title: Tools for investigation of the RNA endonuclease activity of mammalian Argonaute2 protein.
abstract: Mammalian Argonaute2 (Ago2) protein is the key player of RNA-induced silencing complexes (RISCs), regulating gene function through RNA interference. In this paper, a method to investigate the RNA endonuclease activity of Ago2 is reported using electrochemical technique with G-quadruplex-hemin complexes as signal transduction probes. Experimental results reveal that Ago2 may exhibit its slicer activity without any biological partners or ATP in wide pH and temperature ranges; thus, a method to assay the activity of the enzyme is proposed. For purified samples, the endonuclease activity of Ago2 can be quantified in the range from 6.25 to 25 nM with a detection limit of 5.02 nM. In the case of porcine cardiocyte lysates which contain a certain amount of Ago2, a linear correlation can be also obtained between the electrochemical signal and the dilution radio of the lysates. The proposed method shows desirable sensitivity, high selectivity, and excellent reproducibility, implying that this method may hold considerable potential for functional studies of Ago2 and clinical diagnosis in the future.
authors.affiliation: Department of Obstetrics, The First Affiliated Hospital with Nanjing Medical University, 210036 Nanjing, China.
authors.forename: Nana
authors.initials: N
authors.lastname: Yang
authors.forename: Ya
authors.initials: Y
authors.lastname: Cao
authors.forename: Ping
authors.initials: P
authors.lastname: Han
authors.forename: Xuejun
authors.initials: X
authors.lastname: Zhu
authors.forename: Lizhou
authors.initials: L
authors.lastname: Sun
authors.forename: Genxi
authors.initials: G
authors.lastname: Li
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism*; D055664:Electrochemical Techniques; D004566:Electrodes; D004720:Endonucleases / Q000378:metabolism; D054856:G-Quadruplexes; D006427:Hemin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015398:Signal Transduction; D013552:Swine; D013696:Temperature
chemical_list: D060565:Argonaute Proteins; D006427:Hemin; D004720:Endonucleases