Biochemical Characterization of a Uranyl Ion‐Specific DNAzyme

Abstract

Uranyl ion-specific DNAzyme: A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three-way-junction structure with catalytically important nucleotides residing in the bulge. A highly conserved GA mismatch is also crucial for the enzyme's activity.The biochemical characterization of a DNAzyme that is highly specific for uranyl (UO(2) (2+)) ions is described. Sequence alignment, enzyme truncation, and mutation studies have resulted in a conserved sequence that folds into a bulged stem-loop structure. Interestingly, a GA pair next to the scissile site is important for the uranyl ion-specific DNAzyme; this is reminiscent of the GT wobble base pair adjacent to the cleavage site that is crucial for the Pb(II)-specific 8-17 DNAzyme activity. Therefore wobble pairs might be important for formation of metal-specific metal-binding sites in DNAzymes. The DNAzyme binds the uranyl ion with a dissociation constant of 469 nM, which is among the strongest metal-binding affinities in nucleic acid enzymes reported to date. This explains why a catalytic beacon fluorescent sensor based on this enzyme has a detection limit (45 pM) that rivals the most-sensitive analytical instrument. It also has over 1 000 000-fold specificity for the uranyl ion over other metal ions. The DNAzyme can carry out multiple turnover reactions that follow the Michaelis-Menten equation, with a k(cat) of 1.46 min(-1) and a K(M) of 463 nM, similar to that of the 8-17 DNAzyme. The pH profile shows a bell-shaped curve that reaches a maximum at pH 5.5, at which the in vitro selection was carried out; this suggests that in vitro selection can be performed to obtain DNAzymes with optimal performance under specific conditions under which practical applications are required. These findings enrich our fundamental understanding of metal-binding sites in nucleic acids and allow the design of sensors with better performance.

A turn-off fluorescent biosensor for the rapid and sensitive detection of uranyl ion based on molybdenum disulfide nanosheets and specific DNAzyme

“…3B, when the pH = 5.5, DNAzyme has a highest activity to UO 2 2+ . Our results are consistent with the conclusion reported previously [20]. The most likely explanation of this result is that pH has an impact on the uranyl ion speciation in solution, and pH 5.5 produced a uranyl species that is the most effective cofactor for carrying out catalysis of DNAzyme [20].…”

“…So far, a number of highly specific DNAzymes have been isolated and used for detecting metal ions such as Pb 2+ , Zn 2+ , Cu 2+ , Mn 2+ and so on [16][17][18][19]. Not long ago, a uranyl ion-specific DNAzyme was reported by Lu and his co-workers [20]. Encouraged by the discovery of uranyl ion-specific DNAzyme, some DNAzyme-based biosensors have been developed for the rapid and specific detection of UO 2 2+ [21][22][23].…”

Functional nucleic acids for detecting bacteria

“…Therefore, this DNAzyme has been widely used to design sensors for Pb 2 + detection (Li and Lu 2000, Liu and Lu 2003, Xiao et al 2007 ). The DNAzyme (39E) in Figure 1B has a very similar structure but is active only in the presence of UO 2 2 + , uranyl , Brown et al 2009 ). With > 1 million-fold selectivity for uranyl over other metal ions, this DNAzyme has been shown to be highly effective for uranyl detection.…”

Desulfurization Activated Phosphorothioate DNAzyme for the Detection of Thallium

“…5,6 Some (e.g. Na + , 7 Pb 2+ , [8][9][10][11][12] Zn 2+ , 13 Cu 2+ , [14][15][16] UO2 2+ , 17,18 Hg 2+ , 19,20 and lanthanide ions, [21][22][23] ) can act as cofactors in DNAzyme-based catalysis. These interactions are the main basis of the current metal sensing strategies using DNA.…”