Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticles.

5'-Polynucleotide kinase such as T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5'-hydroxyl termini in nucleic acids, playing a crucial role in DNA replication, DNA recombination, and DNA damage repair. Here, we demonstrate for the first time single-molecule detection of PNK based on phosphorylation-directed recovery of fluorescence quenched by Au nanoparticle (AuNP) in combination with lambda exonuclease-mediated cleavage reaction. In the presence of PNK, the γ-phosphate group from adenosine triphosphate (ATP) is transferred to 5'-hydroxyl terminus, resulting in 5'-phosphorylation of the hairpin probe. The phosphorylated hairpin probes may function as the substrates of lambda exonuclease and enable the removal of 5' mononucleotides from the stem, leading to the unfolding of hairpin structure and the formation of binding probes. The resultant binding probes may specifically hybridize with the AuNP-modified capture probes, forming double-strand DNA (dsDNA) duplexes with 5'-phosphate groups as the substrates of lambda exonuclease and subsequently leading to the cleavage of capture probes and the liberation of Cy5 molecules and the binding probes. The released binding probes may further hybridize with new capture probes, inducing cycles of digestion-release-hybridization and consequently the release of numerous Cy5 molecules. Through simply monitoring Cy5 molecules with total internal reflection fluorescence (TIRF)-based imaging, PNK activity can be quantitatively measured. This assay is very sensitive with a limit of detection of 9.77 × 10 U/μL, and it may be further used to screen the PNK inhibitors and measure PNK in cancer cell extracts.