ArticleCell reports methods2026
R-PLA enables enhanced detection of R-loops in mammalian cells using proximity ligation.
Article in Cell reports methods, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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