title: One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification.
abstract: DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Herein, using T4 PNK as a model target, we describe a one-step, highly sensitive, simple and rapid fluorescence approach for monitoring its activity and inhibition. This innovative strategy is inspired by the great amplification capability of ligation-nicking coupled reaction-mediated signal amplification. In the presence of T4 PNK, one of two short oligonucleotides complementary to the loop sequence of molecular beacon (MB) are phosphorylated, and then ligated with the other by DNA ligase. Upon formation of the stable duplex between the ligated DNA and MB, the fluorescence is restored and further significantly amplified through nicking endonuclease assisted cleavage of multiple MBs. Meanwhile, the cleavage of MBs will also generate new nicks to initiate the ligation reaction. Eventually, a maximum fluorescence enhancement is obtained when the ligation and nicking process reached a dynamic equilibrium. As compared to those of the existing approaches except for the assay based on single nanoparticle counting, all limited to 1:1 signal transduction function, the sensitivity (0.00001U/mL) of the proposed strategy is 100-1700 times higher. The application of the sensing system in complex biological matrix and screening of T4 PNK inhibition are demonstrated with satisfactory results. Moreover, this approach is also successfully used to detect biological small molecules such as adenosine triphosphate (ATP), and can be further extended for nicotinamide adenine dinucleotide (NAD(+)) detection.
authors.affiliation: Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.forename: Yongxi
authors.initials: Y
authors.lastname: Zhao
authors.forename: Lin
authors.initials: L
authors.lastname: Qi
authors.forename: Chunhai
authors.initials: C
authors.lastname: Fan
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D017122:Bacteriophage T4 / Q000201:enzymology*; D004247:DNA / Q000737:chemistry; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000255:Adenosine Triphosphate; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011088:DNA Ligases; D000072481:DNA Ligase ATP