Colorimetric Detection and Intracellular Imaging of Polynucleotide Kinase by the Phosphorylation-Induced Assembly of...

Polynucleotide kinase (PNK) that phosphorylates 5'-hydroxyl termini of nucleic acids plays a vital role in DNA replication, recombination, and repair and is regarded as both a critical biomarker and therapeutic target. PNK analysis is based on conversion of 5'-hydroxyl group to 5'-phosphate group catalyzed by the enzyme, which is challenging to be optically detected and generally requires additional enzymes and steps for signal transduction. Besides complicating the assay, it is hard to keep each step with their optimal reaction condition and masks the heterogeneous characteristics of PNK. Herein, we propose a straightforward strategy for visualized detection of PNK. In this strategy, PNK-catalyzed DNA phosphorylation is demonstrated to be able to enhance the overhang binding between strands on gold nanoparticles, which cross-links DNA-modified gold nanoparticles (AuNPs) into network aggregates. The tiny terminal group conversion is thus transduced into bulk nanoparticle aggregation and shows visible color change. It is found that either one strand phosphorylation can stick the duplex and leads to AuNP cross-linking, which influences nanoparticle dispersion state and obtains a high sensitivity comparable to fluorescent or electrochemical methods. The strategy also shows a high specificity toward PNK and can be used for PNK inhibitor analysis. Furthermore, since the aggregation of AuNPs leads to increased light scattering, intracellular imaging of PNK is realized with dark field microscopy by cell endocytosis of DNA-modified AuNPs, showing bright scattering nanoparticle aggregates inside cells. The work proposes a new PNK detection principle and provides a straightforward, convenient, and visual way for PNK analysis.