他RNA伴侣的伴侣和RNA退火活性可以通过在蛋白质存在和不存在的情况下测定RNA碱基配对的动力学来评估。本章介绍了使用电泳凝胶迁移率变动分析（EMSA），停流荧光和荧光各向异性测量RNA退火动力学的协议。EMSA成本低，可以解析天然小RNA和mRNA片段的反应中间体，只要复合物在电泳过程中被捕获的时间足够长（≥ 10 s）。停流荧光可以检测1 ms - 30 s之间的退火反应，最适合测量寡核糖核苷酸的快速退火。荧光各向异性报告复合物的物理尺寸，并且非常适合于监测伴侣循环期间RNA与Hfq的缔合和解离。
Diverse types of RNA binding proteins chaperone the interactions of non-coding RNAs by increasing the rate of RNA base pairing and by stabilizing the final RNA duplex. The E. coli protein Hfq facilitates interactions between small non-coding RNAs and their target mRNAs. The chaperone and RNA annealing activity of Hfq and other RNA chaperones can be evaluated by determining the kinetics of RNA base pairing in the presence and absence of the protein. This chapter presents protocols for measuring RNA annealing kinetics using electrophoretic gel mobility shift assays (EMSA), stopped-flow fluorescence, and fluorescence anisotropy. EMSA is low cost and can resolve reaction intermediates of natural small RNAs and mRNA fragments, as long as the complexes are sufficiently long-lived (≥ 10 s) to be trapped during electrophoresis. Stopped-flow fluorescence can detect annealing reactions between 1 ms – 30 s and is best suited for measuring the rapid annealing of oligoribonucleotides. Fluorescence anisotropy reports the physical size of the complex and is well-suited for monitoring the association and dissociation of RNA from Hfq during the chaperone cycle.