title: A Rapid and Versatile Assay for Ago2-Mediated Cleavage by Using Branched Rolling Circle Amplification.
abstract: Micro RNA (miRNA) research has evolved into an essential part of investigating gene regulation in which deregulation of numerous miRNAs is associated with various cellular dysfunction and diseases. Here, we describe a rapid and homogenous assay for Ago2-mediated target RNA cleavage, based on branched rolling circle amplification (BRCA). In particular, the ability to investigate small molecule binders for inhibition of miRNA function is within the potential of our assay. This method uses no artificial fluorescence labeling of RNA components, which can be an advantage in screening of potential inhibitors. To visualize cleavage of RNA substrate by Ago2, we developed a two-step assay composed of Ago2-mediated cleavage and BRCA-based detection. The assay is cost-effective and practicable and can be performed in 96-well format by using a standard qPCR machine.
authors.affiliation: Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489, Berlin, Germany.
authors.forename: Marlen
authors.initials: M
authors.lastname: Hesse
authors.affiliation: Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489, Berlin, Germany. arenzchr@hu-berlin.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Arenz
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D059367:RNA Cleavage; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D011994:Recombinant Proteins
keywords: Argonaute; RISC; aminoglycoside; branched rolling circle amplification; micro RNA