R-Loop Modification and Quantification by Dot Blot

RNA modifications and their "writer," "eraser," and "reader" proteins are emerging as key regulators of gene expression and DNA repair through dynamically regulating RNA:DNA hybrids, or R-loops, during transcription. Therefore, it is paramount to develop rigorous techniques for accurate analysis of R-loop modifications. A convenient method for analyzing RNA modifications within total RNA is by dot blot with specific RNA modification antibodies; however, analysis of the modification of the RNA moiety within R-loops presents specific challenges. Here, we provide a detailed protocol for the production or purification of DNA containing R-loops in vitro and from cells, and the analysis of the RNA moiety modifications by dot blot. The DNA containing R-loops is treated with either mock or RNase H, which specifically degrades the RNA within RNA:DNA hybrids, to control for the specificity of the signal as originating from R-loops. Known quantities of the mock or RNase H-treated DNA are then spotted on three membranes, each blotted with antibodies that recognize double-stranded DNA, RNA:DNA hybrids, or the specific RNA modification antibodies of interest, such as m⁶A or ac⁴C. Thus, this protocol is useful to both biochemists and cell biologists with scientific interests at the intersection of R-loops and epitranscriptomics. Key features • R-loops produced via in vitro transcription of R-loop-forming DNA sequences can serve as substrates for biochemical assays, quantification standards, or antibody specificity controls. • R-loop-containing DNA can be purified from cells after gene editing or various treatments to analyze how global R-loop levels and modifications are regulated. • This protocol was initially applied to the study of the role of NAT10 and ac⁴C modification of the RNA moiety within R-loops in human cells. • Requires 4-7 days to complete, depending on the origin of R-loops (in vitro transcription vs. from cells).