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Activity, Folding and Z-DNA Formation of the 8-17 DNAzyme in the Presence of Monovalent Ions

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Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Department of Chemical Technology, School of Chemical Engineering, Tianjin University, Tianjin 300072, P. R. China
†University of Illinois at Urbana-Champaign.
‡Tianjin University.
Cite this: J. Am. Chem. Soc. 2009, 131, 15, 5506–5515
Publication Date (Web):March 27, 2009
Copyright © 2009 American Chemical Society

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    The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K+, Rb+, or Cs+ or in the presence of 80 mM, [Co(NH3)6]3+. Only 4 M Li+, NH4+ and, to a lesser extent, Na+ conferred detectable activity. The observed rate constants (kobs ≈ 10−3 min−1 for Li+ and NH4+) are ∼1000-fold lower than that in the presence of 10 mM Mg2+, and ∼200,000-fold slower than that in the presence of 100 μM Pb2+. Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within ∼10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb2+ sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (Kd ∼ 0.35 M for Rb+ and Cs+), while those that confer DNAzyme activity possess stronger affinity (Kd ∼ 0.22 M for Li+, Na+ and NH4+). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb2+; higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn2+ and Mg2+; the Kd values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb2+. These results indicate that Pb2+-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.

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    (1) Activity assay data for 8-17 DNAzyme starting with 2× and 5× enzyme−substrate solution; (2) fluorescence spectra to test direct interaction between fluorophore and metal ions; (3) CD spectra of control sequence; (4) curve fitting used to determine Kd for CD experiments. This material is available free of charge via the Internet at

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