Yang Rong, Li Jianfeng, Wu Yifan, Jiang Xinli, Qu Shuang, Wang Qiang, Liang Hongwei, Zen Ke
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China.
School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
Front Mol Biosci. 2022 Aug 30;9:949181. doi: 10.3389/fmolb.2022.949181. eCollection 2022.
Methylation modification of small RNAs, including miRNA, piRNA, and tsRNA, is critical for small RNA biogenesis and biological function. Methylation of individual small RNA can be defined by liquid chromatography-coupled with mass spectrometry (LC-MS/MS). However, LC-MS/MS analysis requires a high purity of individual small RNA. Due to the difficulty of purifying specific small RNA from tissues or cells, the progress in characterizing small RNA methylation by LC-MS/MS is limited. Here, we report a novel method that can efficiently purify small RNA from human tissues for LC-MS/MS analysis. This method includes two steps: 1) pull down the target small RNA by incubating total small RNAs (18-24 nt) extracted from human tissues with a biotinylated antisense oligonucleotide of the target small RNA, followed by capturing the binding duplex of biotinylated antisense and small RNA streptavidin magnetic beads, and 2) protect the target small RNA by pairing it with a single-strand DNA, which sequence is complementary to the target small RNA, to form a DNA/RNA hybrid double-strand, followed by sequential digestion with exonuclease I, nuclease S1, and DNase I, respectively. Furthermore, employing a mixture of four pairs of synthetic methylated and non-methylated small RNAs, we further refined this two-step method by optimizing the nuclease S1 treatment condition. With this method, we successfully purified miR-21-5p, miR-26-5p, piR-020485, and tsRNA from human lung and sperm tissue samples and analyzed their 2'-O-methylation modification at the 3'-end by LC-MS/MS.
包括微小RNA（miRNA）、Piwi相互作用RNA（piRNA）和转运RNA衍生的小RNA（tsRNA）在内的小RNA的甲基化修饰对于小RNA的生物合成和生物学功能至关重要。单个小RNA的甲基化可以通过液相色谱-质谱联用（LC-MS/MS）来确定。然而，LC-MS/MS分析需要高纯度的单个小RNA。由于从组织或细胞中纯化特定小RNA存在困难，通过LC-MS/MS表征小RNA甲基化的进展有限。在此，我们报告了一种新方法，该方法可以有效地从人类组织中纯化小RNA用于LC-MS/MS分析。该方法包括两个步骤：1）将从人类组织中提取的总小RNA（18 - 24 nt）与目标小RNA的生物素化反义寡核苷酸孵育，下拉目标小RNA，随后用链霉亲和素磁珠捕获生物素化反义寡核苷酸与小RNA的结合双链体；2）通过将目标小RNA与序列与目标小RNA互补的单链DNA配对，保护目标小RNA，形成DNA/RNA杂交双链体，随后分别用核酸外切酶I、核酸酶S1和脱氧核糖核酸酶I进行顺序消化。此外，我们使用四对合成的甲基化和非甲基化小RNA混合物，通过优化核酸酶S1处理条件进一步完善了这种两步法。通过这种方法，我们成功地从人肺和精子组织样本中纯化了miR - 21 - 5p、miR - 26 - 5p、piR - 020485和tsRNA，并通过LC-MS/MS分析了它们3'端的2'-O-甲基化修饰。
