Inhibitory Impact of 3′-Terminal 2′-0-Methylated Small Silencing RNA on Target-Primed Polymerization and Unbiased Amplified Quantification of the RNA in Arabidopsis thaliana
Inhibitory Impact of 3′-Terminal 2′-0-Methylated Small Silencing RNA on Target-Primed Polymerization and Unbiased Amplified Quantification of the RNA in Arabidopsis thaliana
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DOI：
10.1021/acs.analchem.5b01683
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发表时间：
2015-09-01
影响因子：
7.4
通讯作者：
Zhao, Yongxi
中科院分区：
文献类型：
--
作者：
Chen, Feng;Fan, Chunhai;Zhao, Yongxi
3 '-末端2'-O-甲基化存在于多种小分子沉默RNA中，被认为是抑制3' -> 5'降解和3 '-末端尿苷酸化的保护机制。然而，这种修饰对酶促聚合的影响仍然未知。在此，进行了系统的调查，以探讨这一问题。我们发现这些甲基化的小RNA在靶向引发聚合中表现出抑制行为，揭示了传统的基于聚合的方法对这些小RNA的操作的偏倚结果。对相关的机理进行了探讨，认为这可能是由于修饰基团的尺寸较大，且位置靠近3 '-OH。此外，已经提出了两种新的解决方案，每一种都利用碱基堆积杂交和三向连接结构来实现对小RNA的无偏识别。在硫代磷酸酯抗切口的基础上，我们还发展了一种新的扩增策略--硫代磷酸酯保护聚合/双切口扩增，用于拟南芥甲基化小RNA的无偏定量，显示了其在真实的样品分析中的潜力。总的来说，我们的研究揭示了3 '-末端2'-O-甲基化的小RNA的聚合抑制机制的讨论，并提出了新的无偏的解决方案，扩增定量的小RNA在真实的样品。
3'-terminal 2'-O-methylation has been found in several kinds of small silencing RNA, regarded as a protective mechanism against enzymatic 3' -> 5' degradation and 3'-end uridylation. The influence of this modification on enzymatic polymerization, however, remains unknown. Herein, a systematic investigation is performed to explore this issue. We found these methylated small RNAs exhibited a suppression behavior in target-primed polymerization, revealing biased result for the manipulation of these small RNAs by conventional polymerization-based methodology. The related potential mechanism is investigated and discussed, which is probably ascribed to the big size of modified group and its close location to 3'-OH. Furthermore, two novel solutions each utilizing base-stacking hybridization and three-way junction structure have been proposed to realize unbiased recognition of small RNAs. On the basis of phosphorothioate against nicking, a creative amplified strategy, phosphorothioate-protected polymerization/binicking amplification, has also been developed for the unbiased quantification of methylated small RNA in Arabidopsis thaliana, demonstrating its promising potential for real sample analysis. Collectively, our studies uncover the polymerization inhibition by 3'-terminal 2'-O-methylated small RNAs with mechanistic discussion, and propose novel unbiased solutions for amplified quantification of small RNAs in real sample.