title: In situ, real-time monitoring of the 3' to 5' exonucleases secreted by living cells.
abstract: Enzymes containing 3'-5' 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'-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'-5' 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'-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'-5' 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.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Su
authors.forename: Xiaocui
authors.initials: X
authors.lastname: Zhu
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.forename: Xianjin
authors.initials: X
authors.lastname: Xiao
authors.forename: Meiping
authors.initials: M
authors.lastname: Zhao
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D015374:Biosensing Techniques; D005092:Exonucleases / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes; D006147:Guanine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry*; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D054735:Phosphorothioate Oligonucleotides; D006147:Guanine; D005092:Exonucleases