Development of a highly sensitive sensing platform for T4 polynucleotide kinase phosphatase and its inhibitors based...

Dephosphorylation of the 3' termini of nucleic acids, catalyzed by various repair enzymes is important for cellular events, such as DNA replication and recombination. Here, using T4 polynucleotide kinase phosphatase (T4 PNKP) as a model target, a novel fluorescence nanosensor based on the FRET between dye labelled DNA and WS₂ nanosheets has been developed for monitoring the activity and inhibition of T4 PNKP. In this assay, we designed a single-stranded dye labelled probe that forms a self-complementary structure at one end and a 3'-phosphoryl end that served as the substrate for T4 PNKP. Once the phosphorylated probe was hydrolyzed by T4 PNKP, the resulting probe with a 3'-hydroxyl end was immediately elongated to form a double-stranded product by Klenow fragment polymerase (KF polymerase). WS₂ nanosheets were introduced to quench the fluorescence of the single-stranded dye labelled probe without polymerase elongation. The dye labelled double-stranded product preserves most of the fluorescence when mixed with WS₂ nanosheets. Because of the super quenching ability and the high specific surface area of WS₂ nanosheets, the as-proposed platform exhibits an excellent performance with a wide linear range and a low detection limit. Additionally, the effect of its inhibitors has also been investigated. The method not only provides a universal platform for monitoring the activity and inhibition of DNA 3'-phosphatases but also shows great potential for biological process research, drug discovery, and clinical diagnostics.