Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland.
Methods Mol Biol. 2022;2528:1-20. doi: 10.1007/978-1-0716-2477-7_1.
R-loop are physiologically present on genomic DNA of different organisms and play important roles in genome regulation. However, an increase in their abundance and/or size has been suggested to interfere with the DNA replication process, contributing to genome instability. Most available approaches to monitor R-loops are based on antibodies/enzymes that cannot effectively distinguish R-loops from DNA-RNA hybrids and assess R-loop size and frequency in a population of molecules. Electron microscopy has successfully allowed single-molecule visualization of DNA replication and repair intermediates, uncovering key architectural modifications in DNA, induced by genotoxic stress or by the associated cellular response. Here, we describe recent modifications of this visualization workflow to implement partial automation of image acquisition and analysis. Coupling this refined workflow with sample preparation procedures that protect R-loop stability allows for direct visualization of R-loop structures on genomic DNA, independently from probes. Combining single-molecule information and DNA content assessment, this approach provides direct estimations of R-loop frequency, size, and burden on genomic DNA.
R 环存在于不同生物体的基因组 DNA 中，在基因组调控中发挥着重要作用。然而，它们的丰度和/或大小的增加被认为会干扰 DNA 复制过程，导致基因组不稳定。目前大多数监测 R 环的方法都是基于抗体/酶，这些抗体/酶不能有效地将 R 环与 DNA-RNA 杂交区分开来，也无法评估分子群体中 R 环的大小和频率。电子显微镜成功地实现了 DNA 复制和修复中间体的单分子可视化，揭示了由遗传毒性应激或相关细胞反应诱导的 DNA 关键结构修饰。在这里，我们描述了对这种可视化工作流程的最新修改，以实现图像采集和分析的部分自动化。将这种改进的工作流程与保护 R 环稳定性的样品制备程序相结合，允许直接在基因组 DNA 上可视化 R 环结构，而无需探针。将单分子信息和 DNA 含量评估相结合，这种方法可以直接估计基因组 DNA 上 R 环的频率、大小和负担。
