Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing.
Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing.
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DOI：
10.1016/j.ymeth.2017.03.002
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发表时间：
2017-04-15
期刊：
影响因子：
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通讯作者：
Harris ME
中科院分区：
文献类型：
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作者：
Jankowsky E;Harris ME
为了在生物环境中发挥作用，RNA结合蛋白（RBP）必须区分RNA中的替代结合位点。这种区分可以发生在RNA-蛋白质结合反应的基态，在其过渡态，或在两者中。RBP在这些反应状态下的区别程度定义了RBP特异性景观。在这里，我们描述了HiTS-Kin和HiTS-EQ技术，该技术将联合收割机动力学和平衡结合实验与高通量测序相结合，以定量评估大量底物变体在RNA-蛋白质结合反应的基态和过渡态的底物区分。我们讨论了实验设计，实际考虑和数据分析，并概述了HiTS-Kin和HiTS-EQ的组合如何允许RBP特异性景观的映射。
To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes.