Nonselective Intercalation of G-Quadruplex-Targeting Ligands into Double-Stranded DNA Quantified by Single-Molecule...

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• G-quadruplex (G4) targeting ligands often feature planar heteroaromatic groups for intercalation into double-stranded DNA (dsDNA).
• Quantitative data on the binding number (γ) of G4 ligands intercalating into dsDNA are crucial for understanding ligand selectivity but remain scarce.

Purpose of the Study:

• To quantitatively assess the intercalation ability, including dissociation constants (Kd) and binding numbers (γ), of eight common G4 ligands into dsDNA.
• To compare the intercalation behavior of G4 ligands with a known dsDNA intercalator (ethidium bromide) and to elucidate binding mechanisms through computational methods.

Main Methods:

• Utilized a single-molecule stretching assay based on dsDNA lengthening to measure ligand-dsDNA interactions.
• Employed molecular docking and molecular dynamics simulations to calculate binding energies and π-π stacking probabilities.
• Analyzed eight commonly used G4 ligands, including CX-5461, BRACO-19, RHPS4, TrisQ, Phen-DC3, PDS, 360A, and NMM.

Main Results:

• Five ligands (CX-5461, BRACO-19, RHPS4, TrisQ, Phen-DC3) demonstrated avid intercalation into dsDNA (Kd = 0.5–2.1 μM, γ > 0.2 ligands/bp), comparable to ethidium bromide.
• Two bisquinolines (PDS, 360A) exhibited moderate intercalation (Kd = 22.5–48.7 μM, γ < 0.01 ligands/bp at 1 μM ligand concentration).
• Porphyrin NMM showed no significant intercalative binding even at high concentrations (200 μM).

Conclusions:

• Significant variations exist in the dsDNA intercalation affinities of G4 ligands, impacting their potential selectivity.
• The study provides essential quantitative data for rational design and selection of G4 ligands for therapeutic applications.
• Computational simulations support experimental findings, offering insights into the molecular basis of G4 ligand-dsDNA interactions.