Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA.

Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium-quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium-DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet.

Flexibility and curvature of duplex DNA containing mismatched sites as a function of temperature.

The flexibility and curvature of duplex DNAs containing mismatched sites have been monitored as a function of temperature. The diffusion coefficients are dependent on the flexibility and the curvature of the DNA, and these have been determined by NMR-based methods. The diffusion coefficients, D, depend on a Boltzmann term and the viscosity of the solvent, eta, which is also temperature dependent. To analyze the temperature dependence of the diffusion results, the shape function, S(f) = etaD/T, is used. The shape functions do not have the viscosity and temperature dependence of the diffusion coefficients. The presence of mismatched sites significantly enhances the shape function of duplex DNA at all temperatures examined. The observed increases in the shape functions are attributed to the mismatched sites acting as localized flexible joints. The results on the temperature dependence of the shape functions, the optical absorbance, and the proton chemical shifts indicate that local melting at, and adjacent to, mismatched site occurs at a lower temperature than the overall melting of the duplexes. The localized melting gives rise to a considerable increase in the shape function. The contribution of the curvature of the mismatched sites to the enhanced diffusion has been examined. A DNA with mismatches that are in phase with respect to the helical repeat and a DNA which has the mismatches out of phase with respect to the helical repeat have been examined. The results indicate that mismatched sites have modest curvature.

6-Thioguanine alters the structure and stability of duplex DNA and inhibits quadruplex DNA formation.

The ability to chemically synthesize biomolecules has opened up the opportunity to observe changes in structure and activity that occur upon single atom substitution. In favorable cases this can provide information about the roles of individual atoms. The substitution of 6-thioguanine (6SG) for guanine is a potentially very useful single atom substitution as 6SG has optical, photocrosslinking, metal ion binding and other properties of potential utility. In addition, 6-mercaptopurine is a clinically important pro-drug that is activated by conversion into 6SG by cells. The results presented here indicate that the presence of 6SG blocks the formation of quadruplex DNA. The presence of 6SG alters the structure and lowers the thermal stability of duplex DNA, but duplex DNA can be formed in the presence of 6SG. These results indicate that some of the cytotoxic activity of 6SG may be due to disruption of the quadruplex structures formed by telomere and other DNAs. This additional mode of action is consistent with the delayed onset of cytotoxicity.

Determinants of DNA quadruplex structural type: sequence and potassium binding.

There are DNA sequences which adopt the same quadruplex structural type in the presence of sodium as in the presence of sodium and potassium. There are also sequences that appear to have a requirement for the presence of potassium for the adoption of a particular quadruplex structural type. Information about the basis for these potassium effects has been obtained by examining the structures of a set of DNAs with differing numbers of loop residues and different lengths of runs of dG residues in the presence of sodium alone and in the presence of potassium and sodium. On the basis of the results, obtained primarily via solution-state NMR, it appears that very small loops favor parallel stranded quartet structures which do not require the presence of potassium. DNAs with loops of two to four residues and runs of two dG residues can form quadruplex structures of the "edge" or "chair" type in the presence of potassium but not in the presence of sodium alone. When all of the loops contain four residues, a "crossover" or "basket" type structure can be formed in the presence of sodium as well as in the presence of sodium and potassium. Structures with runs of three or four dG residues and with loops from two to four residues can form basket or crossover type structures in the absence of potassium. The presence of a purine in a loop can block both potassium binding and formation of chair type structures. Modeling of the interactions of cations with these quadruplex structures indicates that the potassium ions required for chair type structures interact with a terminal quartet and residues in the adjacent loop.

Damage increases the flexibility of duplex DNA.

It is proposed that much of the recognition of specific types of damaged DNAs is based on accessible structural features, while much of the recognition of damaged DNAs, as a class, is based on flexibility. The more flexible a DNA the faster its diffusion rate. The diffusion rates of each member of a series of damaged duplex DNAs has been found to be significantly faster than that of the corresponding undamaged duplex DNA. The damaged sites studied include apurinic and apyrimidinic a basic sites, thymine glycol and urea. The presence of mismatched sites also increases the diffusion. Thus, damaged DNAs appear to have sufficient flexibility for recognition and the flexibility may allow damaged sites to act as a universal joint or hinge that allows distant sites on the DNA to come together.

Determination of internuclear angles of DNA using paramagnetic-assisted magnetic alignment.

Paramagnetic ions have been used to assist the magnetic alignment of DNA. The anisotropy of the binding sites is sufficient to give rise to significant alignment of the DNA with the observed proton-carbon dipolar couplings spanning a 70-Hz range. The dipolar couplings have been used to determine the positions of the axial and rhombic alignment axes. The positions of the alignment axes relative to the positions of the binding sites of the paramagnetic europium ions have also been determined.

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR in the presence and absence of thrombin.

The interaction of a DNA quadruplex with thrombin has been studied by first determining the sites of manganese binding to the quadruplex in the absence of thrombin. This has been followed by determining if the interactions with thrombin displace the bound manganese. A different DNA quadruplex has also been studied as a control. The refined solution structures of two DNA quadruplexes have been used to predict the electrostatic potentials of these DNAs. The calculated electrostatic potentials have been used to predict the locations of the binding sites of the paramagnetic ion manganese to these DNAs. The enhanced relaxation of DNA protons due to the binding of the paramagnetic metal ion Mn2+ has been used to experimentally determine the locations of the binding sites. The NMR results and the predictions based on the electrostatic potentials both place the binding sites of the manganese in the narrow grooves of these quadruplex DNAs. The predicted locations are spatially close to those experimentally observed, and the predicted and experimental locations also have similar electrostatic potential energy. These results have allowed a validation of the predictions of electrostatic potentials from structure. The 15mer quadruplex has two strong Mn2+ binding sites with one in each narrow groove. Both Mn2+ are released when the 15mer is complexed with thrombin, indicating that both narrow grooves are involved in the 15mer-thrombin interactions. The dimer quadruplex has a different structural motif than the 15mer and the presence of thrombin does not appreciably affect its interactions with Mn2+.