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题名: Real-Time Monitoring of the Activity and Kinetics of T4 Polynucleotide Kinase by a Singly Labeled DNA-Hairpin Smart Probe Coupled with lambda Exonuclease Cleavage
作者: Song, Chen1;  Zhao, Meiping1
刊名: ANALYTICAL CHEMISTRY
发表日期: 2009-02-15
DOI: 10.1021/ac802107w
卷: 81, 期:4, 页:1383-1388
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Physical Sciences
类目[WOS]: Chemistry, Analytical
研究领域[WOS]: Chemistry
英文摘要: We describe a novel method for real-time monitoring of the activity and kinetics of T4 polynucleotide kinase (PNK) by use of a singly fluorophore-labeled DNA-hairpin smart probe (SP) coupled with lambda exonuclease (lambda exo) cleavage. The method was performed in a sealed reaction tube and offered more sensitive, fast, high-throughput, and cost-effective detection. The SP was designed with a fluorophore at the X-end, and the fluorescence was quenched by a GGG-triplet at the 5'-end without any other additional quenchers. The 5'-hydroxyl group of the SP was phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product was promptly cleaved by A exo, which caused significant enhancement of fluorescence. A fast and accurate method for assaying the kinase activity of T4 PNK was developed with a wide linear detection range from 0.022 to 5.6 nM s(-1). The phosphorylation reaction was monitored at varying substrate concentrations at the molecular level, and K(m), V(max), and K(cat) values were all calculated. Furthermore, the effects of ATP concentration and salts were investigated. The developed method can be easily adapted to the detection of many other nucleic acid enzymes and may find widespread applications.
关键词[WOS]: PHOTOINDUCED ELECTRON-TRANSFER ;  RNA REPAIR ENZYME ;  MOLECULAR BEACONS ;  FLUORESCENT DYES ;  STRAND BREAKS ;  PHOSPHORYLATION ;  HYBRIDIZATION ;  POLYMERASE ;  RESIDUES ;  ADDUCTS
语种: 英语
WOS记录号: WOS:000263319000012
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/141516
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn, Beijing Natl Lab Mol Sci BNLMS, Beijing 100871, Peoples R China

Recommended Citation:
Song, Chen,Zhao, Meiping. Real-Time Monitoring of the Activity and Kinetics of T4 Polynucleotide Kinase by a Singly Labeled DNA-Hairpin Smart Probe Coupled with lambda Exonuclease Cleavage[J]. ANALYTICAL CHEMISTRY,2009,81(4):1383-1388.
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