R-loops are also in telomeric regions of chromosomes. R-loops are found in gene promoters 7 and termination 5 sites in mRNA, and along ribosomal RNA 8 as well as transfer RNA 9. Given the many opportunities for their formation, R-loops are not rare, and can be found in 3–5% of the human genome 6, depending on the cell’s transcription status. Additionally, R-loops can form when replication and transcription complexes collide 4, and in antisense transcription 5. Outside of the transcriptional complex, the nascent RNA is close to its DNA template, which is still slightly unwound from being copied, thus the RNA can rehybridize with its template DNA forming R-loops 3. The short RNA-DNA hybrid (<10 bp) is resolved to free the nascent RNA so it can leave the RNA polymerase complex through the exit channel 1, 2. In the transcriptional complex, the nascent RNA is synthesized complementary to the template DNA, and the non-template strand is displaced. R-loops are found in different stages of the lifecycle of RNA. R-loop forms when RNA invades a double-stranded DNA to generate an RNA-DNA hybrid and displaces the other DNA strand. This protocol provides a step-by-step guide to a dot-blot assay that allows a quick comparative assessment of the abundance of R-loop, a three-stranded nucleic acid structure. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. Initially, R-loops were thought to be the by-products of transcription but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation.
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