Bernard Gaugain

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been 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. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been 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 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.

An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.

For studies on the possible involvement of hydrogen bonding in base recognition from the outside of the nucleic acid double helix, 2-methoxy-6-chloro-9-aminoacridine derivatives bearing a carboxamide side chain were examined by 1H NMR spectroscopy. The study of the interaction of these derivatives with CpG or GpC demonstrated that (i) the 2-methoxy-6-chloro-9-aminoacridine ring intercalates preferentially in the minihelix formed by CpG, which indicates a relative pyrimidine-(3'-5')-purine sequence specificity that contrasts with the simple 9-aminoacridine ring wherein Reuben et al. [Reuben, J., Baker, B. M., & Kallenbach, N. R. (1978) Biochemistry 17, 2916-2919] did not observe any sequence preference (ii) the geometry of the intercalated minihelical complex of the 2-methoxy-6-chloro-9-[(5-carbamolypentyl)-amino]acridine with CpG as deduced from isoshielding curves resembles that found in the crystalline complexes of proflavin, with several autocomplementary dinucleoside monophosphates, (iii) the terminal carboxamide group borne by the side chain of 2-methoxy-6-chloro-9-[(5-carbamoylpentyl)amino]acridine (5) intercalated in CpG lies in the small groove and seems to interact through hydrogen bonds with the adjacent guanine.

The DNA binding characteristics of a mono-, di- and trimeric derivative of 9-aminoacridine were studied. The length of the linking carboxamidoalkyl chains was selected to allow bis- or tris-intercalation according to the excluded-site model. Measurements of DNA unwinding angle using closed circular DNA showed that the trimeric derivative behaves as a tris-intercalating agent. Nevertheless the increase of DNA binding affinity on going from dimer to trimer was found to be relatively small. This is probably related to the large structural constraint for DNA binding of the trimeric derivative. The nature of the linking chain for the design of high-affinity DNA poly-intercalating agents appears therefore critical.

Comparative 1H NMR and optical studies of the interaction between poly(dA-dT), ethidium bromide (Et) and ethidium dimer (Et2) in 0.7 M NaCl are reported as a function of the temperature. Denaturation of the complexes followed at both polynucleotide and drug levels leads to a biphasic melting process for poly(dA-dT) complexed with ethidium dimer (t1/2 = 75 degrees C; 93 degrees C) but a monophasic one in poly(dA-dT): ethidium bromide complex (t1/2 = 74 degrees C). In both cases drug signals exhibit monophasic thermal dependence (Et = 81 degrees C; Et2 = 95 degrees C). Evidence is presented showing that the ethidium dimer bisintercalates into poly(dA-dT) in high salt, based on the observation that i) dimer and monomer ring protons exhibit similar upfield shifts upon DNA binding, ii) upfield shifts of DNA sugar protons are twice as large with the dimer than with ethidium bromide. Comparison between native DNA fraction and bound drug fraction indicates that ethidium covers, n = 2.5-3 base pairs. The dimer bisintercalates and covers, n = 5.7 base pairs when the helix fraction is high but as the number of available sites decreases the binding mode changes and the drug monointercalates (n = 2.9).