Probing metal binding in the 8-17 DNAzyme by TbIII 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 microM of Zn(II) was measured to be 3.3+/-0.3 microM. 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 D(2)O and H(2)O 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) approximately 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(III).
Kim
Hee-Kyung
HK
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
Li
Jing
J
Nagraj
Nandini
N
Lu
Yi
Y
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D049449:Luminescence; D008670:Metals / Q000378:metabolism*; D015335:Molecular Probes; D013057:Spectrum Analysis / Q000379:methods*; D013725:Terbium / Q000737:chemistry*
D021881:DNA, Catalytic; D008670:Metals; D015335:Molecular Probes; C407482:RNA-cleaving DNA 8-17; D013725:Terbium; D004247:DNA