Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA.
Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA.
复制标题
DOI：
10.1093/nar/gky496
复制
发表时间：
2018-09-19
影响因子：
14.9
通讯作者：
Nakanishi K
中科院分区：
文献类型：
--
作者：
Dayeh DM;Cantara WA;Kitzrow JP;Musier-Forsyth K;Nakanishi K
最近鉴定和开发的RNA引导的酶可编程切割的目标核酸提供了令人兴奋的可能性，为治疗和生物技术应用。然而，关键的挑战，如昂贵的向导RNA和无法预测的目标识别的效率，特别是对于高度结构化的RNA，仍然有待解决。在这里，我们介绍了一种可编程的RNA限制性内切酶，基于芽殖酵母Argonaute（AGO），编程具有成本效益的23个核苷酸（nt）的单链DNA作为指导。DNA指导提供的优点是可以容易地设计和购买不同的序列，从而能够进行高通量筛选以鉴定靶RNA中的最佳识别位点。使用这种DNA诱导的切割复合物（DISC），用11种不同的指导DNA编程，设计跨越序列，在352-nt的人类免疫缺陷病毒1型5′-非翻译区的切割位点被确定。该分析，加上引物延伸和毛细管电泳，允许检测和相对定量的所有DISC切割位点，同时在一个单一的反应。DISC切割和RNase H切割之间的比较揭示了DISC不仅切割溶剂暴露位点，而且切割在DISC结合后变得更易接近的位点。这项研究证明了DISC系统用于高度结构化的功能性RNA的可编程切割的优势。
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.
登录
查看更多内容
影响因子：
16
作者：
Olovnikov, Ivan;Chan, Ken;Sachidanandam, Ravi;Newman, Dianne K.;Aravin, Alexei A.
通讯作者：
Aravin, Alexei A.
影响因子：
14.9
作者：
Park MS;Phan HD;Busch F;Hinckley SH;Brackbill JA;Wysocki VH;Nakanishi K
通讯作者：
Nakanishi K
影响因子：
7.3
作者：
Nakanishi, Kotaro
通讯作者：
Nakanishi, Kotaro
DOI：
10.1126/science.aaf5573
发表时间：
2016-08-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Abudayyeh OO;Gootenberg JS;Konermann S;Joung J;Slaymaker IM;Cox DB;Shmakov S;Makarova KS;Semenova E;Minakhin L;Severinov K;Regev A;Lander ES;Koonin EV;Zhang F
通讯作者：
Zhang F
影响因子：
4.8
作者：
Dayeh, Daniel M.;Kruithoff, Bradley C.;Nakanishi, Kotaro
通讯作者：
Nakanishi, Kotaro