Competitive Binding of Novel Cyanine Dye AK3-5 and Europium Coordination Complexes to DNA

Abstract

The present study was undertaken to assess the applicability of the novel trimethine cyanine dye AK3-5 as a competitive ligand for the antitumor agents, Eu(III) coordination complexes (EC), in the DNA-containing systems, using the displacement assay as an analytical instrument. The analysis of fluorescence spectra revealed a strong association of AK3-5 with nucleic acids, with the strength of interaction being higher for the double stranded DNA, compared to the single-stranded RNA. The binding parameters of the cyanine dye have been determined in terms of the McGhee & von Hippel neighbouring site-exclusion model and a classical Langmuir model. The AK3-5 association constant in the presence of DNA was found to be equal to 5.1×104 M-1, which is consistent to those of the well-known DNA intercalators. In turn, the binding of the cyanine to the RNA was characterized by a significantly lower association constant ( ~ 3.4×103 M-1) indicating either the external or “partially intercalated” binding mode. The addition of the europium complexes to the AK3-5-DNA system was followed by the fluorescence intensity decrease, with a magnitude of this effect being dependent on the EC structure. The observed fluorescence decrease of AK3-5 in the presence of europium complexes V7 and V9 points to the competition between the cyanine dye and antitumor drugs for the DNA binding sites. The dependencies of the AK3-5-DNA fluorescence intensity decrease vs. europium complex concentration were analyzed in terms of the Langmuir adsorption model, giving the values of the drug association constant equal to 5.4×104 M-1and 3.9×105 M-1 for the europium complexes V7 and V9, respectively. A more pronounced decrease of the AK3-5 fluorescence in the presence of V5 and V10 was interpreted in terms of the drug-induced quenching of the dye fluorescence, accompanying the competition between AK3-5 and Eu(III) complexes for the DNA binding sites. Cumulatively, the results presented here strongly suggest that AK3-5 can be effectively used in the nucleic acid studies and in the dye-drug displacement assays.

Novel cyanine dye as competitive ligand for probing the drug–nucleic acid interactions

“…Next, the dependencies of the AK3-5 fluorescence decrease with increasing concentration of europium complexes were analyzed in terms of the competition model proposed by Gaugain et al (Eqs. 2, 3) to obtain the association constants of the drug binding to DNA or RNA [26,29]. The calculated values of this parameter were estimated to be (1.8±0.4)×10 4 M -1 and (5±1.1)×10 5 M -1 for V7 and V9, respectively, regardless of the DNA concentration in the tested sample.…”

“…The association constants for the dye-DNA and the dye-RNA complexes were found to be (5.1±1.1)×10 4 M -1 , (3±0.6)×10 3 M -1 , respectively, while the site exclusion parameters were equal to 2 for both systems. Based on the analysis of the available information [12,14,16] on the nucleic acid interactions of intercalating agents the following two tendencies can be highlighted: i) the highest possible dye-base pair ratio is 1:2, since, according to the principle of the nearest neighbor exclusion, the binding of one intercalating molecule between the two base pairs hinders the access of the next binding site to another intercalator [12,14,16,29]; ii) the association constants for intercalators do not exceed 10 5 M -1 [12,14,16]. Allowing for the fact that the association constants and the neighbor exclusion parameters recovered here for the novel trimethine dye under study in the presence of the double stranded DNA and single stranded RNA are in accordance with those observed for typical intercalators, we assumed that the examined dyes associate with nucleic acids via intercalating binding mode.…”

“…Recently we demonstrated that the cyanine dye AK3-5 can be employed in the fluorescence displacement assay [29]. Specifically, we have evaluated the ability of the potential pharmacological agents, Eu(III) tris-β-diketonate to coordination complexes to dislodge the AK3-5 dye bound to the double stranded DNA [29].…”

“…Recently we demonstrated that the cyanine dye AK3-5 can be employed in the fluorescence displacement assay [29]. Specifically, we have evaluated the ability of the potential pharmacological agents, Eu(III) tris-β-diketonate to coordination complexes to dislodge the AK3-5 dye bound to the double stranded DNA [29]. In a continuation of our interest in fluorescent dyes suitable for the drug displacement analysis on the nucleic acid, herein we directed our efforts towards investigation of the ability of a novel cyanine dye AK3-5 to serve as a competitor for europium coordination complexes for DNA and RNA binding sites.…”

“…3, Eu(III) coordination complexes produced a dosedependent fluorescence decrease reflecting the competition between the cyanine dye and EC for the binding sites on DNA or RNA. Notably, the magnitude of the fluorescence intensity decrease in the RNA-containing systems, similarly to the DNA-containing systems, was more G. Gorbenko, T. Deligeorgiev pronounced for V5 and V10 compared to other europium complexes under study [29]. Next, the dependencies of the AK3-5 fluorescence decrease with increasing concentration of europium complexes were analyzed in terms of the competition model proposed by Gaugain et al (Eqs.…”