title: Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification.
abstract: Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA. In addition to the primer-binding sequence, the dumbbell template contained another three important parts: 5'-CCGG-3' sequences in double-stranded stems, nicking endonuclease recognition sites and C-rich sequences in single-stranded loops. The introduction of 5'-CCGG-3' sequences allows the dumbbell template to be destroyed by the restriction endonuclease, HpaII, but is not destroyed in the presence of the target MTase-M.SssI MTase. The introduction of nicking endonuclease recognition sites makes the M.SssI MTase-protected dumbbell template-mediated RCA proceed in a multiple primers-like exponential mode, thus providing the RCA with high amplification efficiency. The introduction of C-rich sequences may promote the folding of amplification products into a G-quadruplex structure, which is specifically recognized by the commercially available fluorescent probe thioflavin T. Improved RCA amplification efficiency and specific fluorescent recognition of RCA products provide the M.SssI MTase-sensing platform with high sensitivity. When a dumbbell template containing four nicking endonuclease sites is used, highly specific M.SssI MTase activity detection can be achieved in the range of 0.008-50U/mL with a detection limit as low as 0.0011U/mL. Simple experimental operation and mix-and-detection fluorescent sensing mode ensures that M.SssI MTase quantitation works well in a real-time RCA mode, thus further simplifying the sensing performance and making high throughput detection possible. The proposed MTase-sensing strategy was also demonstrated to be applicable for screening and evaluating the inhibitory activity of MTase inhibitors.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, PR China.
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Li
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, PR China.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Du
authors.affiliation: Department of Chemistry, Tianjin University, Tianjin 300072, PR China.
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Tianjin University, Tianjin 300072, PR China.
authors.forename: Ke-Ke
authors.initials: KK
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Tianjin University, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, PR China. Electronic address: linazhu@tju.edu.cn.
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, PR China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, PR China. Electronic address: kongdem@nankai.edu.cn.
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Kong
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000032:analysis* / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods; D054856:G-Quadruplexes; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D017931:DNA Primers; D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D004720:Endonucleases
keywords: Inhibitor; Methyltransferase; Multiple primers; Real-time; Rolling circle amplification