T4 polynucleotide kinase phosphatase (T4 PNKP) is a bifunctional tool enzyme with 5'-kinase and 3'-phosphatase activities. Considering its important roles in the repair of strand breaks, assay of T4 PNKP activity is of great importance. In this work, we proposed a novel label-free sensing strategy for T4 PNKP activity based on G-quadruplexe-thioflavin T fluorescent indicator. In the assay, we used a single stranded oligonucleotide with 3'-phosphoryl and 5'-phosphoryl ends as enzyme substrate. Upon the addition of T4 PNKP, the 3'-PO3 of the substrate was changed to 3'-OH which initiated the polymerization in the presence of terminal deoxynucleotidyl transferase and G-rich dNTP substrates. The resultant elongated DNA can form G-quadruplex in the inducement of K+, resulting in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. The detection limit of this method is as low as 0.2U/mL. Additionally, the inhibition of T4 PNKP activity by the inhibitor heparin is demonstrated. This method is easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube.
Keywords: Fluorescence assay; Label-free; T4 polynucleotide kinase phosphatase; Thioflavin T.
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