Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2018 Sep 19;46(16):e98. doi: 10.1093/nar/gky496.
The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.
最近发现并开发了 RNA 引导的酶，可用于靶向核酸的可编程切割，这为治疗和生物技术应用带来了令人兴奋的可能性。然而，仍然存在一些关键挑战，例如昂贵的向导 RNA 和无法预测目标识别的效率，尤其是对于高度结构化的 RNA。在这里，我们介绍了一种基于芽殖酵母 Argonaute (AGO) 的可编程 RNA 限制酶，它由具有成本效益的 23 个核苷酸 (nt) 单链 DNA 作为向导编程。DNA 向导具有可以轻松设计和购买多种序列的优势，从而能够进行高通量筛选以鉴定目标 RNA 中的最佳识别位点。使用此 DNA 诱导的切割复合物 (DISC) 编程了 11 个不同的指南 DNA，这些 DNA 设计用于跨越序列，在 352nt 的人类免疫缺陷病毒 1 型 5'-非翻译区中鉴定了切割位点。该测定方法与引物延伸和毛细管电泳相结合，允许在单个反应中同时检测和相对定量所有 DISC 切割位点。DISC 切割和 RNase H 切割的比较表明，DISC 不仅切割溶剂暴露的位点，而且还切割 DISC 结合后变得更容易接近的位点。这项研究证明了 DISC 系统在可编程切割高度结构化、功能性 RNA 方面的优势。
