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Probing Metal Binding in the 8–17 DNAzyme by Tb III Luminescence Spectroscopy
Author(s) -
Kim HeeKyung,
Li Jing,
Nagraj Nandini,
Lu Yi
Publication year - 2008
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200701789
Subject(s) - deoxyribozyme , luminescence , chemistry , metal , metal ions in aqueous solution , substrate (aquarium) , crystallography , stereochemistry , dna , materials science , biochemistry , oceanography , optoelectronics , organic chemistry , geology
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 μ M of Zn II was measured to be 3.3±0.3 μ 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 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 ≈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 .