Biochemical and Biophysical Properties of Positively Supercoiled DNA

Abstract

Here we studied the biophysical and biochemical properties of the positively supercoiled DNA under different conditions. Large amount of positively supercoiled DNA is difficult to obtain compared to the natural-occurring negatively supercoiled DNA. Therefore, the biophysical and biochemical properties have not been fully explored. In this report we utilized Archaeoglobus fulgidus DNA reverse gyrase, which was expressed in E. coli strain BL21(DE3)/pLysS and purified using an optimized purification scheme, to convert the negatively supercoilied plasmid DNA to the positively supercoiled DNA. The positively supercoiled DNA was further purified by the CsCl-EB density-gradient centrifugation. After we obtained a few milligrams of the positively supercoiled DNA template, biochemical and biophysical properties were characterized using different techniques, such as UV melting, circular dichroism, AFM (atomic force microscope), fluorescence spectrometry, DNA topoisomerase assays, and etc. Our results are expected to provide the basis for the utilization of the (+) supercoiled DNA for future biomedical applications, such as the use of (+) supercoiled DNA templates for the screening of anti-gyrase antibiotics.