2 research outputs found
Kinetic stability of intermolecular DNA quadruplexes
No full textFluorescently labeled oligodeoxyribonucleotides containing a single tract of four successive guanines have been used to study the thermodynamic and kinetic properties of short intermolecular DNA quadruplexes. When these assemble to form intermolecular quadruplexes the fluorophores are in close proximity and the fluorescence is quenched. On raising the temperature these complexes dissociate and there is a large increase in fluorescence. These complexes are exceptionally stable in potassium-containing buffers, and possess Tm values that are too high to measure. Tm values were determined in sodium-containing buffers for which the rate of reannealing is extremely slow; the melting profiles are effectively irreversible, and the apparent melting temperatures are dependent on the rates of heating. The dissociation kinetics of these complexes was estimated by rapidly increasing the temperature and following the time-dependent changes in fluorescence. From these data we have estimated the half-lives of these quadruplexes at 37°C. Addition of a T to the unlabeled end of the oligonucleotide increases quadruplex stability. In contrast, addition of a T between the fluorophore and the oligonucleotide leads to a decrease in stability.<br/
A polarized-light spectroscopy study of interactions of a hairpin polyamide with DNA
No full textWe here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding
