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 <-> I-1 <-> I-3 <-> <-> F where U and F represent unfolded and folded conformational ensembles and I-1, I-2, and I-3 are intermediates. Previous kinetic studies have shown that I-1 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 I-1 converts to I-2 with a relaxation time T-1 = 0.1 s at 25 degrees C in 25 mM KCl. The CD spectrum of I-2 is characteristic of an antiparallel quadruplex that could form as a result of intramolecular fold-over of the I-1 hairpins. I-3 is relatively slowly formed (T-2 approximate to 3700 s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I-3 converts to F with T-3 approximate to 750 s. 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[TTAGGG(TTAGGG)(3)A] and d[TAGGG(TTAGGG)(3)TT]}. 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. (C) 2014 Elsevier Ltd. All rights reserved.