Genome-wide in vivo and ex vivo mapping of R-loops using engineered N-terminal hybrid-binding domain of RNase H3 (enDR3)

R-loops are nucleic acid structures composed of RNA/DNA hybrid and a displaced single-stranded DNA, which form during transcription. Defective processing of R-loops threatens genome stability, ultimately leading to severe human diseases. The current knowledge of the mechanisms of R-loop processing and regulation is incomplete. New tools to accurately localize R-loop biology across the genome are of key importance to understand R-loop biology. Here, we propose a new method to capture and sequence RNA/DNA hybrids which can be used to map R-loop locations, providing an essential alternative to currently used approaches, such as immunoprecipitation with S9.6 antibody. Our method relies on an engineered N-terminal hybrid-binding domain from bacterial RNase H3 (enDR3). We developed a tandem form of this domain with specific amino acid substitutions, which enhance affinity and specificity for RNA/DNA hybrids. Moreover, we successfully used this protein for genome-wide R-loop profiling based on DRIPc-seq or ChIP-seq techniques which confirmed that enDR3 is useful for R-loop detection in both in vitro and in vivo methods. The engineered N-terminal RNA/DNA hybrid-binding domain from the bacterial RNase H3 enzyme was used to capture R-loops ex vivo or in vivo from HeLa cells. Additionally, the S9.6 antibody ex vivo was used as a control. This approach allowed a direct comparison of three methods using either two different baits in the same hybrid capture protocol or the same bait using DRIPc or ChIP methodologies in one cell line.