title: Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation.
abstract: Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20U/mL and an extremely low detection limit of 8.6×10(-5)U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics.
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China. Electronic address: xueqingwang1983@163.com.
authors.forename: Qingwang
authors.initials: Q
authors.lastname: Xue
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Lv
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Shuling
authors.initials: S
authors.lastname: Xu
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Yuanfu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Qiaoli
authors.initials: Q
authors.lastname: Yue
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Li
authors.affiliation: Shandong Provincial Key Lab of Test Technology on Food Quality and Safety, Shandong Academy of Agricultural Sciences, Jinan 250100, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Gu
authors.affiliation: Shandong Provincial Key Lab of Test Technology on Food Quality and Safety, Shandong Academy of Agricultural Sciences, Jinan 250100, China.
authors.forename: Shuqiu
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Liaocheng University, Liaocheng 252059, Shandong, China; Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, China. Electronic address: liujifeng111@gmail.com.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Liu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000032:analysis*; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D005453:Fluorescence; D054856:G-Quadruplexes; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D011524:Protoporphyrins; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000032:analysis / Q000037:antagonists & inhibitors / Q000097:blood
chemical_list: D017931:DNA Primers; D011524:Protoporphyrins; C017803:zinc protoporphyrin; D015254:DNA Modification Methylases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: EXPA; Fluorescence assay; Label-free; Methyltransferase; Supramolecular ZnPPIX/G-quadruplex