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 ofN-methylmesoporphyrin IX (NMM) which binds specifically to the G-quadruplex.

Double‐Stranded DNA‐Templated Copper Nanoclusters for Detection of DNA Polymerase Activity

“…The corresponding IC 50 (the half‐maximal inhibition of KF‐) value was 6.23 μM, which was determined from the nonlinear regression analysis of the data in Figure 4. The results indicated that 5‐fluorouracil inhibited DNA polymerase activity effectively like the drugs of daidzein and chloroquine 15,16 …”

“…The results indicated that 5-fluorouracil inhibited DNA polymerase activity effectively like the drugs of daidzein and chloroquine. 15,16…”

“…Apart from the conventional methods to evaluate enzyme activity such as measurement of incorporated radio‐labeled nucleotides coupled with gel electrophoresis, 9,10 fluorescence‐based analytical methods are utilized frequently because of their simplicity, high sensitivity, and efficiency 11 . Consequently, DNA polymerase activity has been detected using fluorophore‐labeled DNA probes, 12 fluorophore‐labeled probes combined with graphene oxide, 13 single‐labeled smart probes, 14 and the fluorescent dye N ‐methyl mesoporphyrin IX coupled with G‐quadruplex 15 . Nevertheless, fluorescence assays still need to be developed further because of the high cost, photobleaching, and problems with the binding specificity and affinity associated with the fluorophore‐labeled probes 16,17 …”

“…11 Consequently, DNA polymerase activity has been detected using fluorophore-labeled DNA probes, 12 fluorophore-labeled probes combined with graphene oxide, 13 single-labeled smart probes, 14 and the fluorescent dye N-methyl mesoporphyrin IX coupled with G-quadruplex. 15 Nevertheless, fluorescence assays still need to be developed further because of the high cost, photobleaching, and problems with the binding specificity and affinity associated with the fluorophore-labeled probes. 16,17 Recently, DNA-templated metal nanoclusters has been used for label-free fluorescence detection assays due to their outstanding optical properties, low toxicity, good biocompatibility and facile synthesis.…”