No abstract

In this study, an assay to quantify the presence of mercuric ions and methyl mercury by double-stranded DNA containing a poly(dT) sequence was developed using a light switch compound, Ru(phen)(2)(dppz)(2+) (1), which is known to intercalate into double-stranded DNA. Upon treatment with mercuric ions, the metal-to-ligand charge transfer (MLCT) emission derived from the intercalation of 1 was reduced due to the formation of DNA duplexes containing T-Hg(2+)-T base pairs by the dehybridization of poly(dT)-poly(dA) duplexes at room temperature. As the concentration of Hg(2+) was increased, the emission of 1 gradually decreased. This label-free method had a detection limit of 5 nM. Other metal ions, such as K(+), Ag(+), Ca(2+), Mg(2+), Zn(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Cd(2+), Cr(3+), Fe(3+), had no significant effect on reducing emission. This emission method can differentiate matched and mismatched poly(dT) sequences based on the dehybridization rate of dsDNA and the rate decreased in the order of T(10)C·A(11)∼ T(10)A·A(11) > T(10)G·A(11) > T(11)·A(11).

Single-stranded oligonucleotides (ssDNA) containing guanine bases in their sequences were adsorbed onto gold nanoparticles (AuNPs) by electrostatic interaction. Cyclic voltammetry of Ru(bpy) 3 2+ in the presence of the ssDNA-AuNP complex resulted in an enhanced anodic current due to the oxidation of the guanine bases of DNA. The current obtained with ssDNA-AuNP appeared much smaller than the corresponding ssDNA alone. This current reduction was due to the decrease in solvent accessibility of the guanines in ssDNA immobilized to AuNPs. A progressive decrease in the current was observed in the titration of AuNPs to ssDNA, and a minimum current was eventually obtained, indicating complete binding of ssDNA. The size dependences of AuNPs on the interaction between ssDNA and AuNP were also studied, and the ssDNA adsorbed to 5 nm AuNPs was more solvent-accessible for the Ru mediator than 13 and 30 nm AuNPs.

A homogeneous assay of the protective antigen in anthrax toxin is reported using two new PA-specific aptamers for selective and sensitive detection, based on reduction in the fluorescence emission according to the formation of the aptamer-PA ternary complex. PA at 1 nM was readily detected using OliGreen as a fluorophore in HEPES buffer. We also demonstrated that the PA detection could be performed in blood serum. The binding interaction between the aptamer and PA was strong enough to dehybridize double-stranded DNA paired completely with 12 bases at room temperature. Moreover, this fluorescence study revealed that the binding sites of the two aptamers were located differently on the PA protein. We believe our approach may lay the groundwork for the real-time detection of PA.

Fenton reaction and iron autoxidation have been debated for the major process in ROS mediated DNA cleavage. We compared both processes on iron oxidation, DNA cleavage, and cyclic voltammetric experiment at different pHs. Both oxidation reactions were preferred at basic pH condition, unlike DNA cleavage. This indicates that iron oxidation and the following steps probably occur separately. The ROS generated from autoxidation seems to be superoxide radical since sod exerted the best inhibition on DNA cleavage when H2O2 was absent. In comparison of cyclic voltammograms of Fe 2+ in NaCl solution and phosphate buffer, DNA addition to phosphate buffer induced significant change in the redox cycle of iron, indicating that iron may bind DNA as a complex with phosphate. Different pulse voltammogram in the presence of ctDNA suggest that iron ions are recyclable at acidic pH, whereas they may form an electrically stable complex with DNA at high pH condition.