DNA bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer. Appendix: Numerical solution of McGhee and von Hippel equations for competing ligands

Abstract

An ethidium homodimer and an acridine ethidium heterodimer were synthesized (Gaugain, et al. (1978)) and their binding to DNA was studied. The dimers intercalate only 1 of their chromophores in DNA. At high salt concentration (Na+ > 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the McGhee and Von Hippel treatment (McGhee and Von Hippel, (1974)). In these conditions the dimers cover 4 base pairs when bound to DNA. Binding affinities were deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. The intrinsic binding constant of the dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 .times. 108 M-1; ethidium bromide K = 1.5 .times. 105 M-1 in 0.2 M Na+). The fluorescence properties of the molecules were also studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore in the acridine ethidium heterodimer when bound to DNA depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acids makes these molecules extremely useful as nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.