发展含有DBN-G-clamp化学修饰的RNA链间交联新技术并应用于microRNA靶标的鉴定

microRNA是调节基因表达以及控制细胞命运和分化极其重要的参与者。而要深入了解miRNA的生物学功能，必须确定单个miRNA的RNA靶标。目前已知的捕获miRNA-RNA靶标相互作用的方法存在着交联效率低，以及难以捕获瞬时的miRNA-RNA相互作用的缺点，因此可能会错失部分潜在的miRNA靶点。因此申请人提出了一种新的技术，该技术基于含有DBN-G-clamp修饰碱基的miRNA核酸探针进行链间共价光交联和Click反应，在生理环境下捕获与其相互作用的RNA靶点。通过高通量测序揭示细胞内miRNA与其靶标的结合模式以及展开调控机制的研究。该方法的建立有潜力为细胞内单个特定miRNA鉴定其作用RNA靶标提供有效的工具。申请人将利用该化学捕获方法，将其应用于在白血病中发挥重要功能的miR-17未知靶标的鉴定。此外，该方法有潜力适用于任何反式非编码RNA的RNA靶标识别。

micro-RNAs, have become extremely important players in regulating gene expression and in controlling cell fate and differentiation. miRNAs are genome-encoded RNA transcripts that are processed and assembled into ribonucleoprotein complexes (micro-RNPs) and bind to the 3 'UTR of specific target RNAs to regulate their translation. In order to understand how miRNAs impact biology, it is essential to identify RNA targets for individual miRNAs. The currently methods for capturing miRNA-target interactions have the disadvantages of low cross-linking efficiency and difficulty in capturing transient miRNA-RNA interactions, so some potential miRNA targets may be lost. We proposed to develop a novel method that has the potential to identify RNA and potential non-coding RNA targets for individual, specific miRNAs in mammalian cells based on an interstrand photo-cross-linking chemistry containing DBN-G-clamp modified bases. This method covalently traps a specific miRNA to its mRNA or non-coding RNA targets. The trapped miRNA-target RNA molecules are then affinity purified to enable RT-PCR and high-throughput sequencing of the target RNAs. Because miRNA has been demonstrated to be important in a large number of cellular processes, including cancer development and progression, we will take advantage of this chemical trapping method and apply it to identify the targets of miR-17 that play important roles in Leukemia and breast cancer cells. Furthermore, this method can be potentially applicable for RNA target identification of any trans-acting non-coding RNAs.