A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe.

ABSTRACT

The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5' hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL-1), a low detection limit (0.01 U·mL-1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics. Graphical abstract A turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK) has been developed. It is based on the use of DNA-templated silver nanoclusters (AgNCs). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.