Amplified detection of T4 polynucleotide kinase activity based on a λ-exonuclease cleavage-induced DNAzyme releasing strategy

The T4 polynucleotide kinase (T4 PNK) plays an essential role in the cellular responses to nucleic acid strand damage. Herein, a simple and sensitive fluorescence approach for monitoring T4 PNK activity was proposed based on a λ-exonuclease (λ-exo) cleavage-induced DNAzyme releasing strategy. A hairpin-shaped DNA probe that contains the sequence of 8-17 DNAzyme as a built-in suppressed catalytic unit was designed. After phosphorylation by T4 PNK followed with the immediate λ-exonuclease (λ-exo) cleavage, the 8-17 DNAzyme unit was successfully released and used for the cyclic cleavage toward the molecular beacon substrate, resulting in an evident fluorescence signal enhancement. With the currently developed λ-exo cleavage reaction and DNAzyme-based platform, the amplified detection of T4 PNK with a low detection limit of 0.005 U mL−1 could be achieved. Furthermore, the inhibition effects of adenosine diphosphate, ammonium sulfate, and sodium hydrogen phosphate have been evaluated. The developed λ-exo cleavage-induced DNAzyme releasing strategy opens a promising avenue for monitoring activity and inhibition of nucleotide kinase, and should be also easily extended for the sensitive detection toward many other nucleic acid enzymes and may find widespread applications in biological process researches, drug discovery, and clinic diagnostics.