Tools for investigation of the RNA endonuclease activity of mammalian Argonaute2 protein.

Nana Yang, Ya Cao, Ping Han, Xuejun Zhu, Lizhou Sun, Genxi Li

Department of Obstetrics, The First Affiliated Hospital with Nanjing Medical University, 210036 Nanjing, China.

Analytical chemistry 2012 Mar 6

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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.

Citation

Nana Yang, Ya Cao, Ping Han, Xuejun Zhu, Lizhou Sun, Genxi Li. Tools for investigation of the RNA endonuclease activity of mammalian Argonaute2 protein. Analytical chemistry. 2012 Mar 6;84(5):2492-7