Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with lambda Exonuclease Assistance

摘要

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 lambda 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 lambda 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(-1) and lysate of about SO cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.