A transition state analogue for an RNA-editing reaction
A transition state analogue for an RNA-editing reaction
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DOI：
10.1021/ja0472073
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发表时间：
2004-09-15
影响因子：
15
通讯作者：
Beal, PA
中科院分区：
文献类型：
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作者：
Haudenschild, BL;Maydanovych, O;Beal, PA
由阿达尔酶催化的RNA中腺苷在C6处的脱氨在相应位置产生肌苷。由于肌苷在翻译过程中被解码为鸟苷，这种修饰可以导致信使RNA中的密码子变化。8-azanebularine在C6-N1双键上的水合作用产生了ADAR催化的水解脱氨基反应的过渡态的极好模拟。在这里，我们报告的合成的亚磷酰胺的8-azanebularine和它的使用在制备的RNA模仿的二级结构中发现的一个已知的编辑位点的谷氨酸受体8亚基前mRNA。含有类似物的RNA的结合特性表明，可通过掺入8-氮杂环丁烷产生阿达尔的紧密结合配体。观察到的高亲和力结合依赖于功能活性位点、ADAR 2的两个双链RNA结合基序（dsRBM）中的一个而不是另一个的存在，以及核苷类似物在已知编辑位点的序列/结构背景中的正确位置。这些结果推进了我们对ADAR催化RNA编辑过程中底物识别的理解，对ADAR-RNA复合物的结构研究具有重要意义。
Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor 8 subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR-RNA complexes.