Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

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.

Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo

DNA nanotechnology has been widely employed for biomedical applications.However,most DNA nanomaterials rely on noncovalent complementary base pairing of short single-stranded DNA oligonucleotides.Herein,we describe a general strategy to construct a long and covalently conjugated branched DNA structure for fast and in situ gelation in vivo.In our design,a short and covalently conjugated branched DNA structure can normally be employed as the DNA primer in the terminal deoxynucleotidyl transferase-dependent enzymatic polymerization system.After enzymatic extension,the DNA aptamer-modified branched DNA structures with the sequences of poly T or poly A can immediately coassemble for in situ encapsulation of the target protein and tumor cell.The fast and in situ gelation system can function in a murine model of local tumor recurrence for targeting residual tumor cells to achieve long-term drug release for efficient tumor inhibition in vivo.This rationally developed DNA self-assembly strategy provides a new avenue for the development of multifunctional DNA nanomaterials.

RNA interference with DNA polymerase and synthesis

RNA can catalyze and participate in many chemical and biochemical reactions. Non-coding RNAs （ncRNA） can regulate cellular transcription and translation reactions. We have demonstrated biochemically that RNA can also interfere with DNA polymerization via transforming DNA polymerase into deoxyribonucleoside triphosphate diphosphatase （dNTP-DPase）. RNA, even with six nucleotides, can transform DNA polymerase into dNTP-DPase, and the dNTP-DPase activity causes the hydrolysis of dNTPs into dNMPs and pyrophosphate. Moreover, we have found that DNA polymerases from several families generally have similar RNA-dependent dNTP-DPase activity. We have also observed that in the presence of RNA, when the dNTP concentrations are relatively low, and that the dNTP-DPase activity can deplete dNTPs and interfere with DNA polymerization Thus, we have discovered for the first time that in the presence of RNA, DNA polymerase can behave as a diphosphatase and inhibit DNA synthesis when dNTP quantity is low. These in vitro observations might imply a plausible role of RNA in vivo, such as suppressing DNA synthesis during a resting phase （Go） of the cell cycle, when RNA quantity is high and dNTP quantity is low.