Highly efficient incorporation of dATP in terminal transferase polymerization forming the ploy (A)n-DITO-1 fluorescent probe sensing terminal transferase and T4 polynucleotide kinase activity

Fluorescent dye DITO-1 has almost no fluorescence in the absence of nucleic acid. G bases in single strand DNA can induce maximum fluorescent enhancement followed by the A bases when it binds the DITO-1. However, the incorporation efficiency of the dATP was higher than dGTP in terminal transferase (TdT) polymerization. As a consequence, ploy (A)n, rather than ploy (G)n via TdT polymerization had the superior photoluminance when it binded DITO-1 fluorescent dye. Here, we developed a high selective and sensitive sensing strategy for assaying TdT and T4 polynucleotide kinase activity (T4 PNK) based on the ploy (A)(n)-DITO-1 fluorescent probe. An increasing amounts of TdT enzyme could promote the distinct incorporation of dATP on the DNA primer and form poly (A)n ssDNA with a difference in length. A good linear relationship between the delta F and the concentrations of TdT in a range of 0.2-50 U/mL was obtained and the detection limit was 0.05 U/mL. Based on the experimental results for TdT, we further expanded the application of this method for detection of a series of concentrations of T4 PNK. The delta F and the logarithm concentrations of T4 PNK in the range of 0.1-10 U/mL showed a good linear response and the detection limit of 0.02 U/mL was obtained. In addition, the detection of T4 PNK in Hela cell lysate was achieved, demonstrating that the proposed method had the potential application in complex system. The ploy (A)(n)-DITO-1 fluorescent probe had the excellent properties of one-step readout, robustness for target detection in complex system, and easiness operation, and showed the great potential in clinical diagnostics, inhibitor screening, and drug discovery.

Automated summary

In this study, a high selective and sensitive sensing strategy for assaying TdT and T4 PNK activity based on the ploy (A)(n)-DITO-1 fluorescent probe was developed. The method showed good linear response and low detection limit for both TdT and T4 PNK, and it was successfully applied for the detection of T4 PNK in Hela cell lysate.