费斯特共振能量转移
细胞内
核酸外切酶
DNA损伤
DNA
生物
稳健性(进化)
细胞生物学
生物物理学
化学
计算生物学
生物化学
基因
荧光
聚合酶
物理
量子力学
作者
Jinhua Shang,Jie Wei,Qing Wang,Jing Wang,Yangjie Zhou,Shanshan Yu,Xiaoqing Liu,Fuan Wang
标识
DOI:10.1016/j.bios.2019.111994
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 Hp was phosphorylated for λ exonuclease (λ 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 Förster 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.
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