Department of Clinical Biochemistry, Faculty of Pharmacy, Nicolaus Copernicus University in Toruń, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
Epigenetic Programme, Babraham Institute, Cambridge, UK.
Methods Mol Biol. 2022;2528:127-143. doi: 10.1007/978-1-0716-2477-7_9.
R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and an unpaired strand of nontemplate DNA that represent a major source of genomic instability and are involved in regulation of several important biological processes in eukaryotic cells. A growing body of experimental evidence suggests that RNA moieties of RNA-DNA hybrids may convey RNA modifications influencing various aspects of R-loop biology. Here we present a protocol for quantitative analysis of RNA modifications on RNA-DNA hybrids using stable-isotope dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry (SID-UPLC-MS/MS). Supplemented by other techniques, this method can be instrumental in deciphering the roles of RNA modifications in R-loop metabolism.
R 环是由 RNA-DNA 杂合体和一条未配对的非模板 DNA 组成的三链核酸结构，代表了基因组不稳定性的主要来源，并参与真核细胞中几个重要生物学过程的调控。越来越多的实验证据表明，RNA-DNA 杂合体的 RNA 部分可能传递 RNA 修饰，影响 R 环生物学的各个方面。本文提供了一种使用稳定同位素稀释超高效液相色谱串联质谱（SID-UPLC-MS/MS）定量分析 RNA-DNA 杂合体上 RNA 修饰的方案。该方法结合其他技术，可以为解析 RNA 修饰在 R 环代谢中的作用提供有力工具。
