Characterization of intermolecular G-quadruplex formation over intramolecular G-triplex for DNA containing three G-tracts.

G-triplex (G3) has been recognized as the popular intermediate during folding of G-quadruplex (G4). This raise the interest to anticipate the ultimate formation of G3 by shorting the G4-forming oligonucleotides with remaining three G-tracts. Some G3 structures have been validated and their stability is also affected by the loop sequences as occurred to G4s. In this work, however, we first found that an intermolecular parallel G4 structure is preferred in K+ for the oligonucleotide of 5′-TGGGTAGGGCGGG-3′ (DZ3) containing only three G-tracts. We screened auramine O (AO) as the appropriate fluorophore with a molecular rotor feature to target this G4 structure. AO binds with DZ3 according to a 1:4 mode as confirmed by isothermal titration calorimetry experiments, suggesting formation of a tetramolecular G4 structure (4erG4). Excimer emission from the labelled pyrene and the DNA melting behaviors at variant pH and in the presence of Ag+ prove this 4erG4 structure, rather than the prevalent intramolecular G3 folding. This work demonstrates that one should be cautious in putatively predicting a G3 structure from an oligonucleotide containing three G-tracts.