Analysis of 2′-O-methylated nucleosides and pseudouridines in ribosomal RNAs using DNAzymes
Analysis of 2′-O-methylated nucleosides and pseudouridines in ribosomal RNAs using DNAzymes
复制标题
DOI：
10.1016/j.ab.2006.11.001
复制
发表时间：
2007-02-01
影响因子：
2.9
通讯作者：
Entian, Karl-Dieter
中科院分区：
文献类型：
--
作者：
Buchhaupt, Markus;Pelfer, Christian;Entian, Karl-Dieter
核糖体RNA(RRNAs)含有大量转录后修饰的核苷，其生理功能尚不清楚。真核生物和古生物中的绝大多数修饰是2‘-O-核糖甲基化核苷和假尿苷。目前鉴定rRNA修饰的方法操作困难，需要昂贵的试剂。在这里，我们报道了一种使用RNA裂解脱氧核酶(DNAzyme)检测2‘-O-核糖甲基化的简便方法，并以酿酒酵母的rRNA为例展示了它的应用。使用10-23类型的DNAzyme，我们可以证明酿酒酵母18S rRNA中的A(973)位或25S rRNA中的G(1450)位只有在这些位置的2‘-O-核糖甲基化缺失时才发生切割。我们还设计了8-17-DNAzyme来检测2‘-O-核糖甲基化。这使得所有的2‘-O-核糖甲基化对于DNAzyme技术来说都是可获得的，作为8-17-DNAzyme的变体，它们一起具有切割几乎任何二核苷酸连接的能力是已知的。此外，我们还发现，假尿苷还降低了邻近磷酸二酯键的DNAzyme的切割效率，因此也可以用DNAzyme进行鉴定。使用DNAzyme的分析提供了一种新的工具来容易地识别rRNA中的核糖甲基化，并将有助于揭示核苷酸修饰的生理功能。(C)2006 Elsevier Inc.保留所有权利。
Ribosomal RNAs (rRNAs) contain a large number of posttranscriptionally modified nucleosides, the physiological function of which is still unclear. The great majority of modifications in eukaryotes and archaea are 2'-O-ribose methylated nucleosides and pseudouridines. The current methods to identify rRNA modifications are difficult to perform and need expensive reagents. Here we report an easy method to detect 2'-O-ribose methylations using RNA-cleaving deoxyribozymes (DNAzymes) and demonstrate its application using rRNA of the yeast Saccharomyces cerevisiae. Using DNAzymes of the 10-23 type, we could show that cleavage at A(973) in the 18S rRNA or at G(1450) in the 25S rRNA from S. cerevisiae occurs only if the 2'-O-ribose methylations at these positions were missing. We also designed 8-17-DNAzymes for the detection of 2'-O-ribose methylations. This makes all 2'-O-ribose methylations accessible to the DNAzyme technique as variants of the 8-17-DNAzyme that together have the ability to cleave nearly any dinucleotide junction are known. Furthermore, we found that pseudouridine also decreases the DNAzyme cleavage efficiency at the adjacent phosphodiester bond and thus can also be identified with DNAzymes. The analysis using DNAzymes provides a new tool to easily identify ribose methylations in rRNAs and will help to unravel the physiological function of nucleotide modifications. (c) 2006 Elsevier Inc. All rights reserved.