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Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signal amplification
Zhao, Yongxi; Chen, Feng; Wu, Yayan; Dong, Yanhua; Fan, Chunhai(樊春海)
2013
Source PublicationBIOSENSORS & BIOELECTRONICS
ISSN0956-5663
Volume42Pages:CONCATENATE(Sheet1!I150,-Sheet1!J150)
AbstractHerein, using DNA adenine methylation (Dam) methyltransferase (MTase) as a model analyte, a simple, rapid, and highly sensitive fluorescence sensing platform for monitoring the activity and inhibition of DNA MTase was developed on the basis of methylation-sensitive cleavage and nicking enzyme-assisted signal amplification. In the presence of Dam MTase, an elaborately designed hairpin probe was methylated. With the help of methylation-sensitive restriction endonuclease DpnI, the methylated hairpin probe could be cleaved to release a single-stranded DNA (ssDNA). Subsequently, this released ssDNA would hybridize with the molecular beacon (MB) to open its hairpin structure, resulting in the restoration of fluorescence signal as well as formation of the double-stranded recognition site for nicking enzyme Nt.BbvCI. Eventually, an amplified fluorescence signal was observed through the enzymatic recycling cleavage of MBs. Based on this unique strategy, a very low detection limit down to 0.06 U/mL was achieved within a short assay time (60 min) in one step, which is superior to those of most existing approaches. 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 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. (C) 2012 Elsevier B.V. All rights reserved.
Indexed BySCI
Language英语
Funding Project应物所项目组
WOS IDWOS:000319951700011
Citation statistics
Cited Times:70[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/13767
Collection中科院上海应用物理研究所2011-2019年
Recommended Citation
GB/T 7714
Zhao, Yongxi,Chen, Feng,Wu, Yayan,et al. Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signal amplification[J]. BIOSENSORS & BIOELECTRONICS,2013,42:CONCATENATE(Sheet1!I150,-Sheet1!J150).
APA Zhao, Yongxi,Chen, Feng,Wu, Yayan,Dong, Yanhua,&Fan, Chunhai.(2013).Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signal amplification.BIOSENSORS & BIOELECTRONICS,42,CONCATENATE(Sheet1!I150,-Sheet1!J150).
MLA Zhao, Yongxi,et al."Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signal amplification".BIOSENSORS & BIOELECTRONICS 42(2013):CONCATENATE(Sheet1!I150,-Sheet1!J150).
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