Interactions of Ligand, Aptamer, and Complementary Oligonucleotide: Studying Impacts of Na(+) and Mg(2+) Cations on Sensitive FRET-Based Detection of Aflatoxin B1.

The effects of magnesium and sodium on the interactions between aptamer, its specific ligand, and short complementary oligonucleotides (cDNAs) differing in affinity of their binding with the aptamer were studied. Aflatoxin B1 (AFB1) and AFB1-binding aptamer were used in the study. Dependencies for the aptamer binding with the fluorophore-labeled AFB1 under varied concentrations of the cations were obtained using fluorescence anisotropy measurements. The increase of the aptamer affinity to AFB1 in the presence of cations was demonstrated using fluorescence anisotropy and isothermal calorimetry. The collected data indicate that 300 mM Mg(2+) (significantly more than the range commonly used in aptamer sensors) provides the best affinity (16.5 ± 2.2 nM) of the aptamer-AFB1 complexation. Sodium decreases the Mg(2+)-modulated affinity at some Na(+)/Mg(2+) ratios. The aptamer affinity with cDNAs increases with concentration of cations, but not in the same way as for AFB1. Based on the characterized conditions for bimolecular interactions, the ligand-induced displacement of cDNAs was studied with the registration of the Forster fluorescence energy transfer (FRET). The most sensitive revealing of AFB1 (IC10% 3.2 ± 0.3 nM) in this three-compound FRET system was demonstrated for cDNA having an equilibrium constant of the aptamer binding close to the constant of the aptamer-AFB1 reaction.