Simultaneous fluorescence imaging of the activities of DNases and 3' exonucleases in living cells with chimeric oligonucleotide probes.

Real-time fluorescence imaging of the activity of nucleases in living cells has been a difficult issue because of unintended degradation of the natural oligonucleotides by nontarget nucleases or interactions with other proteins. In this work, we demonstrate two types of highly selective, sensitive, and robust oligonucleotide probes for simultaneous imaging of the activities of two different nucleases in living cells. The probes consist of the desired substrate structure of the target nuclease and partially phosphorothioate modified backbone labeled with fluorophore and quencher for protection from undesired degradation by other nucleases and signal transduction. Upon reaction with the target nuclease, the initially fluorescence quenched probe was cleaved and the fluorophore was separated from the quencher, giving out strong fluorescence signals. Two nucleases, DNase I and Exonuclease III, were employed as model enzymes to demonstrate the concept. In vitro studies proved that the two probes could discriminate their respective target nucleases in serum with high resistance to other coexisting enzymes. The lower limits of detection for DNase I and Exonuclease III were observed to be 40 U/L and 2.0 U/L, respectively. By labeling the two probes with different fluorophores and quenchers, simultaneous visualization of the activities of DNases and 3' exonucleases was achieved in both HeLa cells and the suspension cells of Arabidopsis thaliana. The developed approaches may greatly facilitate the studies on the intracellular functions of the two nucleases and other related biological processes. The probe design concept may also be further adapted to the detection of many other nucleases.