title: A label-free DNA-templated silver nanocluster probe for fluorescence on-off detection of endonuclease activity and inhibition.
abstract: Endonuclease cleavage of DNA plays an important role in biological and medicinal chemistry. This study aimed to develop a reliable and sensitive method for nuclease activity assay by combining the high specificity of DNA cleavage reactions with ultrahigh fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequences in proximity to silver nanoclusters (Ag NCs). The DNA-templated Ag NC (DNA-Ag NC) probe with endonuclease recognition sequence consists of NC and a G-rich probe. The NC probe was designed by adding Ag NC nucleation sequence at the 5'-end. The G-rich probe is the complementary DNA sequence modified by adding a G-rich overhang sequence at the 3'-end. Thus, the fluorescence of DNA-Ag NC probe was activated because of DNA hybridization. When these DNA-Ag NC probes were exposed to the targeted endonucleases, specific DNA cleavages occurred, and pieces of G-rich DNA fragments separated from Ag NCs, resulting in fluorescence turn-off. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. Detection was demonstrated by assaying EcoRI activity. Under optimized conditions, the fluorescence reduction efficiency was linear with the EcoRI concentration in the range of 5.0×10(-4) U μL(-1) to 3.0×10(-3) U μL(-1), with a detection limit of 3.5×10(-4) U μL(-1), which is much better than or at least comparable with that in previous reports. The potential application of the proposed method for screening endonuclease inhibitors was also demonstrated. The presented assay protocol proved to be convenient, effective, sensitive, and easy in preparing the fluorescent probe.
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, Shaanxi Normal University, PR China; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, PR China.
authors.forename: Yunxia
authors.initials: Y
authors.lastname: Qian
authors.forename: Yaodong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Lu
authors.forename: Yanan
authors.initials: Y
authors.lastname: Cai
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry*; D015246:Deoxyribonuclease EcoRI / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D057230:Limit of Detection; D049329:Nanostructures / Q000737:chemistry*; D012834:Silver / Q000737:chemistry*
chemical_list: D015342:DNA Probes; D004791:Enzyme Inhibitors; D012834:Silver; D015246:Deoxyribonuclease EcoRI
keywords: Activity; Endonuclease; Endonuclease inhibitor; Inhibition; Silver nanoclusters