Mazumdar, Debapriya and Nagraj, N and Kim, HK and Meng, HL and Brown, AK and Sun, Q and Li, W and Lu, Y. (2009) Activity, Folding and Z-DNA Formation of the 8-17 DNAzyme in the Presence of Monovalent Ions. Journal of the American Chemical Society, 131 (15). p. 5506. ISSN 00027863Full text not available from this repository. (Request a copy)
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 (k(obs) approximate to 10(-3) min(-1) for Li+ and NH4+) are similar to 1000-fold lower than that in the presence of 10 mM Mg2+, and similar to 200,000-fold slower than that in the presence of 100 mu M Pb2+. Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within similar to 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 (K-d similar to 0.35 M for Rb+ and Cs+), while those that confer DNAzyme activity possess stronger affinity (K-d similar to 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 K-d 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.
|InterNano Taxonomy:||Nanomanufacturing Processes|
|Collections:||Nanomanufacturing Research Collection|
|Depositing User:||Danielle Federa|
|Date Deposited:||17 Jun 2009 14:24|
|Last Modified:||17 Jun 2009 14:24|
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