Original research articleLabel-free monitoring of DNA polymerase activity based on a thrombin-binding aptamer G-quadruplex

•A novel, real-time fluorescence assay has been developed for the detection of DNA polymerase activity.•This method is highly sensitive with a detection limit of 0.1 U/mL.•This method has been applied to detect DNA polymerase inhibitor.•It is simple and cost-effective without requirement of labeling with a fluorophore-quencher pair.

We have developed a label-free assay for the detection of DNA polymerase activity based on a thrombin-binding aptamer (TBA) G-quadruplex. In the presence of DNA polymerase, the 3′-OH termini of the hairpin substrate are immediately elongated to replace the TBA, which can be recognized quickly by the ThT dye and results in an increase of fluorescence. This method is highly sensitive with a detection limit of 0.1 U/mL. It is simple and cost-effective without any requirement of labeling with a fluorophore-quencher pair. Furthermore, the proposed method can also be applied to analyze the inhibition of DNA polymerase, which clearly indicates that the proposed method can be applied for screening of potential DNA polymerase inhibitors.