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Metal ion as both a cofactor and a probe of metal-binding sites in a uranyl-specific DNAzyme: a uranyl photocleavage study.

Cepeda-Plaza M, Null EL, Lu Y - Nucleic Acids Res. (2013)

Bottom Line: The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand.Another DNAzyme, the 8-17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well.In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

ABSTRACT
DNAzymes are known to bind metal ions specifically to carry out catalytic functions. Despite many studies since DNAzymes were discovered nearly two decades ago, the metal-binding sites in DNAzymes are not fully understood. Herein, we adopt uranyl photocleavage to probe specific uranyl-binding sites in the 39E DNAzyme with catalytically relevant concentrations of uranyl. The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand. Control experiments using two 39E DNAzyme mutants revealed a different cleavage pattern of the mutated region. Another DNAzyme, the 8-17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well. In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated. Together, these experiments suggest that uranyl photocleavage has been successfully used to probe catalytically relevant uranyl-binding sites in the 39E DNAzyme. As uranyl is the cofactor of the 39E DNAzyme as well as the probe, specific uranyl binding has now been identified without disruption of the structure.

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Schematic representation of the 39E DNAzyme (original sequence), summarizing the results from the uranyl photocleavage studies. Bars indicate bases that showed specific binding of uranyl in the photocleavage studies, color code is based on Figure 4.
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gkt694-F5: Schematic representation of the 39E DNAzyme (original sequence), summarizing the results from the uranyl photocleavage studies. Bars indicate bases that showed specific binding of uranyl in the photocleavage studies, color code is based on Figure 4.

Mentions: Previous structural studies of this highly uranyl-specific 39E DNAzyme focused mostly on metal ion-dependent global folding or conformation changes (53). In this work, we use uranyl photocleavage to zoom in on the uranyl-binding site in this DNAzyme. We took advantage of a general footprinting method for mapping nucleic acids and used it to find specific uranyl-binding sites in this DNAzyme by using uranyl concentrations (2.5 nM–1 μM) that are much lower than those used in previous uranyl photocleavage studies (50 μM–1 mM) and more relevant to the DNAzyme activity. This method has produced results with higher resolution than previous folding studies. By adopting a uranyl-mediated photocleavage method, we identified three regions in the 39E DNAzyme involved in binding the uranyl ion: the bulge loop between T23, C25 and A28, the 5′ binding region close to the cleavage site between T2.4 and C3, and the stem loop at G11 and T12 (see Figure 5). It is also shown that uranyl binds specifically at the cleavage site of the substrate strand. The results presented here provide important information about the positions that are crucial for uranyl binding in the 39E DNAzyme.Figure 5.


Metal ion as both a cofactor and a probe of metal-binding sites in a uranyl-specific DNAzyme: a uranyl photocleavage study.

Cepeda-Plaza M, Null EL, Lu Y - Nucleic Acids Res. (2013)

Schematic representation of the 39E DNAzyme (original sequence), summarizing the results from the uranyl photocleavage studies. Bars indicate bases that showed specific binding of uranyl in the photocleavage studies, color code is based on Figure 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814387&req=5

gkt694-F5: Schematic representation of the 39E DNAzyme (original sequence), summarizing the results from the uranyl photocleavage studies. Bars indicate bases that showed specific binding of uranyl in the photocleavage studies, color code is based on Figure 4.
Mentions: Previous structural studies of this highly uranyl-specific 39E DNAzyme focused mostly on metal ion-dependent global folding or conformation changes (53). In this work, we use uranyl photocleavage to zoom in on the uranyl-binding site in this DNAzyme. We took advantage of a general footprinting method for mapping nucleic acids and used it to find specific uranyl-binding sites in this DNAzyme by using uranyl concentrations (2.5 nM–1 μM) that are much lower than those used in previous uranyl photocleavage studies (50 μM–1 mM) and more relevant to the DNAzyme activity. This method has produced results with higher resolution than previous folding studies. By adopting a uranyl-mediated photocleavage method, we identified three regions in the 39E DNAzyme involved in binding the uranyl ion: the bulge loop between T23, C25 and A28, the 5′ binding region close to the cleavage site between T2.4 and C3, and the stem loop at G11 and T12 (see Figure 5). It is also shown that uranyl binds specifically at the cleavage site of the substrate strand. The results presented here provide important information about the positions that are crucial for uranyl binding in the 39E DNAzyme.Figure 5.

Bottom Line: The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand.Another DNAzyme, the 8-17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well.In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

ABSTRACT
DNAzymes are known to bind metal ions specifically to carry out catalytic functions. Despite many studies since DNAzymes were discovered nearly two decades ago, the metal-binding sites in DNAzymes are not fully understood. Herein, we adopt uranyl photocleavage to probe specific uranyl-binding sites in the 39E DNAzyme with catalytically relevant concentrations of uranyl. The results indicate that uranyl binds between T23 and C25 in the bulge loop, G11 and T12 in the stem loop of the enzyme strand, as well as between T2.4 and G3 close to the cleavage site in the substrate strand. Control experiments using two 39E DNAzyme mutants revealed a different cleavage pattern of the mutated region. Another DNAzyme, the 8-17 DNAzyme, which has a similar secondary structure but shows no activity in the presence of uranyl, indicated a different uranyl-dependent photocleavage as well. In addition, a close correlation between the concentration-dependent photocleavage and enzymatic activities is also demonstrated. Together, these experiments suggest that uranyl photocleavage has been successfully used to probe catalytically relevant uranyl-binding sites in the 39E DNAzyme. As uranyl is the cofactor of the 39E DNAzyme as well as the probe, specific uranyl binding has now been identified without disruption of the structure.

Show MeSH