Hg(II) binds to C–T mismatches with high affinity

Binding reactions of Hg(II) and Ag(I) to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and (1)H NMR. Unlike Ag(I), Hg(II) exhibited stoichiometric, site-specific binding of C–T mismatches. The on- and off-rates of Hg(II) binding were approximately 10-fold faster to C–T mismatches (k(on) ≈ 10(5) M(−1) s(−1), k(off) ≈ 10(−3) s(−1)) as compared to T–T mismatches (k(on) ≈ 10(4) M(−1) s(−1), k(off) ≈ 10(−4) s(−1)), resulting in very similar equilibrium binding affinities for both types of ‘all natural’ metallo base pairs (K(d) ≈ 10–150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T–T mismatches exhibited much larger increases in thermal stability upon addition of Hg(II) (ΔT(m) = 6–19°C), as compared to those containing C–T mismatches (ΔT(m) = 1–4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C–Hg(II)–T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs – even in situations where metal binding has little or no impact on the thermal stability of the duplex.