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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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Gel images of uranyl photocleavage reactions of 39E (−1, +5) DNAzyme with the enzyme strand labeled and in the presence of varying concentrations of uranyl.
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gkt694-F2: Gel images of uranyl photocleavage reactions of 39E (−1, +5) DNAzyme with the enzyme strand labeled and in the presence of varying concentrations of uranyl.

Mentions: Previous uranyl photocleavage studies used uranyl concentrations from 50 µM to 1 mM to see a protection effect indicative of protein binding (54,56–57,60,62). These concentrations are significantly higher than what is required for enzymatic activity of the 39E DNAzyme, as it is known to have a high affinity for uranyl with an apparent dissociation constant of 496 nM (45). Therefore, to find the optimal concentrations of uranyl for this study that are relevant to the enzymatic activities, a range of uranyl concentrations from 2.5 nM to 1 mM was tested. At high concentrations of uranyl (500 µM and 1 mM), an even cleavage pattern was obtained across all positions, indicating non-specific uranyl-mediated photocleavage (see Supplementary Figure S1). This result is not surprising, as previous enzymatic assays also showed a largely inactive 39E DNAzyme in the presence of 1 mM uranyl (45). The photocleavage reactions were therefore carried out at lower concentrations (between 2.5 nM and 50 µM), and the data obtained using PAGE are shown in Figure 2. In contrast to the uniform cleavage pattern at high uranyl concentration (e.g. 50 µM), only certain nucleotides are selectively cleaved at low uranyl concentrations. The nucleotides denoted in bold were identified through the use of a GA ladder.


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)

Gel images of uranyl photocleavage reactions of 39E (−1, +5) DNAzyme with the enzyme strand labeled and in the presence of varying concentrations of uranyl.
© Copyright Policy
Related In: Results  -  Collection

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

gkt694-F2: Gel images of uranyl photocleavage reactions of 39E (−1, +5) DNAzyme with the enzyme strand labeled and in the presence of varying concentrations of uranyl.
Mentions: Previous uranyl photocleavage studies used uranyl concentrations from 50 µM to 1 mM to see a protection effect indicative of protein binding (54,56–57,60,62). These concentrations are significantly higher than what is required for enzymatic activity of the 39E DNAzyme, as it is known to have a high affinity for uranyl with an apparent dissociation constant of 496 nM (45). Therefore, to find the optimal concentrations of uranyl for this study that are relevant to the enzymatic activities, a range of uranyl concentrations from 2.5 nM to 1 mM was tested. At high concentrations of uranyl (500 µM and 1 mM), an even cleavage pattern was obtained across all positions, indicating non-specific uranyl-mediated photocleavage (see Supplementary Figure S1). This result is not surprising, as previous enzymatic assays also showed a largely inactive 39E DNAzyme in the presence of 1 mM uranyl (45). The photocleavage reactions were therefore carried out at lower concentrations (between 2.5 nM and 50 µM), and the data obtained using PAGE are shown in Figure 2. In contrast to the uniform cleavage pattern at high uranyl concentration (e.g. 50 µM), only certain nucleotides are selectively cleaved at low uranyl concentrations. The nucleotides denoted in bold were identified through the use of a GA ladder.

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