Sensitive and rapid screening of T4 polynucleotide kinase activity and inhibition based on coupled exonuclease reaction and graphene oxide platform.
Phosphorylation of DNA with 5'-hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption. Determination of nucleotide kinase activity and inhibition is under intense development due to its importance in regulating nucleic acid metabolism. Here, by using T4 polynucleotide kinase (PNK) as a model, which plays an essential role in cellular nucleic acid metabolism, particularly in the cellular responses to DNA damage, we describe a strategy for simply and accurately determining nucleotide kinase activity and inhibition by means of a coupled λ exonuclease cleavage reaction and graphene oxide (GO) based platform. The dye attached dsDNA preserves most of the fluorescence when mixed with GO. While dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, fluorescence is greatly quenched. Because of the super quenching ability and the high specific surface area of GO, the as-proposed platform presents an excellent performance with wide linear range and low detection limit in the cell extracts environment. Additionally, inhibition effects of adenosine diphosphate, ammonium sulfate, and sodium hydrogen phosphate have also been investigated. The method not only provides a universal platform for monitoring activity and inhibition of nucleotide kinase but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.
Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, China.
Lei
L
Lin
Yang
Y
Liu
Xin
X
Zhao
Jinghong
J
Li
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000494:pharmacology; D055162:Biocatalysis; D004305:Dose-Response Relationship, Drug; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology*; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006108:Graphite / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D009696:Nucleic Acids / Q000378:metabolism; D010087:Oxides / Q000737:chemistry*; D010710:Phosphates / Q000494:pharmacology; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D009696:Nucleic Acids; D010087:Oxides; D010710:Phosphates; D000244:Adenosine Diphosphate; D006108:Graphite; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D005092:Exonucleases; C018279:sodium phosphate; D000645:Ammonium Sulfate