title: Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.
abstract: The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL-1and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.
authors: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, 130022, People's Republic of China.
authors: Hua
authors: H
authors: Zhang
authors: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, 130022, People's Republic of China.|University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, People's Republic of China.
authors: Zhen
authors: Z
authors: Zhao
authors: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, 130022, People's Republic of China.|University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, People's Republic of China.
authors: Zhen
authors: Z
authors: Lei
authors: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, 130022, People's Republic of China.
authors: Zhenxin
authors: Z
authors: Wang
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*; D010209:Paper; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D004277:DNA, Single-Stranded; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D005092:Exonucleases