滚动圆复制
底漆(化妆品)
生物传感器
荧光
生物物理学
DNA
化学
组合化学
磷酸二酯键
多核苷酸
生物化学
生物
DNA聚合酶
核糖核酸
基因
量子力学
物理
有机化学
作者
Wanling Cui,Xiaoyang Fan,Wei Zhao,Jinrong Liu,Lei Zheng,Libing Zhou,Junye Zhang,Xiumei Zhang,Xiaoxin Wang
标识
DOI:10.1016/j.saa.2022.121938
摘要
T4 polynucleotide kinase (PNK) plays a key role in maintaining genome integrity and repairing DNA damage. In this paper, we proposed a label-free fluorescent biosensor for amplified detection of T4 PNK activity based on rolling circle amplification (RCA) and catalytic hairpin assembly (CHA). Firstly, we designed a padlock probe with a 5'-hydroxyl terminus for phosphorylation reaction, a complementary sequence of the primer for initiating RCA, and a complementary sequence of the trigger for triggering CHA. T4 PNK catalyzed the phosphorylation reaction by adding a phosphate group to the 5'-hydroxyl terminus of padlock probe, generating a phosphorylated padlock probe. Then it hybridized with the primer to generate a circular probe under the action of ligase. Subsequently, the primer initiated an RCA reaction along the circular probe to synthesize a large molecular weight product with repetitive trigger sequences. The triggers then triggered the cyclic assembly reactions between hairpin probe 1 and hairpin probe 2 to generate a large amount of complexes with free G-rich sequences. The free G-rich sequences folded into G-quadruplex structures, and the N-methylmesoporphyrin IXs were inserted into them to produce an amplified fluorescent signal. Benefiting from high amplification efficiency of RCA and CHA, this fluorescent biosensor could detect T4 PNK as low as 6.63 × 10-4 U mL-1, and was successfully applied to detect its activity in HeLa cell lysates. Moreover, this fluorescent biosensor could effectively distinguish T4 PNK from other alternatives and evaluate the inhibitory effect of inhibitor, indicating that it had great potential in drug screening and disease treatment.
科研通智能强力驱动
Strongly Powered by AbleSci AI