R-PLA enables enhanced detection of R-loops in mammalian cells using proximity ligation

R-loops are key nucleic acid structures involved in many biological processes including DNA damage responses. Here, we develop a method for detecting and visualizing R-loops using fluorescence microscopy by combining S9.6 and RNaseH1 antibodies in a proximity ligation assay (R-PLA). The requirement of two R-loop antibodies to be in close proximity to generate the signal provides enhanced selectivity, and PLA has an amplification step, which increases sensitivity. R-PLA signals are detected as foci, helping to reduce background and increase the ease of quantifying the signal. The ability of R-PLA to produce foci allows for the specific labeling of RNaseH1-associated R-loops at DNA damage sites, telomeres, and stalled replication forks. R-PLA detected increased nucleoplasmic R-loops after DNA damage induction by various methods, including PARP inhibitor (PARPi) and ionizing radiation (IR), conditions known to induce R-loops. R-PLA is also compatible with analyzing RNaseH1-associated R-loops by flow cytometry. Therefore, R-PLA provides a selective and sensitive method for detecting R-loops associated with RNaseH1 and potentially other R-loop factors, in mammalian cells.