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Kinetic Stability of Intermolecular DNA Quadruplexes

School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, United Kingdom

Correspondence: Address reprint requests to Keith R. Fox, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK. Tel.: 44-23-8059-4374; Fax: 44-23-8059-4459; E-mail: K.R.Fox{at}soton.ac.uk.

Fluorescently 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 T_m values that are too high to measure. T_m 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.

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