A highly sensitive fluorescence method for the detection of T4 polynucleotide kinase phosphatase based on polydopamine nanotubes

T4 polynucleotide kinase phosphatase (T4 PNKP) plays a critical role in various cellular events, such as DNA damage repair, replication, and recombination. Here, we have described a novel biosensor to detect the activity of T4 PNKP based on polydopamine nanotubes (PDANTs) mediated fluorescence resonance energy transfer (FRET). A FAM-labelled (6-carboxyl-fluorescein) hairpin DNA probe with 30-phosphoryl terminal was designed as the substrate for T4 PNKP. With the addition of PDANTs, the fluorescence of FAM-labelled hairpin DNA probe could be quenched because of the high adsorption of hairpin DNA on PDANTs. When T4 PNKP dephosphorylated the DNA probe, a double-stranded DNA (dsDNA) product was obtained by Klenow fragment polymerase (KF polymerase) on its 3'-hydroxyl terminal, which could retain most of the fluorescence due to the week adsorption of dsDNA on PDANTs. The developed method demonstrates the sensitivity for T4 PNKP assay in the range from 0.05 to 1.5 U mL(-1) with the detection limit of 0.005 U mL(-1), which endows the proposed strategy with high enough sensitivity for practical detection in cell lysates. With the advantages mentioned above, this novel sensitive strategy has the potential in the study of DNA damage repair mechanisms. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

This study presents a novel biosensor based on PDANTs-mediated FRET for detecting the activity of T4 PNKP. The fluorescence quenching and retention are utilized to measure the dephosphorylation activity of T4 PNKP, showing high sensitivity for practical detection in cell lysates.