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Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

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Published on: October 20, 2018

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

Anthony Sanchez¹, Jullian O Perren¹, Meili Aiello¹

¹Department of Radiation Oncology, Emory University, School of Medicine, Winship Cancer Institute, Atlanta, GA 30322, USA.

Cell Reports Methods

|April 8, 2026

Summary

Researchers developed R-PLA, a new method to detect R-loops (nucleic acid structures) using antibody proximity. This sensitive and selective technique visualizes R-loops at DNA damage sites and other cellular locations.

Keywords:

CP: cell biology CP: molecular biology DNA damage R-loops RNA-DNA hybrids RNaseH1 fluorescence microscopy proximity ligation

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Published on: September 2, 2019

Area of Science:

Molecular Biology
Genetics
Cell Biology

Background:

R-loops are nucleic acid structures crucial for DNA damage responses.
Existing methods for R-loop detection may lack selectivity or sensitivity.

Purpose of the Study:

To develop a novel, highly selective, and sensitive method for detecting and visualizing R-loops.
To characterize RNaseH1-associated R-loops in mammalian cells.

Main Methods:

Development of a proximity ligation assay (R-PLA) using S9.6 and RNaseH1 antibodies.
Detection of R-PLA signals as foci via fluorescence microscopy.
Compatibility assessment with flow cytometry for R-loop analysis.

Main Results:

R-PLA demonstrated enhanced selectivity and sensitivity in detecting R-loops.
R-PLA successfully labeled RNaseH1-associated R-loops at DNA damage sites, telomeres, and stalled replication forks.
Increased nucleoplasmic R-loops were detected after DNA damage induction (PARPi, IR).

Conclusions:

R-PLA is a selective and sensitive method for detecting RNaseH1-associated R-loops.
The technique facilitates R-loop visualization and quantification in mammalian cells.
R-PLA holds potential for studying R-loop dynamics in various biological contexts.