The Interaction of the Silicotungstate Anion with Nucleic Acids and Preparation of Closed Circular DNA from HeLa Cell Mitochondria

Abstract

This report describes an investigation of the interaction of the silicotungstate anion with DNA in acidic solutions. The properties of equilibrium density gradients of solutions of sodium silicotungstate were determined. It was found that the silicotungstate anion binds to protonated sites in DNA with a subsequent increase in buoyant density. Denatured DNA binds silicotungstate at a higher pH than native DNA. Equilibrium dialysis measurements show that protonation must precede binding of the silicotungstate, and that the mechanism of binding between the protonated species and the anion is not simply counterion binding as required by electrical neutrality. A "local insolubility" mechanism is proposed for the binding. The application of this system to the investigation of the properties of DNA, particularly the detection of short single stranded regions in molecules, has been explored. The DNA from λ bacteriophage has been investigated with the hope of separating the linear form (with single stranded ends) from the two circular forms (hydrogen bonded and covalently closed.) This study was troubled by the decyclization of the hydrogen bonded rings in the low salt banding medium, and by difficulties in the preparation of closed circular λ DNA.