Ethidium bromide-(dC-dG-dC-dG)2 complex in solution: intercalation and sequence specificity of drug binding at the tetranucleotide duplex level.

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Abstract
The binding of ethidium bromide (EtdBr) [a trypanocidal drug] to the dC-dG-dC-dG self-complementary duplex was monitored at the resolvable drug and nucleic acid protons and backbone phosphates at high nucleotide/drug (N/D) ratios by NMR spectroscopy in aqueous solution. Averaged resonances (25.degree.-95.degree.) were observed for the nuclei acid and drug nonexchangeable protons in the presence of excess tetranucleotide (N/D = 24), indicative of rapid exchange relative to the chemical shifts in the free and complexed states. Complex formation results in upfield shifts for the base protons at the terminal and internal base pairs and an increase in the transition midpoint for the duplex-to-strand conversion. Upfield chemical shift changes of 1.2 ppm at the Watson-Crick guanine N-1 proton(s) on complex formation (N/D = 24) were observed with slow exchange between (dC-dG-dC-dG)2 and EtdBr(dC-dG-dC-dG)2 relative to this chemical shift difference at -5.degree.. The EtdBr phenanthridine ring protons shift upfield by about 0.9 ppm (H-2, H-4, H-7, H-9) and > 0.5 ppm (H-1, H-10) on complex formation, with the chemical shifts vs. temperature plots (25.degree.-95.degree.) monitoring the dissociation of the EtdBr.cntdot.(dC-dG-dC-dG)2 structure. These upfield shifts at the exchangeable and nonexchangeable base protons and phenanthridine ring (but not side chain) protons demonstrate intercalation of the phenanthridine ring of EtdBr into the dC-dG-dC-dG duplex in solution. The intercalation model may be supported by the observation of downfield shifts (up to 1 ppm) at the internucleotide phosphate(s) of the tetranucleotide duplex on addition of EtdBr at low temperatures. Stronger binding of EtdBr to the self-complementary dC-dG-dC-dG(2 dC-dG intercalation sites) and dC-dC-dG-dG (1 dC-dG site) duplexes was observed compared to the dG-dG-dC-dC (no dC-dG sites) as monitored by UV absorbance changes at 480 nm. These studies extend to the tetranucleotide duplex level earlier observations that EtdBr exhibits a selectivity for formation of complexes to dinucleoside monophosphates with a pyrimidine (3''-5'') purine sequence in the crystal and in solution. The experimental proton NMR upfield shifts at the phenanthridine protons on formation of the EtdBr.cntdot.dC-dG-dC-dG)2 complex compare favorably with calculated values (atomic diamagnetic anisotropy and ring current contributions) based on the overlap geometry for EtdBr intercalated into the pyrimidine (3''-5'') purine dinucleoside monophosphate duplex in the crystal.