Folding Landscape of a Parallel G‑Quadruplex

Circular dichroism and stopped-flow UV spectroscopies were used to investigate the thermodynamic stability and the folding pathway of d­[TGAG₃TG₃TAG₃TG₃TA₂] at 25 °C in solutions containing 25 mM KCl. Under these conditions the oligonucleotide adopts a thermally stable, all-parallel G-quadruplex topography containing three stacked quartets. K⁺-induced folding shows three resolved relaxation times, each with distinctive spectral changes. Folding is complete within 200 s. These data indicate a folding pathway that involves at least two populated intermediates, one of which seems to be an antiparallel structure that rearranges to the final all-parallel conformation. Molecular dynamics reveals a stereochemically plausible folding pathway that does not involve complete unfolding of the intermediate. The rate of unfolding was determined using complementary DNA to trap transiently unfolded states to form a stable duplex. As assessed by 1D-¹H NMR and fluorescence spectroscopy, unfolding is extremely slow with only one observable rate-limiting relaxation time.