Label-Free Fluorescent Detection of T4 PNK Using a DNA Mimic Green Fluorescent Protein-Based Turn-On Biosensor

A new label‑free turn‑on fluorescent biosensor is established for highly sensitive and specific detection of T4 polynucleotide kinase (T4 PNK) activity by integrating DNA mimic green fluorescent protein (DMGFP) with λ exonuclease (λ exo) cleavage for the first time. The sensing mechanism relies on T4 PNK‑catalyzed 5'‑phosphorylation of hairpin HP‑DNA1, which triggers sequential λ exo digestion and releases ssDNA1. The liberated ssDNA1 hybridizes with DNA2 to assemble the active DMGFP structure, yielding drastic fluorescence enhancement upon binding DFHBI‑1T. Under optimized conditions, the sensor exhibits a linear response from 0 to 0.5 U/mL with a low detection limit of 0.01 U/mL and outstanding specificity toward T4 PNK. It enables quantitative evaluation of concentration‑dependent inhibition by (NH₄)₂SO₄ and Na₂HPO₄, and accurately quantifies endogenous polynucleotide kinase Phosphatase (PNKP) in HeLa cell lysates. Featuring label‑free operation, rapid response, and low background, this DMGFP‑based strategy provides a reliable and versatile platform for T4 PNK activity analysis, inhibitor screening, and clinical diagnosis, showing great potential in nucleic acid metabolism research and biomedical applications.