Highly sensitive detection of DNA phosphorylation by counting single nanoparticles

DNA phosphorylation is a vital process in the repair, replication, and recombination of nucleic acids. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive, simple, and economical method for DNA phosphorylation detection and T4 polynucleotide kinase (T4 PNK) activity assay based on marking DNA phosphorylation/biotinylation events by the attachment of fluorescent nanoparticles. Enzyme activity of T4 PNK is measured down to a limit of 5 x 10(-6)U/ml, which is 400 times lower than previous reports. We also studied DNA phosphorylation specificity with different DNA substrates. Furthermore, T4 PNK inhibition by the inhibitor ADP and activation by the activator spermine are shown, demonstrating the potential for high-throughput screening for inhibitors and activators.