Abstract
Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expression levels and catalytic activities of topoisomerases in cell-free systems and human cells. Using our newly designed biosensors, we tricked DNA topoisomerases within their catalytic cycles to switch on fluorescence and resume new rounds of catalysis. Considering that human topoisomerases have been widely recognized as biomarkers for multiple cancers and identified as promising targets for several anticancer drugs, we believe that these DNA-based biosensors and our design strategy would greatly benefit the future development of clinical tools for cancer diagnosis and treatment.Graphical
Highlights
The catalytic action of most cellular enzymes involves the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products
Westergaard group reported in 1985, human topoisomerase I does not act on all DNA sequences indiscriminately but preferentially binds to particular tracts (e.g., Duplex 1 shown in Fig. 1a and b) in the macronuclear DNA of the eukaryote, Tetrahymena thermophila [29]
Intracellular levels of topoisomerase I (topo I) were measured by a commercially available Enzyme-linked Immunosorbent Assay (ELISA) kit in our study as well, results of which
Summary
The catalytic action of most cellular enzymes involves the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. As the fluorophore on Probe 1 is located close to the Validation of our newly designed biosensors in cell‐free systems To determine whether the DNA-based biosensors are accessible and photoswitchable by topo I’s catalytic action as originally designed, fluorescence spectroscopic examinations were conducted (Fig. 2).
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