Adaption of an autonomously cascade DNA circuit for amplified detection and intracellular imaging of polynucleotide kinase with ultralow background

摘要

Polynucleotide kinase (PNK) plays a crucial role in phosphorylation-related DNA repair, while its real time tracking and monitoring is unexplored for limited sensitivity and robustness of current PNK sensing strategies in complex biological environment. Herein, we proposed a concatenated hybridization chain reaction (Con-HCR)-based PNK sensing platform for ultra-sensitive PNK assay and intracellular imaging. In the presence of PNK, the 5'-hydroxyl termini of hairpin H-p was phosphorylated for X exonuclease (lambda Exo)-mediated DNA cleavage reaction. This leads to the generation of initiator I for stimulating the subsequent Con-HCR-motivated assembly of branched dsDNA nanostructures with tremendously amplified Forster resonance energy transfer (FRET) signal. Owing to the multiple-responsive recognitions of PNK/exonuclease and the dual amplification of Con-HCR, the proposed method realized the sensitive and selective PNK assay as well as the screening of PNK inhibitors. This FRET-based signal transduction provides a straightforward and accurate procedure for analyzing PNK from complex cell lysate. Furthermore, the robust feature of the present system enabled their extensive application in monitoring the varied expression levels of intracellular PNK in living cells. In view of these remarkable characters, our Con-HCR-mediated PNK sensing strategy shows more potential applications in clinical diagnosis and new drug discovery researches.