Recognition of the hybrid G-quadruplex from Tetrahymena thermophila telomeric sequence by the tri-star ligand TrisQO

Telomeric G-rich DNA sequences can form G-quadruplex (G4) structures implicated in biological processes and diseases, notably cancer. Targeting such sequences with small-molecule ligands can induce and stabilize G4 formation, offering potential for therapeutic development. Here, we investigate the interaction of the 25-nt telomeric sequence from Tetrahymena thermophila (TET25) with trisquinolizinium G4 ligand, TrisQ, and its hitherto unknown oxide (TrisQO) through X-ray crystallographic and solution studies. We crystallized TET25–TrisQO complex, formed by spontaneous oxidation of TrisQ, and solved the structure at a resolution of ∼1.66 Å. In the crystal, TET25 adopts a hybrid topology and forms an interlaced dimer incorporating the symmetry-generated G1′ into the 5′-tetrad, which is different from the 5′-snapback hybrid structure adopted by unliganded TET25. TrisQO binds at the 5′-tetrad and forms a dimer with its symmetry-generated copy. The carbonyl oxygen, which differentiates TrisQO from TrisQ, is crucial for binding to TET25 as it forms a hydrogen bond with loop nucleotide T21. Notably, the presence of TET25 G4 catalyzes the oxidation of TrisQ into TrisQO in ambient conditions. In solution, both TrisQ and TrisQO cause a shift in topology of TET25 from hybrid to parallel when annealed with the DNA, but do not remodel the preformed hybrid topology. The TET25–TrisQ complex is characterized by binding stoichiometry of 1:1 with a moderate-to-strong binding affinity, Ka = (5 ± 2) 10^6 M^−1. The TET25–TrisQO complex is characterized by binding stoichiometry of 1:4 with a tight binding, although the affinity could not be quantified. Our findings contribute to the growing body of data on G4–small molecule interactions and offer insight into how G4-driven small structural modifications in a ligand can modulate G4 affinity and specificity.