Effect of molecular crowders on ligand binding kinetics with G-quadruplex DNA probed by fluorescence correlation spectroscopy

Abstract

Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect-a nearly three-fold decrease-observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.

Keywords: G-quadruplex DNA; fluorescence correlation spectroscopy; ligand interaction kinetics; saccharide molecular crowder.