title: One-strand oligonucleotide probe for fluorescent label-free "turn-on" detection of T4 polynucleotide kinase activity and its inhibition.
abstract: Thioflavin T (ThT), as one of the most exciting fluorogenic molecules, boasts the "molecular-rotor" ability to induce DNA sequences containing guanine repeats to fold into G-quadruplex structures. It has been demonstrated to sense this change by its remarkable fluorescence enhancement. In this work, taking T4 polynucleotide kinase (PNK) as a model, the ThT/G-quadruplex based platform and λexonuclease (λexo) cleavage reaction were combined to design a label-free "turn-on" strategy for fast, simple and accurate detection of T4 PNK activity and its inhibition. In the presence of T4 PNK, the designed thioflavin T based molecular beacon (TMB) DNA probe could be phosphorylated and then digested by the cleavage of λexo, releasing the G-quartets. These then bound to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation, for the "turn-on" detection of T4 PNK. In comparison to traditional methods, the proposed TMB probe is convenient, requiring no sophisticated labeling and separation processes and displaying high analytical performance. It exhibits a satisfying detection result for the activity of T4 PNK with a low detection limit of 0.001 U mL(-1). This is not only meaningful for further research on disease-related biochemical processes, but also valuable for molecular-target therapies.
authors.affiliation: Anhui Key Laboratory of Chem-biosensing, College of Chemistry and Materials Science, Center for Nanoscience and Nanotechology, Anhui Normal University, Wuhu, 241000, P.R. China. xjzhang@mail.ahnu.edu.cn wangyuz@mail.ahnu.edu.cn.
authors.forename: Fu
authors.initials: F
authors.lastname: Zhou
authors.forename: Guangfeng
authors.initials: G
authors.lastname: Wang
authors.forename: Dongmin
authors.initials: D
authors.lastname: Shi
authors.forename: Yue
authors.initials: Y
authors.lastname: Sun
authors.forename: Liang
authors.initials: L
authors.lastname: Sha
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Qiu
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhang
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D005453:Fluorescence; D006801:Humans; D057230:Limit of Detection; D015345:Oligonucleotide Probes / Q000737:chemistry*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D015345:Oligonucleotide Probes; D011116:Polynucleotide 5'-Hydroxyl-Kinase