R-Loop Analysis by Dot-Blot

Abstract

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. 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. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. 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. 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. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. 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.

Figure 1:. Specificity of S9.6 as shown by dot-blot loaded with nucleic acids from human fibroblasts.

Nucleic acid samples from human fibroblasts were either mock treated or treated with RNase T1, RNase H, or RNase III and then loaded onto nylon membranes in a dilution series of 200, 100, 50, 25, 12.5, and 6.25 ng per 2 μL dot. Membranes were then probed with S9.6 antibody (A), or dsDNA antibody (B).

Figure 2:. Quantification of S9.6 R-loop staining.

50 ng samples from Figure 1 were selected for quantification with ImageJ. S9.6 signal was divided by dsDNA signal intensity, then normalized to the mock sample following the steps outlined in section 7.

Figure 3:. S9.6 dot-blot using oligonucleotide controls.

S9.6 antibody dot-blot against a dilution series of synthetic oligonucleotides as dsRNA, dsDNA, or RNA-DNA hybrid. S9.6 binds specifically to RNA-DNA hybrids in a dose-dependent manner.