Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides have been developed for their potential applications in nanotechnology. We recently found that a natural T:T mismatched base pair bound with Hg2+ ion to form a novel T-Hg-T base pair. Here, we examined the thermodynamic properties of the binding between Hg2+ and each of the single and double T:T mismatched base pair duplex DNAs by isothermal titration calorimetry. Hg2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio with 10(6) M-1 binding constant, which was significantly larger than those for nonspecific metal ion-DNA interactions. In the Hg2+-double T:T mismatched base pair interaction, the affinity for the second Hg2+ binding was significantly larger than that for the first Hg2+ binding. The positively cooperative binding may be favorable to align multiple Hg2+ in duplex DNA for the application of the metal-mediated base pairs in nanotechnology. (C) 2011 Elsevier B.V. All rights reserved.