Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate–DNA interactions for the analysis of lipoplex formation in gene therapy

Supramolecular Self-Assembling Cyanine as an Alternative to Ethidium Bromide Displacement in DNA-Drug Model Interactions during High Throughput Screening

“…23,24 During HTS, the compound library might be dispensed in microwells containing DNA. The robotics/liquid handling system dispenses cyanine to the wells and fluorescence is measured for rapid analyses of DNA-drug interactions.…”

“…In studies with EB, polyamines were used for DNA-model drug interactions or as potential gene delivery vehicles. 9,11,12,[22][23][24] Similar to DNA-drug interactions, cyanines can intercalate or self-assemble upon DNA double helix. 7,10,25 Despite extensive studies, the exact mechanism of polyamines binding to DNA remains to be finalized; 9,21 our studies might aid in the further elucidation of these interactions.…”

“…These factors contribute to delocalized and sequence-dependent binding of polyamines to DNA. 9,11,20,23,24,26,27 Given the variety of factors influencing DNA-spermine interactions, the ability of our supramolecular self-assembling cyanine to displace the polyamine was remarkable. Dynamics of the formation and disassembly of the complex detected through fluorescence might arise from fluorescence quench by spermine or a competition between the polyamine and the dye for the same or similar binding sites on DNA.…”

“…Reciprocal of IC50 transforming into Ka 11,12,28,29 assumes that spermine and cyanine compete for the same/similar binding sites on DNA, like the widely employed EB. 2,6,[12][13][14][22][23][24] However, this Ka is only an estimate, since affinity is influenced by several factors (vide supra), and we did not factor the Ka of cyanine for DNA during affinity calculations. 12,28,29 Our Ka of ~10 6 M -1 is within the 10 5 to 10 11 M -1 range for DNA-drug interactions using EB 2 and similar to the 1.4 × 10 6 M -1 Ka for spermine binding to λDNA at 17 mM ionic strength 26 compared to the 20 mM NaCl we used.…”

Interaction of antimutagenic 1,4-dihydropyridine AV-153-Na with DNA and DNA-damaging molecules and its impact on DNA repair activity

“…Fluorescence intensity of the DNA-EtBr complex was recorded at 600 nm using an indirect excitation wavelength of EtBr at 260 nm (Geall & Blagbrough 2000). Quenching constants were calculated using linear Stern-Volmer equation as described (Geethanjali et al 2015).…”