title: Highly sensitive detection of T4 polynucleotide kinase activity by coupling split DNAzyme and ligation-triggered DNAzyme cascade amplification.
abstract: In current study, a dual strategy for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity was proposed, which combined split DNAzyme-based background reduction with ligation-triggered DNAzyme cascade for signal amplification. The 8-17 DNAzyme is split into two separate oligonucleotide fragments, which can be separately hybridized to the template DNA to form a ligatable nick after one of the fragments is phosphorylated at the 5at the yl by T4 PNK. With the further addition of Escherichia coli DNA ligase, the two oligonucleotides can be ligated to produce the activated 8-17 DNAzyme, the amount of which is positively related to the activity of T4 PNK. The signal amplification can be achieved through the cyclic cleavage of 8-17 DNAzyme toward the molecular beacon substrate, resulting in an evident fluorescence signal enhancement. The current dual strategy can significantly improve the detection sensitivity of the sensing systems, resulting in a detection limit of 0.001 U mL(-1) for T4 PNK activity, which is superior or comparable to the reported methods. Furthermore, the inhibition effects of adenosine diphosphate and sodium hydrogen phosphate on T4 PNK activity have also been demonstrated with satisfactory results. The current method may be further developed as a universal protocol for monitoring activity and inhibition of nucleotide kinase, and may show the huge potentials in biological process researches, drug discovery, and clinic diagnostics.
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China. Electronic address: shufengliu77@yahoo.com.
authors.forename: Shufeng
authors.initials: S
authors.lastname: Liu
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Ming
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Lin
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China.
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Wang
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China.
authors.forename: Chuanbin
authors.initials: C
authors.lastname: Cheng
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.affiliation: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People's Republic of China. Electronic address: wangli@qust.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D021881:DNA, Catalytic / Q000737:chemistry*; D004789:Enzyme Activation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D021881:DNA, Catalytic; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase
keywords: 8–17 DNAzyme; DNA ligation; Phosphorylation; T4 polynucleotide kinase