Fluorescence Based Investigation of Temperature-Dependent Pb-Specific 8-17E DNAzyme Catalytic Sensor.

The 8-17E DNAzyme is a temperature-dependent DNA metalloenzyme catalyzing RNA trans esterification in the presence of Pb metal ions. Labeling the stems of the substrate and DNAzyme with the Cy3 and Cy5 respectively, the considered DNAzyme was studied by the fluorescence spectroscopy. The temperature-dependent variability of the Pb-specific 8-17E DNAzyme catalytic sensor was investigated trough a number of successive temperature fluctuations from 4 to 25 °C to obtain information. Investigating underlined biochemical system reveals that in this sensor, free single strands Enzyme (Cy5-E) and Substrate (Cy3-S) have higher fluorescence intensities than hybridized forms, suggesting that the fluorophores are in a contact quenched. Increasing the temperature has three effects: 1) Fluorescence intensities for the free fluorophores were reduced, 2) stability of the hybridized form was reduced and cleavage of substrate in presence of Pbwas occurred, and 3) conformation of ES hybridized form was changed (before cleavage). As a result of conformation changes in ES, S was more affected than E in the ES. Pb ion shows quenching effect on both fluorophores and in the absence of N(g) purge the effect was more considerable. A main goal that we had in mind was to find if significantly lower concentrations of Pb and ES, compared to previous reports, can generate any observable cleavage in substrate. Analysis of the cleavage reaction for 50 nM ES indicates that S is cleaved at 25 °C in presence of N(g) and 0.5 μM Pb, while in same condition no apparent change occurs in the 4 or 10 °C. The rapid, sensitive and low cost strategy presented here can be applicable to study temperature-dependent behavior of other nucleic acid-based biosensors.