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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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Predicted secondary structures of the trans-cleaving 39E DNAzyme used in this study. (a) The 39E DNAzyme consisting of a DNAzyme strand (in green) and a substrate strand (in black). The substrate strand contains a single riboadenosine at the cleavage site (in red). (b) A 39E DNAzyme variant used for the uranyl-mediated photocleavage experiments. The scissile riboadenosine was changed to deoxyriboadenosine to prevent DNAzyme-based cleavage. In addition, the arms of the DNAzyme-substrate at both ends are either extended (in blue) or deleted (in gray), making both arms more symmetrical to aid data collection and analysis. These changes do not affect the enzymatic activity.
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gkt694-F1: Predicted secondary structures of the trans-cleaving 39E DNAzyme used in this study. (a) The 39E DNAzyme consisting of a DNAzyme strand (in green) and a substrate strand (in black). The substrate strand contains a single riboadenosine at the cleavage site (in red). (b) A 39E DNAzyme variant used for the uranyl-mediated photocleavage experiments. The scissile riboadenosine was changed to deoxyriboadenosine to prevent DNAzyme-based cleavage. In addition, the arms of the DNAzyme-substrate at both ends are either extended (in blue) or deleted (in gray), making both arms more symmetrical to aid data collection and analysis. These changes do not affect the enzymatic activity.

Mentions: The DNAzyme that has been most studied so far is the 8–17 DNAzyme, which catalyzes the cleavage of a substrate strand containing a single ribonucleotide at the cleavage site in the presence of divalent metal ions, such as Mg2+, Ca2+ and Zn2+, showing the highest activity with Pb2+ (33,34). Extensive studies of the 8–17 DNAzyme have been carried out using various biochemical and biophysical methods (34–43), and a lock-and-key mechanism commonly observed in protein enzymes has been found to be responsible for the high selectivity for Pb2+. Another DNAzyme, called the 39E DNAzyme (Figure 1a), is even more selective, exhibiting over million-fold selectivity for uranyl over other metal ions (44,45), as compared with the ∼100-fold selectivity of Pb2+ over the next-best competing metal ion (Zn2+) for the 8–17 DNAzyme. Despite the demonstrated high selectivity and sensing applications (44,46–52), the source of the selectivity of the 39E DNAzyme for uranyl over other metal ions is unknown. To gain deeper insights into this selectivity, we have carried out a biochemical study to obtain conserved sequences responsible for the uranyl binding and enzymatic activity (45), and a fluorescence resonance energy transfer study to elucidate uranyl-dependent global folding of the 39E DNAzyme (53). Although these results contribute to our understanding of metal specificity, they provide information only on the global scale and do not provide detailed information about the metal-binding site. To overcome this limitation, we herein take advantage of uranyl-mediated photocleavage, commonly used as a general technique to probe the structure of nucleic acids and nucleic acid–protein complexes, to elucidate important uranyl-binding sites relevant to catalysis.Figure 1.


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)

Predicted secondary structures of the trans-cleaving 39E DNAzyme used in this study. (a) The 39E DNAzyme consisting of a DNAzyme strand (in green) and a substrate strand (in black). The substrate strand contains a single riboadenosine at the cleavage site (in red). (b) A 39E DNAzyme variant used for the uranyl-mediated photocleavage experiments. The scissile riboadenosine was changed to deoxyriboadenosine to prevent DNAzyme-based cleavage. In addition, the arms of the DNAzyme-substrate at both ends are either extended (in blue) or deleted (in gray), making both arms more symmetrical to aid data collection and analysis. These changes do not affect the enzymatic activity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814387&req=5

gkt694-F1: Predicted secondary structures of the trans-cleaving 39E DNAzyme used in this study. (a) The 39E DNAzyme consisting of a DNAzyme strand (in green) and a substrate strand (in black). The substrate strand contains a single riboadenosine at the cleavage site (in red). (b) A 39E DNAzyme variant used for the uranyl-mediated photocleavage experiments. The scissile riboadenosine was changed to deoxyriboadenosine to prevent DNAzyme-based cleavage. In addition, the arms of the DNAzyme-substrate at both ends are either extended (in blue) or deleted (in gray), making both arms more symmetrical to aid data collection and analysis. These changes do not affect the enzymatic activity.
Mentions: The DNAzyme that has been most studied so far is the 8–17 DNAzyme, which catalyzes the cleavage of a substrate strand containing a single ribonucleotide at the cleavage site in the presence of divalent metal ions, such as Mg2+, Ca2+ and Zn2+, showing the highest activity with Pb2+ (33,34). Extensive studies of the 8–17 DNAzyme have been carried out using various biochemical and biophysical methods (34–43), and a lock-and-key mechanism commonly observed in protein enzymes has been found to be responsible for the high selectivity for Pb2+. Another DNAzyme, called the 39E DNAzyme (Figure 1a), is even more selective, exhibiting over million-fold selectivity for uranyl over other metal ions (44,45), as compared with the ∼100-fold selectivity of Pb2+ over the next-best competing metal ion (Zn2+) for the 8–17 DNAzyme. Despite the demonstrated high selectivity and sensing applications (44,46–52), the source of the selectivity of the 39E DNAzyme for uranyl over other metal ions is unknown. To gain deeper insights into this selectivity, we have carried out a biochemical study to obtain conserved sequences responsible for the uranyl binding and enzymatic activity (45), and a fluorescence resonance energy transfer study to elucidate uranyl-dependent global folding of the 39E DNAzyme (53). Although these results contribute to our understanding of metal specificity, they provide information only on the global scale and do not provide detailed information about the metal-binding site. To overcome this limitation, we herein take advantage of uranyl-mediated photocleavage, commonly used as a general technique to probe the structure of nucleic acids and nucleic acid–protein complexes, to elucidate important uranyl-binding sites relevant to catalysis.Figure 1.

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
Related in: MedlinePlus