In Situ, Real-Time Monitoring of the 3鈥?to 5鈥?Exonucleases Secreted by Living Cells

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• 作者：Xin Su ; Xiaocui Zhu ; Chen Zhang ; Xianjin Xiao ; Meiping Zhao
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：June 5, 2012
• 年：2012
• 卷：84
• 期：11
• 页码：5059-5065
• 全文大小：330K
• 年卷期：v.84,no.11(June 5, 2012)
• ISSN：1520-6882

文摘

Enzymes containing 3鈥?5鈥?exonuclease activities play vital roles in maintaining genome stability. Though a wide variety of methods have been developed for detection of these enzymes, few of them can be directly applied for in situ and real-time monitoring of the secretion of these active substances by living cells. Taking advantages of the free 3鈥?end of stacked guanine-quenched photoinduced electron transfer fluorescent probes, here we demonstrate a novel assay capable of in situ and real-time monitoring of the 3鈥?5鈥?exonucleases secreted by living cells. The detection limit of the new method achieved as low as 0.04 U/mL, allowing direct monitoring of the target enzymes in an extracellular environment without preconcentration steps. False positive signals caused by other nonspecific enzymes were easily ruled out by the use of a control probe with the 3鈥?end modified with exonuclease-resistant phosphorothioate guanines. Using Alexa Fluor 488 as the fluorophore, the probe is adaptable to a wide range of pH conditions. The approach was successfully applied for in situ, real-time monitoring of the 3鈥?5鈥?exonucleases secreted by suspension cells of Arabidopsis thaliana. It also holds great potential for in situ and real-time detection of many other DNA end-processing enzymes produced by other types of cells.