Accessibility of Telomeric Overhangs to Stabilizing Small-Molecule Ligands

Human chromosomes terminate in 50-300 nucleotide (nt) long single-stranded telomeric overhangs composed of repeating d(TTAGGG) sequences, which can fold into tandem G-quadruplex (GQ) structures that protect chromosome ends. Stabilization of GQs by small-molecule ligands inhibits telomerase activity, motivating extensive efforts to develop GQ-targeting anti-cancer therapeutics. However, how interactions between successive GQs and bound ligands influence small-molecule accessibility remains poorly understood. Here, we employ single-molecule fluorescence microscopy and stepwise photobleaching analysis to quantify the binding stoichiometry of a fluorescently-labeled oxazole telomestatin derivative (L1Cy5-7OTD) to telomeric overhangs capable of forming 1-6 GQs (30-162 nt long), spanning much of the physiologically relevant range. We find that longer overhangs accommodate more ligands on average but exhibit consistently lower binding stoichiometry than the theoretical maximum, saturating at six molecules even in constructs with twelve binding sites. This trend was further supported by experiments showing increased L1Cy5-7OTD binding when the inter-GQ spacer was extended from 3-nt to 9-nt. This effect was independently confirmed by ensemble fluorescence enhancement experiments utilizing N-methyl mesoporphyrin IX (NMM) as a ligand. Complementary modeling with a one-dimensional lattice model describing equilibrium ligand binding to partially ordered telomeric overhangs revealed positive cooperativity between folding of successive GQs, negative cooperativity between ligands bound opposing (top and bottom) faces of successive GQs, and reduced binding affinity to GQs located at the junction of double stranded and single stranded telomeres. Together, these findings demonstrate how telomeric overhang architecture governs ligand accessibility and provide mechanistic insight to guide the rational design of GQ-targeting anticancer agents.