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Updated: Feb 19, 2026
Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions
Published on: August 30, 2024
Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA
Cristina Pérez-Arnaiz1, Natalia Busto1, Javier Santolaya2
1Department of Chemistry, University of Burgos, 09001 Burgos, Spain.
Background:
Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in >80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence.
Methods:
UV-Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics.
Results:
TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na+ or K+. T-Jump kinetic experiments show that the rates of formation and dissociation of these complexes in the ms time scale differ by one order of magnitude. MD simulations reveal that, in K+ buffer, "hybrid 1" conformation yields kinetic constants on interaction with TMPyP4 one order lower than "hybrid 2". The binding involves π-π stacking with external loop bases.
Conclusions:
For the first time we show that for a particular buffer TMPyP4 interacts in a kinetically different way with the two Tel22 conformations even if the complexes formed are thermodynamically indistinguishable.
General Significance:
G-quadruplexes, endowed with technological applications and potential impact on regulation mechanisms, define a new research field. The possibility of building different conformations from same sequence is a complex issue that confers G-quadruplexes very interesting features. The obtaining of reliable kinetic data constitutes an efficient tool to determine reaction mechanisms between conformations and small molecules.
Insights
Small molecule ligands like TMPyP4 bind differently to G-quadruplex structures, impacting telomerase activity in cancer. This study reveals distinct kinetic interactions with specific G-quadruplex conformations.
Area of Science:
- Biochemistry and Molecular Biology
- Chemical Biology
- Structural Biology
Background:
- G-quadruplex helices are therapeutic targets due to their role in telomerase inhibition, crucial for over 80% of cancer cells.
- Small molecule ligands, such as TMPyP4, are investigated for their ability to stabilize G-quadruplexes and inhibit telomerase.
- Understanding ligand-G-quadruplex interactions is key to developing novel cancer therapeutics.
Purpose of the Study:
- To investigate the differential binding kinetics of the G-quadruplex ligand TMPyP4 with distinct conformations of a human telomeric sequence (Tel22).
- To elucidate the molecular mechanisms underlying these interactions using biophysical and computational methods.
- To highlight the importance of kinetic data in understanding ligand-G-quadruplex interactions.
Main Methods:
- Utilized UV-Vis spectroscopy, Fluorescence Resonance Energy Transfer (FRET) melting assays, and Isothermal Titration Calorimetry (ITC) for thermodynamic characterization.
- Employed Time-resolved Fluorescence lifetime and Temperature-jump (T-Jump) relaxation kinetics to study ms-timescale dynamics.
- Conducted Molecular Dynamics (MD) simulations to provide atomic-level insights into the binding processes.
Main Results:
- TMPyP4 formed two distinct complexes with two different Tel22 conformations under Na+ or K+ conditions.
- T-Jump experiments revealed a one-order of magnitude difference in the formation and dissociation rates of these complexes.
- MD simulations showed that in K+ buffer, the 'hybrid 1' conformation exhibited significantly lower kinetic constants with TMPyP4 compared to the 'hybrid 2' conformation, involving π-π stacking interactions.
Conclusions:
- Demonstrated for the first time that TMPyP4 interacts kinetically differently with two Tel22 conformations within the same buffer.
- These kinetic differences persist even when the resulting complexes are thermodynamically indistinguishable.
- Emphasized the critical role of kinetic analysis in differentiating ligand interactions with G-quadruplex conformational variants.
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