Issue 22, 2022

Phosphorothioated and phosphate-terminal dumbbell (PP-TD) probe-based rapid detection of polynucleotide kinase activity

Abstract

Polynucleotide kinase (PNK), a bifunctional enzyme with 5′-kinase and 3′-phosphatase activities, plays an important role in DNA repair and is associated with various diseases. Here, we developed a primer-free, sensitive, and isothermal quantitative assay to detect PNK activity. In the presence of PNK, the 3′-phosphate group of the substrate was digested with 3′-OH, initiating the amplification reaction. Elongated dsDNA binds to the dsDNA-specific fluorescent dye EvaGreen, leading to a significant enhancement in fluorescence intensity. The limit of detection (LOD) of this method was 7.7 × 10−7 U μL−1, which is comparable or even superior to that of previously reported methods. This approach also showed good quantitative ability in complex cell lysates, indicating potential for biological sample analysis. Additionally, this facile and sensitive assay can be used to screen for PNK inhibitors. The proposed method provides a promising platform for sensitive PNK activity monitoring and inhibition screening for drug discovery and clinical treatment.

Graphical abstract: Phosphorothioated and phosphate-terminal dumbbell (PP-TD) probe-based rapid detection of polynucleotide kinase activity

Supplementary files

Article information

Article type
Paper
Submitted
30 Aug. 2022
Accepted
02 Okt. 2022
First published
04 Okt. 2022

Analyst, 2022,147, 4986-4990

Phosphorothioated and phosphate-terminal dumbbell (PP-TD) probe-based rapid detection of polynucleotide kinase activity

X. Jiang, X. Shen, J. Talap, D. Yang, S. Zeng, H. Liu and S. Cai, Analyst, 2022, 147, 4986 DOI: 10.1039/D2AN01431A

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