Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

Abstract

The fluorescein properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form I.degree.) conformation in order to determine whether qualitative or quantitative differences in fluorecence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of .apprx. 8 nm was observed forHO 33342 complexed with Form I or Form I.degree.. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form I.degree. DNA; however, when Form I and Form I.degree.were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different from those of HO 33342 complexed with Form I.degree. DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.