A renewable enzyme-driven dissipative DNA strand displacement strategy for T4 polynucleotide kinase activity assay and inhibitor screening

Development of dissipation-based sensing systems would provide enormous opportunities for reliable bioanalysis owing to their oscillatory signal response pattern. Herein, a fluorescence biosensor was developed for the detection of T4 polynucleotide kinase (T4 PNK) activity and inhibitor screening, leveraging an enzyme-driven dissipative strand displacement reaction. The sensing system involves the T4 PNK-catalyzed phosphorylation of a fuel strand and then a dissipative strand displacement reaction facilitated by a fuel consumption unit, lambda exonuclease (λ-Exo). As the strand displacement system progressively reverted to its initial state, a characteristic oscillatory fluorescence signal was generated, absolutely discriminating target T4 PNK against other non-specific enzymes. To achieve accurate quantification, a calibration relationship was established between T4 PNK activity and the area under the fluorescence kinetic curve (AUC) rather than relying solely on fluorescence intensity. By adjusting the amount of λ-Exo, the sensing performance toward T4 PNK could be tuned. A low detection limit toward T4 PNK could be obtained as 0.48 U mL^-1 and 0.9 U mL^-1 at λ-Exo amounts of 15 and 2 U mL^-1, respectively. Furthermore, the inhibitory effects of two compounds were successfully evaluated using this method. Significantly, the reversibility of this dissipative system highlights its potential in consecutive analysis or dynamic monitoring. This strategy not only provides a promising approach for T4 PNK-related biological and medical research but also sheds new light on the application of dissipative systems in quantitative analysis.