Metal‐Ion‐Dependent Folding of a Uranyl‐Specific DNAzyme: Insight into Function from Fluorescence Resonance Energy Transfer Studies

Abstract

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO22+)-specific 39E DNAzyme in the presence of Mg2+, Zn2+, Pb2+, or UO22+. At pH 5.5 and physiological ionic strength (100 mm Na+), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg2+ or Zn2+. However, no folding occurred in the presence of Pb2+ or UO22+; this is analogous to the “lock-and-key” catalysis mode first observed in the Pb2+-specific 8–17 DNAzyme. However, Mg2+ and Zn2+ exert different effects on the 8–17 and 39E DNAzymes. Whereas Mg2+ or Zn2+-dependent folding promoted 8–17 DNAzyme activity, the 39E DNAzyme folding induced by Mg2+ or Zn2+ inhibited UO22+-specific activity. Group IIA series of metal ions (Mg2+, Ca2+, Sr2+) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr2+ (1.12 Å) is comparable to that of Pb2+ (1.20 Å), but contrary to Pb2+, Sr2+ induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb2+ and UO22+. Under low ionic strength (30 mm Na+), all four metal ions (Mg2+, Zn2+, Pb2+, and UO22+), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA-backbone phosphates in addition to playing specific catalytic roles. Mg2+ at low (<2 mm) concentration promoted UO22+-specific activity, whereas Mg2+ at high (>2 mm) concentration inhibited the UO22+-specific activity. Therefore, the lock-and-key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock-and-key mode only at ionic strengths of 100 mm or greater.

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

“…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.…”

“…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.…”

Fluorescent nanoprobes for sensing and imaging of metal ions: Recent advances and future perspectives

“…In addition to aptamers described above, DNAzymes have been investigated due to their high metal ion specificity [283-289]. Various optical sensors for metal ions were developed using graphene and DNAzymes [290-294].…”

“…In light of these drawbacks, there is increasing interest in developing new sensing techniques for the detection of metal ions that have higher sensitivity and selectivity, shorter response times, and lower costs [14]. Among the current sensor approaches, fluorescence detection [3,15-21] is the most common analytical tool used in the detection of metal ions due to its capabilities for sensitivity, selectivity, reproducibility, and rapid, real-time monitoring. The critical element that achieves this goal is the fluorescent probe which offers a real-time, noninvasive way to image metal ions in their native environments with high spatial and temporal resolution [3].…”

Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode

“…'8-17' DNAzyme consisting of a substrate strand and a catalytic strand has a specific cleavage site of Pb 2 þ , which has been widely used for the detection of lead ion based on different signal-transduction principles (Lu et al, 2015;Zhou et al, 2014; Mazumdar et al, 2009). The Lu's group designed a series of fluorescent/colorimetric sensors for the monitoring of lead ions by using Pb 2 þ -specific DNAzyme owing to their easy-to-use and small-in-size (He and Lu, 2011;Xiang and Lu, 2014;Lan et al, 2010). The lead-dependent DNAzyme with a two-step mechanism was described in detail in this work (Brown et al, 2003).…”