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Updated: Jul 15, 2026
Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
Published on: April 4, 2025
Fluorescence-based melting assays for studying quadruplex ligands
Anne De Cian1, Lionel Guittat, Markus Kaiser
1Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, USM 503, INSERM UR 565, CNRS UMR 5153, 43 rue Cuvier, 75231 Paris Cedex 05, France.
Abstract:
The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for T(m) calculation, complicating a direct comparison of the results obtained by different laboratories.
Insights
Telomeric G-quadruplex DNA can inhibit telomerase. Researchers analyzed FRET methods for measuring quadruplex ligand stabilization, finding variations complicate comparisons between labs.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Telomeric G-rich DNA forms quadruplex structures that inhibit telomerase.
- Telomerase reactivation is crucial in cancer, making telomeres a target for oncology.
- Telomere ligands and telomerase inhibitors are explored as anticancer agents.
Purpose of the Study:
- To analyze the Förster Resonance Energy Transfer (FRET) method for assessing quadruplex ligand stabilization and selectivity.
- To identify factors influencing the measurement of human telomeric G-quadruplex stabilization.
Main Methods:
- Utilized FRET assays to evaluate ligand interactions with the human telomeric G-quadruplex.
- Investigated the impact of fluorescent tags, buffer conditions, and melting temperature (Tm) calculation methods on stabilization measurements.
Main Results:
- Stabilization values are sensitive to the choice of fluorescent tags.
- Incubation buffer composition significantly affects measured stabilization.
- The method used for Tm calculation introduces variability.
Conclusions:
- Direct comparison of quadruplex ligand stabilization data across different laboratories is challenging due to methodological variations.
- Standardization of FRET-based assays is needed for reliable evaluation of telomere-targeting anticancer agents.
