title: Sensitive detection of DNA methyltransferase using the dendritic rolling circle amplification-induced fluorescence.
abstract: The analysis of DNA methylation and MTase activities is very important in the early clinical diagnosis of cancer, on purposes of providing insights into the mechanism of gene repression and developing novel drugs of treating methylation-related diseases. Combining the dendritic rolling circle amplification and Mg2+-dependent DNAzyme with a function of catalyzing the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses, a new fluorescent method for DNA methyltransferase detection was reported. In the presence of DNA methyltransferases (MTase), the methylation-responsive sequence of double-stranded DNA probe was methylated and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved hybrid DNA probe then functioned as a signal primer to initiate the dendritic rolling circle amplification reaction, containing a circular DNA and a structurally tailored hairpin structure. Subsequently, the circular nucleic acid template produced a complementary sequence to the Mg2+-dependent DNAzyme and a sequence identical to the loop region of the co-added hairpin structure. At last, a fluorescence readout signal was afforded by the DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate. This method enabled the analysis of the target MTase with a detection limit up to 0.36 U mL-1, and a dynamic range was obtained from 1.0 to 10 U mL-1. Moreover, the proposed strategy was successfully applied in real sample assay. With this assay, the inhibitors of MTase were evaluated and screened which might be helpful for the discovery of anticancer drugs.
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Weiling
authors.initials: W
authors.lastname: Song
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Yawen
authors.initials: Y
authors.lastname: Luan
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Guo
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Peng
authors.initials: P
authors.lastname: He
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China. Electronic address: zhangxr7407@126.com.
authors.forename: Xiaoru
authors.initials: X
authors.lastname: Zhang
mesh_terms: D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000032:analysis*; D015342:DNA Probes; D021881:DNA, Catalytic; D005453:Fluorescence; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation
chemical_list: D015342:DNA Probes; D021881:DNA, Catalytic; D015254:DNA Modification Methylases
keywords: DNA; DNA methyltransferase; DNAzyme; Dendritic rolling circle amplification; Fluorescence