HgII 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 H-1 NMR. Unlike Ag-I, Hg-II exhibited stoichiometric, site-specific binding of C-T mismatches. The on-and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (k(on) approximate to 10(5) M-1 s(-1), k(off) approximate to 10(-3) s(-1)) as compared to T-T mismatches (k(on) approximate to 10(4) M-1 s(-1), k(off) approximate to 10(-4) s(-1)), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (K-d approximate to 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 (Delta T-m = 6-19 degrees C), as compared to those containing C-T mismatches (Delta T-m = 1-4 degrees 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.