Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation

Maika Malig, Stella R. Hartono, Jenna M. Giafaglione, Lionel A. Sanz, Frederic Chedin

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop ‘footprints’ at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation.

Original languageEnglish (US)
Pages (from-to)2271-2288
Number of pages18
JournalJournal of Molecular Biology
Issue number7
StatePublished - Mar 27 2020


  • DNA:RNA immunoprecipitation
  • Nondenaturing bisulfite conversion
  • R-loops
  • S9.6 antibody
  • SMRT-sequencing

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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