A novel microchip electrophoresis laser induced fluorescence detection method for the assay of T4 polynucleotide kinase activity and inhibitors

Abstract

T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5′-hydroxyl termini in nucleic acids, which play a crucial role in DNA recombination, replication and damage repair. Here, a microchip electrophoresis laser induced fluorescence (MCE-LIF) method based on biochemical reaction was developed for the detection of T4 PNK activity and inhibitors. In this method, the single strand DNA (ssDNA) was hybridized with the 5-carboxyfluorescein (FAM) labeled single strand DNA (ssDNA-FAM) to form FAM labeled double-stranded DNA (dsDNA-FAM). In the presence of T4 PNK and adenosine triphosphate (ATP), T4 PNK catalyzes the transfer of γ-phosphate residues from ATP to the 5-hydroxyl terminal of dsDNA-FAM. The phosphorylated dsDNA-FAM can be gradually hydrolyzed by λexo to produce a FAM labeled single nucleotide fragment. Then the FAM labeled single nucleotide fragment and the unhydrolyzed dsDNA-FAM were separated by MCE, and two electrophoresis peaks appeared in the electrophoretogram. The detection of T4 PNK activity and inhibitors was realized by measuring the peak height of the FAM labeled single nucleotide fragment in electrophoretogram. This assay is very sensitive with a limit of detection of 0.002 U/mL, and it can be further used to screen the T4 PNK inhibitors.