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 an RNA/DNA hybrid and a displaced single-stranded DNA that form during transcription. Their defective processing has been implicated in genome instability, which is associated with severe human diseases. Despite their biological significance, the mechanisms regulating R-loops remain incompletely understood, underscoring the need for improved tools to accurately map R-loops across the genome. Here, we propose enDR3, a novel tool designed to capture and sequence RNA/DNA hybrids for precise R-loop localization. enDR3 is an engineered tandem fusion of the N-terminal hybrid-binding domain from bacterial RNase H3, modified with specific amino acid substitutions to enhance its affinity and specificity for RNA/DNA hybrids. This engineered protein provides an important alternative to the widely used S9.6 antibody-based immunoprecipitation methods. We successfully applied enDR3 for genome-wide R-loop profiling using DNA/RNA immunoprecipitation with complementary DNA conversion and chromatin immunoprecipitation techniques, followed by sequencing. Our results confirm that enDR3 enables robust R-loop detection in both in vitro and in vivo, providing a versatile tool for advancing R-loop biology.