Probing Metal Binding in the 8-17 DNAzyme by Tb-III Luminescence Spectroscopy

Metal-dependent cleavage activities of the 8-17 DNAzyme were found to be inhibited by Tb-III ions, and the apparent inhibition constant in the presence of 100 mu m of Zn-II was measured to be 3.3 +/- 0.3 mu M. The apparent inhibition constants increased linearly with increasing Zn-II concentration, and the inhibition effect could be fully rescued with addition of active metal ions, indicating that Tb-III is a competitive inhibitor and that the effect is completely reversible. The sensitized Tb-III luminescence at 543 nm was dramatically enhanced when Tb-III was added to the DNAzyme-substrate complex. With an inactive DNAzyme in which the GT wobble pair was replaced with a GC Watson-Crick base pair, the luminescence enhancement was slightly decreased. In addition, when the DNAzyme strand was replaced with a complete complementary strand to the substrate, no significant luminescence enhancement was observed. These observations suggest that Tb-III may bind to an unpaired region of the DNAzyme, with the GT wobble pair playing a role. Luminescence lifetime measurements in D2O and H2O suggested that Tb-III bound to DNAzyme is coordinated by 6.7 +/- 0.2 water molecules and two or three functional groups from the DNAzyme. Divalent metal ions competed for the Tb-III binding site(s) in the order Co-II > Zn-II > Mn-II > Pb-II > Ca-II approximate to Mg-II. This order closely follows the order of DNAzyme activity, with the exception of Pb-II. These results indicate that Pb-II, the most active metal ion, competes for Tb-III binding, differently from other metal ions such as Zn-II, suggesting that Pb-II may bind to a different site from that for the other metal ions including Zn-II and Tb-II.