title: A label-free cyclic assembly of G-quadruplex nanowires for cascade amplification detection of T4 polynucleotide kinase activity and inhibition.
abstract: Several fluorescence methods have been developed for sensitive detection of PNK activity based on signal amplification techniques, but they need fluorescently labeled DNA probes and superabundant assistant enzymes. We have addressed these limitations and report here a label-free and enzyme-free amplification strategy for sensitively and specifically studying PNK activity and inhibition via hybridization chain reaction (HCR). First, the phosphorylation of hairpin DNA H1 by T4 PNK makes it be specifically digested by lambda exonuclease (λ exo) from 5' to 3' direction to generate a single-stranded initiator which can successively open hairpins H2 and H3 to trigger an autonomous assembly of long DNA nanowires. Meanwhile, an intermolecular G-quadruplex is formed between H2 and H3, thereby providing fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) which is a highly quadruplex-selective fluorophore. So, the PNK activity can be facilely and sensitively detected by using NMM as a signal probe which provides a low background signal to improve the overall sensitivity, resulting in the detection limit of 3.37 × 10(-4) U mL(-1). More importantly, its successful application for detecting PNK activity in a complex biological matrix and studying the inhibition effects of PNK inhibitors demonstrated that it provides a promising platform for screening PNK inhibitors as well as detecting PNK activity. Therefore, it is a highly sensitive, specific, reliable and cost-effective strategy which shows great potential for biological process research, drug discovery, and clinical diagnostics.
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Materials Science, Shaanxi Normal University, Xi'an 710062, China. jinyan@snnu.edu.cn.
authors.forename: Zhilu
authors.initials: Z
authors.lastname: Shi
authors.forename: Xiafei
authors.initials: X
authors.lastname: Zhang
authors.forename: Rui
authors.initials: R
authors.lastname: Cheng
authors.forename: Baoxin
authors.initials: B
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D054856:G-Quadruplexes; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D008652:Mesoporphyrins / Q000737:chemistry; D053770:Nanowires / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D015342:DNA Probes; D004791:Enzyme Inhibitors; D008652:Mesoporphyrins; C031468:mesoporphyrin IX; D000244:Adenosine Diphosphate; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase