Kang Taek Jin, Suga Hiroaki
Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan.
Nucleic Acids Res. 2007;35(12):4186-94. doi: 10.1093/nar/gkm438. Epub 2007 Jun 12.
Here we report in vitro selection 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-gammaS. 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 a trans-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 the trans-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.
在此，我们报告了一种新型核酶的体外筛选，该核酶催化形成2'-5'磷酸二酯键的5'-核苷酸转移反应。在富含用于掺入ATP-γS的5'-三磷酸依赖性活性的文库中，该核酶作为唯一序列被检索到。通过一系列突变研究，最初选择的由109个核苷酸（nt）组成的核酶被小型化为45-nt的M4核酶，并基于此小型核酶构建了一个反式作用系统。我们研究中最具挑战性的任务之一是确定在5'-ppp位点发生的化学反应。我们利用了各种分析方法，包括对反式作用系统产生的产物进行基质辅助激光解吸电离飞行时间（MALDI-TOF）分析，并阐明该化学反应为形成2'-5'磷酸二酯键的3'→5'单核苷酸延伸。有趣的是，M4核酶不加选择地接受各种带有5'-单磷酸、二磷酸和三磷酸的嘌呤核苷酸作为底物。M4核酶的这种显著能力将引导我们开发一种用于用独特化学基团对RNA进行5'-修饰的新工具。
