Detecting R-Loop Formation Using a Plasmid-Based In Vitro Transcription Assay

Abstract

R-loops are three-stranded nucleic acid structures that consist of a DNA-RNA hybrid and a displaced single-stranded DNA. Since it was first reported by Ronald Davis and colleagues over 40 years ago, the study of R-loops has become an increasingly expanded area of research. Numerous factors have been identified to modulate the dynamic formation and resolution of R-loops, which are critical for proper controls of gene expression and genome stability. Along the lines of these discoveries, various biochemical and cellular assays have been developed to detect R-loop changes in vitro and in vivo. In this chapter, we describe a protocol for measuring R-loop formation using a plasmid-based in vitro transcription assay. The R-loop formed is then detected and quantified by using gel mobility, antibody staining, and DNA-RNA immunoprecipitation (DRIP)-qPCR assays. Unlike the helicase assay that uses short R-loop substrates, this assay system introduces DNA topology and active transcription as additional variables that impact R-loop formation, thus, more closely recapitulating in vivo situations. Furthermore, this method can be adopted for investigation of cis-elements and trans-acting factors that influence R-loop formation.

Keywords: DNA/RNA immunoprecipitation-qPCR; Dot-blot; Gel electrophoresis; In vitro transcription; R-loop.