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One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification
Chen, Feng; Zhao, Yongxi; Qi, Lin; Fan, Chunhai
2013
Source PublicationBIOSENSORS & BIOELECTRONICS
ISSN0956-5663
Volume47Pages:CONCATENATE(Sheet1!I243,-Sheet1!J243)
AbstractDNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Herein, using T4 PNK as a model target, we describe a one-step, highly sensitive, simple and rapid fluorescence approach for monitoring its activity and inhibition. This innovative strategy is inspired by the great amplification capability of ligation-nicking coupled reaction-mediated signal amplification. In the presence of T4 PNK, one of two short oligonucleotides complementary to the loop sequence of molecular beacon (MB) are phosphorylated, and then ligated with the other by DNA ligase. Upon formation of the stable duplex between the ligated DNA and MB, the fluorescence is restored and further significantly amplified through nicking endonuclease assisted cleavage of multiple MBs. Meanwhile, the cleavage of MBs will also generate new nicks to initiate the ligation reaction. Eventually, a maximum fluorescence enhancement is obtained when the ligation and nicking process reached a dynamic equilibrium. As compared to those of the existing approaches except for the assay based on single nanoparticle counting, all limited to 1:1 signal transduction function, the sensitivity (0.00001 U/mL) of the proposed strategy is 100-1700 times higher. The application of the sensing system in complex biological matrix and screening of T4 PNK inhibition are demonstrated with satisfactory results. Moreover, this approach is also successfully used to detect biological small molecules such as adenosine triphosphate (ATP), and can be further extended for nicotinamide adenine dinucleotide (NAD(+)) detection. (C) 2013 Elsevier B.V. All rights reserved.
Indexed BySCI
Language英语
Funding Project应物所项目组
WOS IDWOS:000320481900032
Citation statistics
Cited Times:35[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/13847
Collection中科院上海应用物理研究所2011-2019年
Recommended Citation
GB/T 7714
Chen, Feng,Zhao, Yongxi,Qi, Lin,et al. One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification[J]. BIOSENSORS & BIOELECTRONICS,2013,47:CONCATENATE(Sheet1!I243,-Sheet1!J243).
APA Chen, Feng,Zhao, Yongxi,Qi, Lin,&Fan, Chunhai.(2013).One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification.BIOSENSORS & BIOELECTRONICS,47,CONCATENATE(Sheet1!I243,-Sheet1!J243).
MLA Chen, Feng,et al."One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification".BIOSENSORS & BIOELECTRONICS 47(2013):CONCATENATE(Sheet1!I243,-Sheet1!J243).
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