Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, QC, Canada.
Methods Mol Biol. 2022;2528:31-37. doi: 10.1007/978-1-0716-2477-7_3.
Early evidence for R-loop formation in vivo came from the study of Escherichia coli topA (topoisomerase I; topo I) null mutants. Assays with plasmids to detect RNase HI-sensitive hypernegative supercoiling or R-looped DNA were used in vitro and in vivo to demonstrate R-loop formation. In addition, these R-loop-dependent topological modifications of plasmid DNA were shown to correlate with severe growth and gene expression inhibition in topA null mutants that could be corrected by RNase HI overproduction. However, direct evidence for R-loop formation on chromosomal DNA from E. coli cells was only obtained recently by using the S9.6 antibody to detect RNA-DNA hybrids in dot-blot experiments. Here, we present a protocol for such experiments with a special emphasis on the procedure used for bacterial genomic DNA extraction and preparation including treatment with appropriate ribonucleases to eliminate RNA-RNA hybrids (that are also recognized by S9.6) as well as single-stranded RNA (ssRNA), in order to obtain a signal that is specific to stable RNA-DNA hybrids generated. Furthermore, we recommend that the results of such experiments be correlated with RNase HI-sensitive phenotypes.
体内 R 环形成的早期证据来自于对大肠杆菌 topA（拓扑异构酶 I；topo I）缺失突变体的研究。使用检测 RNase HI 敏感的超负超螺旋或 R 环 DNA 的质粒进行的测定在体外和体内证明了 R 环的形成。此外，这些依赖 R 环的质粒 DNA 的拓扑修饰与 topA 缺失突变体中的严重生长和基因表达抑制相关，这种抑制可以通过 RNase HI 的过度产生得到纠正。然而，最近才通过使用 S9.6 抗体在斑点印迹实验中检测 RNA-DNA 杂交，从大肠杆菌细胞的染色体 DNA 中获得了 R 环形成的直接证据。在这里，我们提出了一种用于此类实验的方案，特别强调了用于细菌基因组 DNA 提取和制备的程序，包括用适当的核糖核酸酶处理以消除 RNA-RNA 杂交（S9.6 也能识别）以及单链 RNA（ssRNA），以便获得对生成的稳定 RNA-DNA 杂交体特异的信号。此外，我们建议将此类实验的结果与 RNase HI 敏感表型相关联。
