Spectrofluorometry of dyes with DNAs of different base composition and conformation

The increase in fluorescence, upon interaction with several fluorescent dyes was found to depend on the base composition of DNA. 4',6-Diamidino-2-phenylindole-2 HCl and Hoechst 33258 which bind to AT base pairs show a logarithmic relation. This relation is linear when DNAs interact with mithramycin, chromomycin A3, and olivomycin, which bind to GC base pairs. Deviations from these relationships were observed for T2 DNA, containing hydroxymethylcytosine, and for 2C DNA, containing hydroxymethyluracil. On the basis of these data, a simple technique is proposed for determination of base composition. The presence of abnormal bases can be monitored by the use of given fluorophores. Fluorescence intensities were not modified upon linearization of covalently closed circular plasmid pBR322. Denaturation of lambda DNA was accompanied by a decrease of fluorescence, when complexed with the five dyes tested.