Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer.

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ >> Zn2+ >>Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.

A lead-dependent DNAzyme with a two-step mechanism.

A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) >> Zn(2+) >> Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.

New highly sensitive and selective catalytic DNA biosensors for metal ions.

While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a 'negative selection' strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a new class of metal ion fluorescent sensors that are the first examples of catalytic DNA biosensors for metal ions. The sensors combine the high selectivity of catalytic DNA with the high sensitivity of fluorescent detection, and can be applied to the quantitative detection of metal ions over a wide concentration range and with high selectivity. The use of DNA sensors in detection and quantification of lead ions in environmental samples such as water from Lake Michigan has been demonstrated. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fiber and microarray technology for device manufacture. Thus, the DNA sensors explained here hold great promise for on-site and real-time monitoring of metal ions in the fields of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring.