Double strand DNA-templated copper nanoparticle as a novel fluorescence indicator for label-free detection of polynucleotide kinase activity

Phosphorylation of DNA by polynucleotide kinase (PNK) takes an important role in DNA damage repair, replication and recombination. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, we present a label-free fluorescent method for PNK activity assay using double strand DNA (dsDNA) -templated copper nanoparticles (CuNPs) as a fluorescent indicator. Upon the PNK reaction, the dsDNA template is phosphorylated and then digested by λ exonuclease immediately, prohibiting the formation of fluorescent CuNPs due to the lack of dsDNA template. This homogeneous PNK activity assay does not require any other additional modifications of DNA substrate or complex design, making the proposed strategy simple, cost-effective and high throughput. The proposed strategy is sensitive, selective and exhibits a good assay performance in complex biological samples. The strategy presented here opens a new avenue for PNK assay and nucleic acid phosphorylation related research.

► A simple PNK activity assay using dsDNA-templated CuNPs as a fluorescent indicator was presented.
► Any other additional modifications of DNA substrate or complex design were not required for this method.
► PNK activity was sensitively and selectively detected.
► In complex biological samples, this method exhibited a good assay performance.