Enhancement of the thermal stability of G-quadruplex structures by urea.

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5'-GGGTGGGTGGGTGGG-3']), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

The Pressure Dependence of the Stability of the G-quadruplex Formed by d(TGGGGT).

The G-quadruplex (GQ), a tetrahelix formed by guanine-rich nucleic acid sequences, is a potential drug target for several diseases. Monomolecular GQs are stabilized by guanine tetrads and non-guanine regions that form loops. Hydrostatic pressure destabilizes the folded, monomolecular GQ structures. In this communication, we present data on the effect of pressure on the conformational stability of the tetramolecular GQ, d[5'-TGGGGT-3']4. This molecule does not have loops linking the tetrads; thus, its physical properties presumably reflect those of the tetrads alone. Understanding the properties of the tetrads will aid in understanding the contribution of the other structural components to the stability of GQ DNA. By measuring UV light absorption, we have studied the effect of hydrostatic pressure on the thermal stability of the tetramolecular d[5'-TGGGGT-3']4 in the presence of sodium ions. Our data show that, unlike monomolecular GQ, the temperature at which d[5'-TGGGGT-3']4 dissociates to form the constituent monomers is nearly independent of pressure up to 200 MPa. This implies that there is no net molar volume difference (∆V) between the GQ and the unfolded random-coil states. This finding further suggests that the large negative ∆V values for the unfolding of monomolecular GQ are due to the presence of the loop regions in those structures.

Dimethyl sulfoxide (DMSO) is a stabilizing co-solvent for G-quadruplex DNA.

We report the effect of dimethyl sulfoxide (DMSO) on the stability of the four-stranded structures formed by the oligodeoxyribonucleotides d[5'-AGGG(TTAGGG)3-3'] (HTel), d[5'-(GGGT)3GGG-3'] (G3T), d[5'-GGTTGGTGTGGTTGG-3] (TBA), d[5'-GGGGTTTTGGGG-3'] (Oxy-1.5), and d[5'-TGGGGT-3'] (TG4T). In these measurements, influence of the co-solvent was assessed by the change in the mid-point of the heat-induced unfolding, Tm, by monitoring the change in the UV absorption of the sample. Increasing concentrations of DMSO led to an increase in the Tm from the folded to unfolded states. We have also studied the effect of the denaturant urea and mixtures of urea and DMSO on the stability of the intramolecular HTel and the intermolecular TG4T G-quadruplexes. Consistent with earlier data, we found that urea destabilized the folded G-quadruplex structure; the Tm decreases with increasing urea concentration. However, in solutions containing both urea and DMSO, we observed that the two co-solvents off-set the destabilizing and stabilizing effect, respectively, of one another. That is, in solutions containing urea, increasing concentrations of DMSO led to the increase of the Tm of the G-quadruplex structure. This effect is observed in solutions containing sodium, potassium, or ammonium as the ion that stabilizes the folded G-quadruplex structure. The complementary effect of the two co-solvents presumably arises from differential interactions between urea and DMSO and the oligonucleotide or the cations involved in the stabilization of the G-quadruplexes. These results highlight the importance of co-solutes and co-solvents in systems containing guanine-rich DNA, particularly experimental processes that require DMSO.

Volumetric Interplay between the Conformational States Adopted by Guanine-Rich DNA from the c-MYC Promoter.

The kinetic and thermodynamic stabilities of G-quadruplex structures have been extensively studied. In contrast, systematic investigations of the volumetric properties of G-quadruplexes determining their pressure stability are still relatively scarce. The G-rich strand from the promoter region of the c-MYC oncogene (G-strand) is known to adopt a range of conformational states including the duplex, G-quadruplex, and coil states depending on the presence of the complementary C-rich strand (C-strand) and solution conditions. In this work, we report changes in volume, ΔV, and adiabatic compressibility, ΔKS, accompanying interconversions of G-strand between the G-quadruplex, duplex, and coil conformations in the presence and absence of C-strand. We rationalize these volumetric characteristics in terms of the hydration and intrinsic properties of the DNA in each of the sampled conformational states. We further use our volumetric results in conjunction with the reported data on changes in expansibility, ΔE, and heat capacity, ΔCP, associated with G-quadruplex-to-coil transitions to construct the pressure-temperature phase diagram describing the stability of the G-quadruplex. The phase diagram is elliptic in shape, resembling the classical elliptic phase diagram of a globular protein, and is distinct from the phase diagram for duplex DNA. The observed similarity of the pressure-temperature phase diagrams of G-quadruplexes and globular proteins stems from their shared structural and hydration features that, in turn, result in the similarity of their volumetric properties. To the best of our knowledge, this is the first pressure-temperature stability diagram reported for a G-quadruplex.