Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications.
Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications.
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DOI：
10.1261/rna.079454.122
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发表时间：
2023-03
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影响因子：
--
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动质体中不可翻译的线粒体转录物通过需要尿苷插入/删除的 RNA 编辑过程在转录后被解密。这种独特的逐步过程是由编辑体介导的，编辑体是一种多蛋白复合物，是一种经过验证的药物靶标，在解决动质体疾病未得到满足的医疗需求方面具有很大的兴趣。为了实现这一目标，已经开发了几种体外 RNA 编辑测定方法，尽管在发现有效抑制剂方面取得的成功有限。本手稿描述了三种基于锤头核酶 (HHR) FRET 报告基因的 RNA 编辑测定的开发，用于预切割删除、插入和连接测定，绕过限速内切核酸切割步骤，提供有关 U 删除、U 插入和连接活性的信息。与之前发表的全轮体外 RNA 编辑测定相比，这些测定在更短的孵育时间内表现出更高的编辑效率，同时需要显着更少的纯化编辑体和 10,000 倍的 ATP。此外，报告核酶序列的修改使得多重基于核酶的插入/删除编辑（RIDE）测定成为可能，该测定同时监视适合 HTS 的 U 插入和删除编辑。这些测定可用于寻找具有化疗应用的新型化合物或作为研究编辑体机制的探针。
Untranslatable mitochondrial transcripts in kinetoplastids are decrypted post-transcriptionally through an RNA editing process that entails uridine insertion/deletion. This unique stepwise process is mediated by the editosome, a multiprotein complex that is a validated drug target of considerable interest in addressing the unmet medical needs for kinetoplastid diseases. With that objective, several in vitro RNA editing assays have been developed, albeit with limited success in discovering potent inhibitors. This manuscript describes the development of three hammerhead ribozyme (HHR) FRET reporter-based RNA editing assays for precleaved deletion, insertion, and ligation assays that bypass the rate-limiting endonucleolytic cleavage step, providing information on U-deletion, U-insertion, and ligation activities. These assays exhibit higher editing efficiencies in shorter incubation times while requiring significantly less purified editosome and 10,000-fold less ATP than the previously published full round of in vitro RNA editing assay. Moreover, modifications in the reporter ribozyme sequence enable the feasibility of multiplexing a ribozyme-based insertion/deletion editing (RIDE) assay that simultaneously surveils U-insertion and deletion editing suitable for HTS. These assays can be used to find novel chemical compounds with chemotherapeutic applications or as probes for studying the editosome machinery.
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DOI：
10.1038/nrmicro.2016.193
发表时间：
2017-02-27
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Field MC;Horn D;Fairlamb AH;Ferguson MA;Gray DW;Read KD;De Rycker M;Torrie LS;Wyatt PG;Wyllie S;Gilbert IH
通讯作者：
Gilbert IH
DOI：
10.1073/pnas.0901754106
发表时间：
2009-07-28
影响因子：
11.1
作者：
Li, Feng;Ge, Peng;Simpson, Larry
通讯作者：
Simpson, Larry
DOI：
10.1073/pnas.0604476103
发表时间：
2006-09-19
影响因子：
11.1
作者：
Kang, Xuedong;Gao, Guanghan;Simpson, Larry
通讯作者：
Simpson, Larry
影响因子：
5.3
作者：
Cruz-Reyes, J;Zhelonkina, A;Sollner-Webb, B
通讯作者：
Sollner-Webb, B
影响因子：
64.5
作者：
BLUM, B;BAKALARA, N;SIMPSON, L
通讯作者：
SIMPSON, L