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Updated: Mar 7, 2026
Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
Published on: April 4, 2025
Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations
Federica Moraca1, Jussara Amato2, Francesco Ortuso1
1Dipartimento di Scienze della Salute, University of Catanzaro "Magna Græcia," I-88100 Catanzaro, Italy.
Abstract:
G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG3(T2AG3)3]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy ([Formula: see text] = -10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands.
Insights
This study reveals how berberine binds to G-quadruplex DNA structures using funnel-metadynamics. This method accurately predicts binding energy, aiding in the design of new cancer drugs targeting G-quadruplexes.
Area of Science:
- Biochemistry
- Computational Biology
- Structural Biology
Background:
- G-quadruplexes (G4s) are DNA structures found in gene promoter regions and telomeres.
- G4 formation impacts DNA replication and can lead to apoptosis, making them targets for cancer therapy.
- Understanding ligand interactions with G4s is crucial for developing targeted cancer treatments.
Purpose of the Study:
- To elucidate the binding mechanism of the anticancer alkaloid berberine to the human telomeric G-quadruplex (d[AG3(T2AG3)3]).
- To compute the binding free-energy landscape of berberine to the G4 structure.
- To validate a novel free-energy method, funnel-metadynamics (FM), for investigating ligand/DNA interactions.
Main Methods:
- Utilized funnel-metadynamics (FM) simulations with explicit representation of ions and water molecules.
- Investigated the binding mechanism and free-energy landscape of berberine to the human telomeric G4.
- Performed steady-state fluorescence binding assays to validate computational results.
Main Results:
- Identified two lowest-energy ligand binding modes for berberine on the G4 structure.
- Discovered prebinding sites that facilitate the final binding mode.
- Calculated an accurate absolute ligand/DNA binding free energy of -10.3 ± 0.5 kcal/mol.
- Demonstrated good agreement between theoretical FM predictions and experimental binding assays.
Conclusions:
- Funnel-metadynamics (FM) is a powerful method for accurately investigating ligand/DNA interactions.
- The study provides a detailed understanding of berberine's binding mechanism to telomeric G-quadruplex DNA.
- FM can be a valuable tool for the rational design of novel G-quadruplex-targeting ligands for cancer therapy.
