title: A label-free assay for T4 polynucleotide kinase/phosphatase activity and its inhibitors based on poly(thymine)-templated copper nanoparticles.
abstract: DNA 3'-phosphatase takes an important role in DNA damage repair, replication and recombination. Here, we present a novel label-free fluorescent assay for T4 polynucleotide kinase/phosphatase (T4 PNKP) activity and its inhibitor screening by using poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as a fluorescent indicator. In this assay, we designed a simple T-rich hairpin primer with a 3'-phosphoryl end, which can serve as both the substrate for T4 PNKP and DNA template for the formation of fluorescent CuNPs. Once the phosphorylated hairpin primer was hydrolyzed by T4 PNKP, the resulting hairpin primer with a 3'-hydroxyl end was immediately elongated to form a long double-strand product by DNA polymerase, which prohibited the formation of fluorescent CuNPs due to the lack of poly T single-stranded DNA template. This new strategy provides a sensitive, selective, and cost-effective manner for T4 PNKP analysis, which holds a great potential in the study of DNA damage repair mechanisms.
authors.affiliation: College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China.
authors.forename: Zhen-Zhen
authors.initials: ZZ
authors.lastname: Dong
authors.affiliation: College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Qiao
authors.affiliation: College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China. Electronic address: jiage0630@hnu.edu.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Ge
authors.affiliation: College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China.
authors.forename: Ai-Ling
authors.initials: AL
authors.lastname: Liu
authors.affiliation: College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China; State Key Laboratory for Chemo/biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China. Electronic address: zhaohui.li@zzu.edu.cn.
authors.forename: Zhao-Hui
authors.initials: ZH
authors.lastname: Li
mesh_terms: D055162:Biocatalysis; D045744:Cell Line, Tumor; D003300:Copper / Q000737:chemistry*; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry*; D013941:Thymine / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D011108:Polymers; D003300:Copper; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D045643:DNA Repair Enzymes; D013941:Thymine
keywords: Fluorescence; Label-free; Poly(thymine)-templated fluorescent copper nanoparticles; T4 polynucleotide kinase/phosphatase