1] Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. [2].
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
Nat Methods. 2014 Jun;11(6):683-8. doi: 10.1038/nmeth.2970. Epub 2014 May 8.
RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.
RNA 与蛋白质的相互作用在基因调控中起着至关重要的作用，但目前缺乏大规模定量分析这些相互作用的方法。我们通过改造高通量 DNA 测序仪，开发了一种高通量测序-RNA 亲和力分析（HiTS-RAP）检测方法，用于定量分析荧光标记蛋白与锚定在测序 cDNA 模板上的数百万个 RNA 的结合情况。使用 HiTS-RAP，我们测量了 GFP 结合和 NELF-E 结合适体突变文库对其各自靶标的亲和力，并确定了相互作用的关键区域。突变对 NELF-E 结合适体的亲和力具有累加效应，其相互作用主要取决于单链 RNA 基序，而 GFP 适体的亲和力则不然，其相互作用主要取决于二级结构。
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