Detection and characterization of R Loop structures

Abstract

R loops are special three stranded nucleic acid structures that comprise a nascent RNA hybridized with the DNA template strand, leaving a non-template DNA single-stranded. More specifically, R loops form in vivo as G-rich RNA transcripts invade the DNA duplex and anneal to the template strand to generate an RNA:DNA hybrid, leaving the non-template, G-rich DNA strand in a largely single-stranded conformation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012). DNA-RNA hybrids are a natural occurrence within eukaryotic cells, with levels of these hybrids increasing at sites with high transcriptional activity, such as during transcription initiation, repression, and elongation. RNA-DNA hybrids influence genomic instability, and growing evidence points to an important role for R loops in active gene expression regulation (Ginno et al., Mol Cell 45, 814–825, 2012; Sun et al., Science 340: 619–621, 2013; Bhatia et al., Nature 511, 362-365, 2014). Analysis of the occurrence of such structures is therefore of incre sing relevance and herein we describe methods for the in vivo and in vitro identification and characterization of R loops in mammalian systems.