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Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
Published on: April 4, 2025
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Single-molecule visualization of DNA G-quadruplex formation in live cells
Marco Di Antonio1,2, Aleks Ponjavic1,3,4, Antanas Radzevičius1
1Department of Chemistry, University of Cambridge, Cambridge, UK.
Nature Chemistry
|July 22, 2020
Summary
Scientists developed a new fluorescent probe to observe DNA G-quadruplexes (G4s) in living cells. This probe reveals that G4s dynamically form and change during the cell cycle, impacting gene expression.
Area of Science:
- Molecular Biology
- Genetics
- Biophysics
Background:
- DNA G-quadruplexes (G4s) are increasingly linked to gene expression regulation.
- Understanding G4 dynamics in living cells is crucial for elucidating their biological functions.
- Existing methods often perturb G4 folding, limiting in vivo studies.
Purpose of the Study:
- To develop and utilize a novel G4-specific fluorescent probe for real-time, single-molecule detection of G4s in living cells.
- To investigate the dynamic nature of G4 formation and its dependence on cellular processes.
- To assess the impact of cellular conditions on G4 folding and stability.
Main Methods:
- Development of a G4-specific fluorescent probe (SiR-PyPDS).
- Live-cell single-molecule fluorescence imaging at low probe concentrations (20 nM) to minimize perturbation.
- Time-dependent chemical trapping assays to study G4 unfolding dynamics.
- Correlation of G4 formation with cell cycle progression and inhibition of transcription/replication.
Main Results:
- SiR-PyPDS enabled real-time, single-molecule visualization of individual G4 structures in living cells.
- G4s were observed to fluctuate dynamically between folded and unfolded states.
- G4 formation demonstrated cell-cycle dependence.
- Inhibition of transcription and replication disrupted G4 formation.
Conclusions:
- The study provides robust evidence for dynamic G4 formation in living cells.
- The developed probe allows for non-perturbing, detailed investigation of G4s in their native cellular environment.
- G4 dynamics are intrinsically linked to fundamental cellular processes like cell cycle, transcription, and replication.
