A label-free colorimetric assay for sensitive screening of T4 polynucleotide kinase activity and inhibition based on enzyme-aided cyclic strand displacement amplification and PNA-gold nanoparticle platform.

The phosphorylation of nucleic acids mediated by 5'-polynucleotide kinase (PNK) exerts a crucial regulatory function in a wide range of significant cellular activities. Nevertheless, the current approaches for detecting PNK require expensive labeled probes and complex instrumentation, making it impossible to achieve real-time, on-site, and rapid analysis. Here, we take T4 PNK as a model and establish a novel colorimetric strategy for the detection of PNK activity and its inhibition by means of a coupled enzyme-assisted cyclic strand displacement amplification (SDA) and peptide nucleic acid (PNA)-gold nanoparticle (AuNP) based platform. The inspiration for this innovative strategy comes from the high stability, strong binding ability, and potent regulatory effect of PNA probes on AuNPs. Under the catalysis of PNK, the 5'-hydroxyl end of the hairpin-shaped DNA (hpDNA) is initially phosphorylated and subsequently digested by λ exonuclease (λ exo). This results in the release of a single-stranded DNA, which serves as a triggering factor to initiate the strand displacement reaction (SDR). The replaced PNA probe adheres to the surface of AuNPs, inducing their aggregation and causing a remarkable color change. Meanwhile, the double-stranded SDR product releases the SDR trigger with the aid of a nicking enzyme, triggering the next round of the SDR cycle and achieving highly efficient and controllable signal amplification. This assay is simple to operate and does not require bulky and expensive instruments or complex labeled probes. Compared with existing colorimetric methods, the detection sensitivity has been greatly improved, reaching 3.52 × 10 U/mL. Additionally, the method has demonstrated satisfactory results when applied to intricate biological matrices and the screening of T4 PNK inhibitors. Therefore, the proposed strategy holds significant potential for real-time analysis, high-throughput detection, and PNK-related drug screening.