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 ...

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.

The kinetics of opening of the DNA quadruplex formed by the human telomeric repeat have been investigated using real-time fluorescence resonance energy transfer (FRET) measurements with a peptide nucleic acid (PNA) trap. It has been found that this opening is zero-order with respect to PNA, indicating that the initial step is a rate-limiting internal rearrangement of the quadruplex. A study of the temperature dependence of the rate of quadruplex opening was performed and the activation energy of the process estimated to be 98 +/- 8 kJ mol(-1).

We have investigated the interaction of the intramolecular human telomeric DNA G-quadruplex with a hemicyanine-peptide ligand, by studying the rate of quadruplex opening with a complementary DNA oligonucleotide. By employing a minimal kinetic model, the relationship between the observed rate of quadruplex opening and the ligand concentration has enabled estimation of the dissociation constant. A van't Hoff analysis revealed the enthalpy and entropy changes of binding to be −77 ± 22 kJ mol −1 and −163 ± 75 J mol −1 K −1 , respectively. Arrhenius analyses of the rate constants of opening free and bound quadruplex gave activation energies of 118 ± 2 and 98 ± 10 kJ mol −1 , respectively. These results indicate that the presence of the ligand has only a small effect on the activation energy, suggesting that the unbinding of the ligand occurs after the transition state for quadruplex unfolding.

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