Folding and unfolding pathways of the human telomeric G-quadruplex

Sequence analogs of human telomeric DNA such as d[AGGG(TTAGGG)3] (Tel22) fold into monomeric quadruplex structures in the presence of a suitable cation. To investigate the pathway for unimolecular quadruplex formation, we monitored the kinetics of K(+)-induced folding of Tel22 by circular dichroism (CD), intrinsic 2-aminopurine fluorescence, and fluorescence resonance energy transfer (FRET). The results are consistent with a four-step pathway U ↔ I1 ↔ I2 ↔ I3 ↔ F where U and F represent unfolded and folded conformational ensembles and I1, I2, and I3 are intermediates. Previous kinetic studies have shown that I1 is formed in a rapid pre-equilibrium and may consist of an ensemble of "prefolded" hairpin structures brought about by cation-induced electrostatic collapse of the DNA. The current study shows that I1 converts to I2 with a relaxation time τ1=0.1s at 25 °C in 25 mM KCl. The CD spectrum of I2 is characteristic of an antiparallel quadruplex that could form as a result of intramolecular fold-over of the I1 hairpins. I3 is relatively slowly formed (τ2≈3700s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I3 converts to F with τ3≈750s. Identical pathways with different kinetic constants involving a rapidly formed antiparallel intermediate were observed with oligonucleotides forming mixed parallel/antiparallel hybrid-1 and hybrid-2 topologies {e.g. d[TTGGG(TTAGGG)3A] and d[TAGGG(TTAGGG)3TT]}. Aspects of the kinetics of unfolding were also monitored by the spectroscopic methods listed above and by time-resolved fluorescence lifetime measurements using a complementary strand trap assay. These experiments reveal a slow, rate-limiting step along the unfolding pathway.