In vitro selection and characterization of highly selective metal -dependent DNAzymes with potential biosensor applications

Since the discovery of nucleic acids with catalytic functions, much research has been dedicated to understanding the mechanisms of so-called RNAzymes and DNAzymes. Due to the limited chemical functionalities present in RNA and DNA relative to protein enzymes, most RNAzymes and DNAzymes depend on divalent metal ions for catalysis. The structural and functional roles of metal ions in these systems has been studied using a variety of biochemical, spectroscopic, and crystallographic techniques. However, the exact role of metal ions is still uncertain.;A Pb2+-dependent DNAzyme (called 17E) with RNA cleavage activity was converted by our lab into a biosensor for Pb2+, a highly toxic metal ion in biological systems. In order to provide some insight into the catalytic mechanism of this DNAzyme and the role of Pb2+ ions, a detailed biochemical characterization of the DNAzyme was carried out in the presence of Pb2+. The DNAzyme has the highest affinity for Pb2+ (KD, app = 14 muM), but also shows considerable activity with Zn2+ (KD, app = 0.97 mM) and Mg 2+ (KD, app = 10.5 mM). The observed rate constant increases in a log-linear fashion with increasing pH, suggesting a single-deprotonation in the rate-limiting step. The affects of enzyme concentration, buffer, and sequence variants were analyzed. Studies of the reaction products, the stability and activity of 17E in human serum, and 17E activity with monovalent cations alone were also carried out.;Unlike RNAzymes, DNAzymes have not been found in Nature. Instead they have been isolated using the in vitro selection technique. We have used this technique to extend the repertoire of metal ions utilized by DNAzymes and fine-tune the metal specificity. A pool of DNAzymes were thus obtained that specifically require UO22+. The DNAzyme with the highest activity was then truncated to form a trans-cleaving system with a strong apparent UO22+ binding affinity of approximately 500 nM. Biochemical studies of this system were carried out, and preliminary fluorescence experiments have shown that this DNAzyme has the potential to be utilized as a biosensor for UO22+ in water.;A separate in vitro selection experiment was carried out in the presence of Pb2+ in order to compare the secondary structures of the resulting DNAzymes with the structure of the 8-17-like DNAzymes, which were selected through three independent experiments in the presence of: (1) Zn2+, (2) Mg2+, and (3) histidine plus Mg2+. The DNAzymes obtained from this experiment did not appear to contain the secondary structure of the 8-17 catalytic core. The reason for this is unclear, although one possibility is that the larger random region in the initial pool may have allowed for more sequence and structural diversity in the initial pool. The DNAzymes examined displayed higher specificity and lower sensitivity for Pb2+ compared to the 8-17 DNAzyme. The sensitivity may be improved through more stringent selection conditions. This work may eventually lead to an improved Pb2+ biosensor that does not display significant activity with contaminating metal ions.