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Updated: Sep 8, 2025

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Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy.

Henriette Stoy1, Joel Luethi1,2,3, Fabienne K Roessler1,2,4

  • 1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|June 15, 2022
PubMed
Summary
This summary is machine-generated.

This study introduces an enhanced electron microscopy workflow to visualize R-loops, structures that can cause genome instability. The improved method allows for direct, probe-independent measurement of R-loop frequency, size, and genomic burden at the single-molecule level.

Keywords:
Automated workflowDNA content assessmentDNA replicationDNA–RNA hybridDirect visualizationElectron microscopyR-loopsSingle-molecule analysis

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biophysics

Background:

  • R-loops are DNA:RNA hybrids with crucial roles in genome regulation.
  • Increased R-loop abundance or size can lead to genome instability by interfering with DNA replication.
  • Current methods struggle to accurately quantify R-loop characteristics like size and frequency.

Purpose of the Study:

  • To develop and refine an electron microscopy (EM) workflow for direct visualization and quantification of R-loops.
  • To overcome limitations of antibody/enzyme-based methods in distinguishing R-loops and assessing their molecular properties.
  • To enable accurate measurement of R-loop frequency, size, and genomic burden at the single-molecule level.

Main Methods:

  • Modified electron microscopy workflow with partially automated image acquisition and analysis.
  • Sample preparation techniques to preserve R-loop stability during visualization.
  • Coupling single-molecule EM visualization with DNA content assessment.

Main Results:

  • Direct visualization of R-loop structures on genomic DNA without reliance on probes.
  • Successful implementation of automated image analysis for enhanced efficiency.
  • Accurate, single-molecule based estimation of R-loop frequency, size, and overall burden on genomic DNA.

Conclusions:

  • The refined EM workflow provides a powerful, probe-independent method for studying R-loops.
  • This approach offers direct insights into R-loop dynamics and their impact on genome stability.
  • Enables precise quantification of R-loops, advancing our understanding of their physiological and pathological roles.