How Do Molecular Crowders Influence Ligand Binding Kinetics with G-Quadruplex DNA? The Role of Bound Water

Understanding the kinetics of ligand interaction with G-quadruplex DNA (GqDNA) in a crowded cell-like environment is of paramount importance in biology and pharmacology, as it elucidates the effect of molecular crowders on reaction rates governing these interactions─a process that largely remains unexplored. In this study, we investigate the binding/unbinding kinetics of a G-quadruplex stabilizing benzophenoxazine ligand, cresyl violet (CV), with a human telomeric hybrid GqDNA structure using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. Experiments are conducted with and without 10% and 20% (w/v) ethylene glycol (EG), PEG200 and PEG6000 crowders. The steady-state fluorescence results reveal a reduction in the ligand binding affinity to GqDNA as the size and concentration of the crowders increase. FCS data further demonstrate that the crowder-induced reduction in binding affinity is primarily driven by the viscosity-induced decrease in the association rate (<i>k</i>₊) and a competing excluded volume effect, as well as a concomitant increase in the dissociation rate (<i>k</i>_-) of the ligand. Atomistic MD simulations highlight the key role of strong electrostatic forces between the G-tetrad and π-stacked ligand, along with long-lived water-mediated hydrogen-bond bridges, in stabilizing the ligand/GqDNA complex in the absence of crowders. However, in the presence of EG/PEG crowders, the ligand binding mode is disrupted by hydrogen-bond interactions of the crowders with the ligand, causing rotation of the ligand's molecular plane relative to the G-tetrad. This disruption weakens the π-stacking electrostatic forces between the ligand and the G-tetrad and breaks the long-lived water-mediated hydrogen-bond bridges between the ligand and GqDNA, destabilizing the ligand/GqDNA complex. The current investigation underscores the prominent role of hydrogen-bond interactions of EG/PEG crowders, along with other factors, in affecting the stability of the ligand/GqDNA interaction in a crowded milieu.