Highly sensitive detection of T4 polynucleotide kinase activity by coupling split DNAzyme and ligation-triggered DNAzyme cascade amplification

• A novel and amplified fluorescence assay for T4 PNK activity was developed.
• It combines split DNAzyme-based background reduction and ligation-triggered DNAzyme cascade signal amplification.
• An impressive low detection limit of 0.001 U mL−1 for T4 PNK activity can be achieved.
• The inhibition effects of various inhibitors toward T4 PNK activity were evaluated.

In current study, a dual strategy for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity was proposed, which combined split DNAzyme-based background reduction with ligation-triggered DNAzyme cascade for signal amplification. The 8–17 DNAzyme is split into two separate oligonucleotide fragments, which can be separately hybridized to the template DNA to form a ligatable nick after one of the fragments is phosphorylated at the 5at the yl by T4 PNK. With the further addition of Escherichia coli DNA ligase, the two oligonucleotides can be ligated to produce the activated 8–17 DNAzyme, the amount of which is positively related to the activity of T4 PNK. The signal amplification can be achieved through the cyclic cleavage of 8–17 DNAzyme toward the molecular beacon substrate, resulting in an evident fluorescence signal enhancement. The current dual strategy can significantly improve the detection sensitivity of the sensing systems, resulting in a detection limit of 0.001 U mL−1 for T4 PNK activity, which is superior or comparable to the reported methods. Furthermore, the inhibition effects of adenosine diphosphate and sodium hydrogen phosphate on T4 PNK activity have also been demonstrated with satisfactory results. The current method may be further developed as a universal protocol for monitoring activity and inhibition of nucleotide kinase, and may show the huge potentials in biological process researches, drug discovery, and clinic diagnostics.

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