Construction of a simple, localized and homogeneous fluorescence detection platform for T4 PNK activity based on tetrahedral DNA nanostructure-mediated primer exchange reaction

T4 polynucleotide kinase (T4 PNK), which acts as a bifunctional enzyme, is widely involved in important phosphorylation and dephosphorylation processes. Sensitive and specific detection of T4 PNK activity will help to elucidate its critical importance in disease diagnosis and treatment. The conventional detection method uses hairpin or single-strand primers modified with phosphorylation group as the substrates for T4 PNK hydrolysis. However, considering to the large spatial distances and disordered reaction orientation among the substrates and enzymes, the efficiency and sensitivity of the method are somewhat limited. Here, we propose a simple and sensitive signal amplification strategy, integrating the tetrahedral DNA nanostructure with primer exchange reaction (TDN-PER), which ensures different reaction direction and improves the local reaction concentrations. Compared to dispersed single PER, localized TDN-PER provides more primers for initiating signal amplification. Benefiting from the unique structural transformation from dispersion to localization, this strategy provided ultrahigh sensitivity, achieving a detection limit of 1.8 × 10−5 U/mL, while maintaining excellent accuracy and specificity in complex human serum and cell lysates environments. The method successfully evaluated the inhibitory effects of (NH4)2SO4 and Na2HPO4 on T4 PNK activity. Finally, the practical application capability of this strategy was verified in the detection of cell-free extracts, providing a promising analytical platform for T4 PNK-related biological and medical research.