摘要
DNA methylation, catalyzed by DNA methyltransferases(MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein(Sc GFP)/graphene oxide(GO) interaction, coupled with methylation-initiated template-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3′-terminal(DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase(Td T), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with Sc GFP and form the Sc GFP/DNA nanocomplex. Such nanocomplex can effectively protect Sc GFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of Sc GFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of Sc GFP/GO. The method was successfully used to detect the activity of DNA adenine methylation(Dam) MTase with a wide linear range(0.1–100 U/m L) and a low detection limit of 0.1 U/m L. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect Td T activity.
DNA methylation, catalyzed by DNA methyltransferases （MTases）, is a key component of genetic regulation, and DNA MTa- ses have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein （ScGFP）/graphene oxide （GO） interaction, coupled with methylation-initiated tem- plate-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3＇-terminal （DNA-1） was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleo- tidyl transferase （TdT）, DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA frag- ments quickly bind with ScGFP and form the ScGFP/DNA nanocomplex. Such nanocomplex can effectively protect ScGFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of ScGFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of ScGFP/GO. The method was successfully used to detect the activity of DNA adenine methylation （Dam） MTase with a wide linear range （0.1-100 U/mL） and a low detection limit of 0.1 U/mL. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was success- fully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect TdT activity.
基金
supported by the National Basic Research Program (2011CB911002)
the National Natural Science Foundation of China (21190044, 21475037, 21222507, 21175036)
the fundamental research funds for the central universities
