title: Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.
abstract: DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity.
authors.affiliation: Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, 1417466191, Tehran, Iran.
authors.forename: Hanie Ahmadzade
authors.initials: HA
authors.lastname: Kermani
authors.affiliation: Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, 1417466191, Tehran, Iran. smhosseini@khayam.ut.ac.ir.
authors.forename: Morteza
authors.initials: M
authors.lastname: Hosseini
authors.affiliation: Department of Biotechnology, Payame Noor University, 19395-4697, Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Dadmehr
authors.affiliation: Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, 1417466191, Tehran, Iran.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Ganjali
mesh_terms: D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000737:chemistry*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D057230:Limit of Detection; D049329:Nanostructures / Q000737:chemistry*; D012834:Silver / Q000737:chemistry*
chemical_list: D012834:Silver; D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases
keywords: DNA methyltransferase enzyme; Fluorescence assay; M.SssI activity; Silver nanoclusters