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

Abstract

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using singlemolecule 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 kcal⅐mol ؊1 and ؊52.3 ؎ 1.4 cal⅐mol ؊1 ⅐K ؊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 kcal⅐mol ؊1 and ؊23.0 ؎ 0.8 cal⅐mol ؊1 ⅐K ؊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 BT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale. DNA sequences with stretches of multiple guanines can form four-stranded structures called quadruplexes (1). The evidence is growing that quadruplexes may be important for several biological mechanisms (1-8). Evidence supports the occurrence of quadruplexes in the cell nucleus (9), and, furthermore, a recent study suggests that particular quadruplexes may have the potential to control gene expression (10). The intramolecular DNA quadruplex based on the human telomeric repeat sequence, d(TTAGGG), has been the subject of extensive biophysical and biological studies. It has been demonstrated, in vitro, that formation of this structure impedes the extension mechanism of human telomerase (3). Furthermore, stabilization of this quadruplex may be a natural or unnatural mechanism to influence the regulation of teleomeres at the ends of chromosomes. Because telomerase function and telomere maintenance are critical for the division of cancer cells, the human telomeric intramolecular quadruplex (HTIQ) is being seriously considered as a potential molecular target for the development of novel anticancer agents (7,8). Detailed structural studies have provided evidence for two distinct folds for the HTIQ in the presence of sodium and potassium ions (11,12). An NMR spectroscopic study, where the dominant monovalent cation was sodium, showed that the HTIQ exists predominantly with an antiparallel arrangement of strands with a diagonal loop across a terminal tetrad and edgewise loops (11) (see Fig. 7A, which is published as supporting information on the PNAS web site). In contrast, a recent x-ray crystal structure of the same oligonucleotide, in the presence of potassium, shows all four strands to be parallel ...

Extreme conformational diversity in human telomeric DNA

“…If the same is true for the unimolecular construct studied here, F 1 would correspond to the parallel conformation and F 2 to the antiparallel conformation. Circular dichroism measurements of the same DNA sequence and labeling also suggested that at room temperature and at 100 mM K ϩ the structure is mostly antiparallel (20), further supporting our assignment. Thus, our data suggest that the human telomeric DNA in physiological conditions (37°C, 140 mM K ϩ ) (28) is primarily folded with comparable populations of parallel and antiparallel conformations.…”

“…This observation also makes it very unlikely that F 1 and F 2 are different only in the dye properties. Even though fluorescent labeling does have some effects in that it slightly increased the stability of the G-quadruplex in 100 mM K ϩ (20), distinct conformations seen here are not likely to be caused by mere changes in dyes because interconversion between them requires G-quadruplex unfolding.…”

“…More recently, it was shown by NMR studies that parallel and antiparallel forms are in dynamic equilibrium at 100 mM K ϩ (19). Earlier single-molecule fluorescence resonance energy transfer (FRET) measurements of freely diffusing human telomeric DNA molecules detected two populations of differing FRET values (20). The two forms did not interconvert during the time a molecule spends in the excitation volume of a confocal microscope (Ͻ1 ms), and both conformations could be made into duplex structures in minutes by means of hybridization to a complementary strand, suggesting that the dynamic interconversion between different conformations may occur in the time window between 1 ms and a few minutes.…”

“…Its 3Ј end is modified by biotin for immobilization. The complementary stem strand is labeled with tetramethylrhodamine internally via amino-modified C6 dT (20). DNA was annealed with 2:1 mixture of the stem and the G-quadruplex strands by heating the mixture to 95°C and then cooling down to room temperature slowly.…”

Fluorescence of unmodified oligonucleotides: A tool to probe G‐quadruplex DNA structure

“…[9][10][11][12][13][14] Fluorescence resonance electron transfer (FRET) in molecular beacons has been exploited in the detection of folded and unfolded states of oligonucleotides. [15][16][17] These changes have been coupled with sensing capabilities to detect a variety of analytes. FRET also has been used to detect movements of nanomachines.…”