Double-Strand Gated Biosensor for Ultrasensitive T4 PNK Detection via λ-Exonuclease-Driven Background Suppression and Dimer G-Triplex Signal Amplification

Accurate monitoring of polynucleotide kinase (PNK) activity is essential for DNA repair studies and early clinical diagnostics. Conventional λ-exonuclease (λ-exo)-based biosensors often suffer from high background signals and false‑positive results, mainly due to nonspecific hydrolysis and unintended phosphorylation. To address these issues, we developed a double- strand gated (DSG) biosensing platform that integrates high-order dimer G-triplex (DG-3) structures as signal reporters, significantly reducing background interference and enabling robust signal amplification. In this DSG strategy, λ-exo cleavage requires both 5'-phosphorylation by PNK and double-stranded DNA (dsDNA) synthesis mediated by the Klenow fragment polymerase. This dual- verification mechanism effectively eliminates false positives resulting from nonenzymatic hydrolysis or phosphatase interference. The DG-3 structure, formed via Klenow-mediated extension, is fully released upon λ-exo cleavage, generating strong fluorescence upon binding with thioflavin T (ThT). Furthermore, DNA nanoflowers (DNFs) fabricated through rolling circle replication exhibit a unique three-dimensional architecture that spatially confines nucleic acid probes via nanotechnology. This configuration increases the local concentration of probes and enzymes, promoting molecular interactions and accelerating enzymatic kinetics. It also protects nucleic acid probes from nuclease degradation, thereby improving biosensing stability and sensitivity. The proposed strategy allows for the detection of PNK activity within a linear range of 10-4-1 U/mL, with a detection limit of 3.15 × 10-5 U/mL. This method has been successfully applied to assess PNK activity in cell extracts and to screen for potential inhibitors. With its high sensitivity, specificity, and biostability, the platform enables rapid and accurate PNK detection, demonstrating considerable potential for biochemical analysis and inhibitor discovery.