Engineering a Unimolecular DNA-Catalytic Probe for Single Lead Ion Monitoring

The binding of proteins and small molecules by DNA is well established, but more recently, DNA molecules have been selected to catalyze biochemical reactions. These catalytic DNAs, or DNAzymes, can be activated by metal ions. In this paper, we take advantage of DNA molecular engineering to improve the properties of DNAzymes by designing a unimolecular probe for lead ion (Pb2+)-catalyzed reaction, achieving in turn, the ability to monitor a single Pb2+ in solution "by fluorescence microscopy. Specifically, by applying a unimolecular design, a leaving substrate DNA strand labeled with a fluorophore is linked to a hairpin 8-17 DNAzyme sequence labeled with a quencher. The hairpin structure and the substrate are connected using poly T, which brings the quencher into close proximity with the fluorophore in the inactive state. The intramolecular 'linkage of the two strands assures efficient quenching of the fluorescence, generating almost zero background. In the presence of Pb2+, however, the leaving substrate fragment is cleaved at the RNA site by the enzyme, releasing a fluorescent fragment for detection with repetitive cycling for signal amplification. The resulting high sensitivity with a quantifiable detection range from 2 nM to 20 mu M was achieved with a high selectivity in excess of 80-fold for Pb2+ over other metal ions. The limit of detection is about 167 times better than the previously reported similar probes (Liu, J; Lu, Y. Anal. Chem. 2003, 75, 6666-6672) and 1600 times better compared to the Pb2+ detection limit obtained from atomic spectroscopy. Thus, this probe could provide a simple, yet rapid and sensitive measurement for Pb2+. Furthermore, we used this probe to monitor single Pb2+ reaction kinetics. Given this degree of sensitivity and selectivity, our new probe design may prove useful in the development of other nucleic acid-based probes for intracellular, toxicological, and environmental monitoring.