Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer

doi:10.1073/pnas.2433350100

Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

Edited by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved October 2, 2003 (received for review June 3, 2003)

Abstract

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 ± 0.4 kcal·mol^–1 and –52.3 ± 1.4 cal·mol^–1·K^–1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 ± 0.2 kcal·mol^–1 and –23.0 ± 0.8 cal·mol^–1·K^–1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 k _B T based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.

Footnotes

↵ ‡ To whom correspondence may be addressed. E-mail: dk10012{at}cam.ac.uk or sb10031{at}cam.ac.uk.
↵ † L.Y. and J.J.G. contributed equally to this work.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: HTIQ, human telomeric intramolecular quadruplex; FRET, fluorescence resonance energy transfer; TMR, tetramethylrhodamine.
↵ § The difference between the melting temperatures of I and IV means that we cannot rule out the possibility that the presence of Cy5 may influence the relative populations of the two conformations.
↵ ¶ Another possibility considered for the observed populations was a “hinging motion” of the quadruplex/duplex linker, which is a stable “stacked” structure resulting from π interaction between the terminal base pair of the duplex and a terminal G tetrad. Molecular modeling predicted that the energy of such a stacked conformer was ≈10 kcal·mol^–1 higher than that of the extended structure presented in Fig. 6A. We expect that a change from a stacked to an extended conformation would be accompanied by an increase in entropy due to the greater freedom of the linker in this conformation. However, our results show that the entropy difference between the species changes sign in different buffer conditions, which is inconsistent with this model.
↵ ∥ The measured quantum yield of TMR in II·IV is 0.81, leading to a calculated Förster radius, R ₀, of 7.1 nm, assuming an orientational factor, κ², of 2/3. This value is higher than commonly measured for the TMR/Cy5 pair, largely because of the high quantum yield of TMR in this system.
↵ †† Our assignment of the two conformations is insensitive to R ₀ for values >4 nm.