Interplay of Depletion Interaction and Viscosity Drives Non-monotonic Ligand Binding to DNA G-Quadruplex under Crowding

Study of ligand binding to G-quadruplex (G4) DNA under molecular crowding is essential for understanding its implications in cellular contexts; however, the underlying mechanism remains largely unresolved. We investigate the binding of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP4) to parallel c-MYC G4-DNA under ethylene glycol/poly(ethylene glycol) (EG/PEG) crowding using steady-state and fluorescence correlation spectroscopy (FCS). The ligand binding affinity to G4 (K) exhibits a pronounced non-monotonic, U-shaped dependence on crowder size─first decreasing due to viscosity-induced hindrance and then recovering for larger PEGs. Kinetic analysis of FCS data reveals that this arises from competing effects of crowder-induced viscosity and depletion interactions on ligand association (kon) and dissociation (koff) rates. A modified Collins–Kimball–Debye formalism combined with hybrid molecular dynamics (MD) simulations shows that larger crowders create polymer-free depletion cavities that accelerate ligand–G4 association, counteracting viscous drag. These results provide a generalizable framework connecting polymer physics to nucleic acid–ligand recognition that will be useful for G4-targeted drug design in crowded cellular milieu.