In vitro selection of a 5'-purine ribonucleotide transferase ribozyme.
In vitro selection of a 5'-purine ribonucleotide transferase ribozyme.
复制标题
5-嘌呤核糖核苷酸转移酶核酶的体外选择。
DOI：
10.1093/nar/gkm438
复制
发表时间：
2007
影响因子：
14.9
通讯作者：
Suga,Hiroaki
中科院分区：
文献类型：
--
作者：
Kang,TaekJin;Suga,Hiroaki
在这里，我们报告了一种新型核酶的体外选择，该核酶催化形成2 ‘ -5 ’磷酸二酯键的5 ' -核苷酸转移反应。该核酶作为唯一的序列在与ATP-γS结合的过程中富集了5 ' -三磷酸依赖活性。最初选择的由109个核苷酸（nt）组成的核酶通过一系列的突变研究被小型化为45-nt M4核酶，并在此基础上构建了微型核酶转导作用体系。我们研究中最具挑战性的任务之一是确定5 ' -ppp位点发生的化学反应。我们利用MALDI-TOF等多种分析方法对反式反应体系生成的产物进行了分析，并阐明了其化学性质为3′→5′单核苷酸延伸形成2′-5′磷酸二酯键。有趣的是，M4核酶可以混杂地接受多种嘌呤核苷酸，这些嘌呤核苷酸含有5 ' -单磷酸、二磷酸和三磷酸作为底物。M4核酶的这种非凡能力将引导我们开发一种新的工具，用于具有独特化学基团的rna的5 '修饰。
Here we reportin vitroselection of a novel ribozyme that catalyzes the 5′-nucleotidyl transfer reaction forming the 2′–5′ phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5′-triphosphate-dependent activities in incorporating ATP-γS. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme atrans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5′-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by thetrans-acting system and elucidated the chemistry to be 3′→5′ mononucleotide extension forming the 2′–5′ phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5′-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5′-modification of RNAs with unique chemical groups.