Amplified Intracellular Imaging of Polynucleotide Kinase via a Self-Stacking Autocatalytic DNA Circuit

Polynucleotide kinase (PNK) plays a pivotal role in nucleic acid metabolism and is closely associated with genome instability and disease progression. However, current PNK detection methods are constrained by the low sensitivity and complex probe composition. Herein, we report a self-stacking autocatalytic assembly circuit (AAC) for the sensitive and specific detection of PNK activity in live cells. In this system, PNK phosphorylates a 5′-hydroxylated hairpin substrate, enabling its selective degradation by λ-exonuclease to release a single-stranded DNA trigger. This DNA trigger further initiates a catalytic hairpin assembly reaction among three rationally designed probes, generating Y-shaped DNA junctions that expose new recombinational trigger sequences. The regenerated triggers drive a self-sustaining amplification cycle, enabling the rapid signal generation with low background. The AAC system, requiring minimal DNA components, enables the sensitive and robust detection of PNK both in vitro and in living cells and supports intracellular PNK activity monitoring and inhibitor screening. This strategy provides a robust and efficient tool for low-abundance biomarker analysis and holds broad potential in biomedical research and diagnostic applications.