Journal of Molecular Biology
Volume 426, Issue 8, 17 April 2014, Pages 1629-1650
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Folding and Unfolding Pathways of the Human Telomeric G-Quadruplex

https://doi.org/10.1016/j.jmb.2014.01.009Get rights and content
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Highlights

  • Folding and unfolding pathways for the human telomere quadruplex are defined by kinetic studies using CD, FRET, and 2-aminopurine fluorescence.

  • Folding and unfolding reactions are slow and complex with sequentially formed intermediates.

  • Spectra of reaction intermediates are captured for the first time.

  • Folding to the final quadruplex form occurs through antiparallel chair and triplex forms.

Abstract

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 = 0.1 s 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  3700 s) 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  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[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.

Abbreviations

2-AP
2-aminopurine
FRET
fluorescence resonance energy transfer
6-Fam
6-carboxyfluorescein
Tamra
5-carboxytetramethylrhodamine
SVD
singular value decomposition

Keywords

kinetics
stopped-flow
folding intermediates
triplex
circular dichroism

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