A comparative study of ethidium bromide complexes with dinucleotides and DNA: direct evidence for intercalation and nucleic acid sequence preferences

Abstract

The relative fluorescence quantum yields of ethidium bromide solutions with deoxyribodinucleoside monophosphates have been measured and found to correlate with similar data for the binding of ethidium to DNA. The fluorescence lifetime of ethidium has been measured both in solution and in polycrystalline 2:2 ethidium complexes with the ribodinucleoside monophosphate C-G. In aqueous solutions containing an excess of nucleotide, the experimental decay curve could be accurately fit using a single exponential decay with a lifetime of 23 ns, whereas the cocrystalline complexes exhibited two exponential decays with lifetimes of 4 and 10 ns. Detailed 1H nuclear magnetic resonance experiments have shown that in the presence of excess nucleotide the solution complex is one in which ethidium is intercalated between two Watson-Crick G-C base pairs. The observation of two spectroscopically distinct ethidiums in the 2:2 cocrystalline complexes is consistent with the structure of the 2:2 5-iodoC-G complexes determined by X-ray cocrystallographic techniques [Tsai, C. C., Jain, S. C., and Sobell, H. M. (1977), J. Mol. Biol. 114, 301-31 51 in which one ethidium was intercalated between two G-C base pairs while the second ethidium was stacked on the end of the miniature double-helical complex. We have also observed that ethidium will stack on the end of the miniature double-helical complexes at both the dinucleotide (this manuscript) and the deoxytetranucleotide levels [Kastrup, R. V., Young, M. A., and Krugh, T. R. (1978), Biochemistry 17, following paper in this issue] when there is an excess of ethidium compared to the number of favorable intercalation sites. Thus, the combined use of optical and nuclear magnetic resonance spectroscopic studies has provided a link between the solution studies and the solid-state studies. Ethidium exhibits a clear preferential binding to pyrimidine-purine deoxydinucleotides as compared to their purine-pyrimidine sequence isomers. 1H nuclear magnetic resonance experiments show that ethidium forms a miniature intercalated complex with either the self-complementary deoxydinucleotides pdC-dG, dC-dG, and pdT-dA or with a mixture of the complementary dinucleotides dT-dG plus dC-dA. The selfaggregation of ethidium was studied by monitoring the concentration dependence of the phenanthridinium protons. Ethidium aggregation can be adequately represented by dimer formation with a dimerization constant Kd ~= 95 M-' at 3 degrees C in 0.1 M NaCI. The dimerization constant was also measured at 25 degrees C (Kd ~= 70 M-I) and at both 3 and 25 degrees C in the absence of salt (Kd ~= 64 and 48 M-I, respectively). A photoncounting spectrofluorimeter was used to measure the binding of ethidium bromide to calf thymus DNA by itself and in the presence of actinomycin D and actinomine. An analysis of the binding data indicates that ethidium bromide and actinomycin D do not compete for the same binding sites (at low r values), which suggests that these drugs preferentially bind to different sequences of DNA, as suggested by the model studies with the oligonucleotides. On the other hand, ethidium bromide does compete with actinomine, which is again consistent with the visible spectral titrations of the model compounds since actinomine appears to bind equally well to both pdG-dC and pdC-dG.