Label-free simple fluorescence assay of DNA polymerase using the G-quadruplex structure

Abstract

In this study, the G-quadruplex structure was used to detect the enzymatic activity and inhibition of the Klenow fragment exo⁻ (KF⁻) in the presence of N-methyl mesoporphyrin IX (NMM) which binds specifically to the G-quadruplex. We found that KF⁻ synthesized complementary strands of the template DNA, leading to a decrease in fluorescence intensity in the presence of NMM due to the difficulty in G-quadruplex formation. Thus, KF⁻ activity was sensitively detected using NMM. In addition, daidzein, the major phytoestrogen in soy, inhibited DNA synthesis mediated by KF⁻, and the half maximal inhibitory concentration (IC₅₀) was determined to be 15.6 μM by nonlinear regression analysis.