一种结合SELEX与二代测序技术筛选RNA结合蛋白特异识别序列新方法的建立

Screening specific recognition motif of RNA-binding proteins by SELEX in combination with next-generation sequencing technique

摘要:

RNA结合蛋白通过特异识别RNA底物发挥重要的生物学作用。指数富集的配体系统进化 (Systematic evolution of ligands by exponential enrichment，SELEX) 技术是一种体外筛选核酸底物的基本方法，SELEX技术通过重复多轮筛选从随机核酸序列库中筛选出特异性与靶物质高度亲和的核酸底物，本研究将利用该技术与二代高通量测序 (NGS) 相结合，体外合成含有20个随机碱基的RNA文库，将所要研究的蛋白构建到带有可被链亲和酶素磁珠捕获的SBP标记的载体上去，显著提高筛选效率，仅需1轮筛选即可获得所需RNA底物motif。通过该方法获得了人的hnRNP A1的UP1结构域特异识别AGG和AG二种RNA序列，并通过EMSA实验证实其可以与获得的RNA motif结合。这一方法的建立对于研究RNA结合蛋白识别底物的序列特异性，并进一步了解其在生物体内的调控机制有重要意义。

Abstract:

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.