Chen Yu, Mandic Jana, Varani Gabriele
Department of Chemistry and Department of Biochemistry, University of Washington, Seattle WA, USA.
Nucleic Acids Res. 2008 Nov;36(19):e128. doi: 10.1093/nar/gkn559. Epub 2008 Sep 12.
RNA-binding proteins (RBPs) perform many essential functions in the post-transcriptional control of gene expression. If we were able to engineer RBPs with new specificity, it would also become possible to develop new tools to control and investigate gene expression pathways. Molecular evolution methods such as phage display have been introduced to achieve this goal, but the large interface between these proteins and RNA relative to the size of library that can be constructed limits the efficacy of this method. In order to increase the diversity of libraries used for selection of RBPs, we applied the emulsion-based in vitro compartmentalization (IVC) method to select RBPs with defined specificity. A new approach was developed to link genotype and phenotype by fusing the target RBP to zinc finger proteins (ZFPs) that bind to a cognate DNA sequence inserted upstream of the promoter. The expressed fusion protein (ZFP-RBP) binds to its encoding DNA with high affinity via the ZFP target-binding site. After breaking the emulsion, the RBP can be selected based on its affinity for a biotinylated RNA bait. We demonstrate the effectiveness of this method that should enable the selection of RBPs with new specificity or improved affinity.
RNA结合蛋白（RBPs）在基因表达的转录后调控中发挥着许多重要功能。如果我们能够设计出具有新特异性的RNA结合蛋白，那么开发用于控制和研究基因表达途径的新工具也将成为可能。诸如噬菌体展示等分子进化方法已被引入以实现这一目标，但相对于可构建文库的大小而言，这些蛋白质与RNA之间的大界面限制了该方法的效率。为了增加用于选择RNA结合蛋白的文库的多样性，我们应用基于乳液的体外区室化（IVC）方法来选择具有特定特异性的RNA结合蛋白。通过将目标RNA结合蛋白与锌指蛋白（ZFPs）融合，开发了一种将基因型与表型联系起来的新方法，锌指蛋白可与插入启动子上游的同源DNA序列结合。表达的融合蛋白（ZFP-RBP）通过ZFP靶标结合位点与其编码DNA高亲和力结合。打破乳液后，可基于RNA结合蛋白对生物素化RNA诱饵的亲和力来选择该蛋白。我们证明了该方法的有效性，该方法应能够选择具有新特异性或更高亲和力的RNA结合蛋白。
