Construction And Application Of Biosensing System For T4 Polynucleotide Kinase Detection Based On DNAzyme

DNA phosphorylation that occurred in the cells is of great significance to DNA damage repair.As a functional enzyme responsible for 5'-OH phosphorylation,T4 polynucleotide kinase(T4 PNK)plays a key role in the phosphorylation of DNA.Many studies have shown that the abnormal phosphorylation caused by the abnormal activity of T4 PNK is closely related to the occurrence of some serious human diseases.Because of the important biological and medical functions of T4 PNK,development of a sensitive detection method for assessing the activity of T4 PNK and its application in complex biological systems has great value for clinical diagnosis and drug development.Based on DNAzyme,we constructed two signal amplification biosensors for T4 PNK monitoring in vitro and living cells.The main contents are described as follows:1.DNAzyme-rGO coupled fluorescence assay for T4 PNK activity in vitro and intracellular imaging.Accurate monitoring of T4 PNK helps to quickly and accurately obtain important information for diagnosis and treatment of diseases.Based on the RNA cleavage activity and signal amplification function of DNAzyme,the discrimination of rGO on the nucleic acid strands with different length and the excellent fluorescence quenching ability,we constructed a DNAzymerGO coupled fluorescence method for T4 PNK detection.Under optimal conditions,there was a reliable relationship between fluorescence intensity and T4 PNK concentration within the range of 0.001 U/mL-5 U/mL.Meanwhile,this method with good selectivity showed high sensitivity with a low detection limit of 0.001 U/mL.Moreover,6 potential activators were obtained by using this method to investigate the effect of natural compounds on the T4 PNK activity,which also maintained stimulatory effect on the T4 PNK level in living cells.This DNAzyme-rGO sensing platform for T4 PNK detection is expected to be used in clinical diagnosis,prognostic evaluation and targeted drug screening.2.Construction of a new fluorescence method for monitoring T4 PNK activity in vitro,natural compound screening and intracellular imaging.The overexpression of T4 PNK can increase ROS production and activate the inflammation pathway,leading to chronic inflammation.Based on the connection function of T4 DNA Ligase,the hydrolysis function of RNase H and the ability of DNAzyme to cleavage RNA,we proposed a novel fluorescence method assisted by multiple enzymes for in vitro monitoring of T4 PNK,natural compound screening and intracellular imaging.The method exhibited highly specificity toward T4 PNK,and the detection limit was 0.0021 U/mL.By investigating the effects of natural compounds on T4 PNK activity in vitro,we found that NS17 can inhibit the activity of T4 PNK in a concentration-dependent manner.Meanwhile,this method was reliabilly used to monitor the regulating function of NS17 on T4 PNK activity in living cells.Moreover,molecular study indicated that the T4 PNK inhibitor NS17 can reduce inflammation by eliminating intracellular ROS level,killing inflammatory cells and down-regulating COX-2 expression.In our point,this new multi-enzyme-as sis ted fluorescence platform provide a new alternative for screening inhibitors against T4 PNK,which can be used for clinical diagnosis and therapy evaluation of inflammatory diseases.