Kinetic analysis of target RNA binding and slicing by human Argonaute 2 protein

Sarah Willkomm, Tobias Restle*

*Korrespondierende/r Autor/-in für diese Arbeit


    Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels. Here, we present techniques to kinetically characterize recombinant Argonaute 2-mediated guide and target binding as well as target RNA slicing. We focus on fluorescence-based steady-state and in particular pre-steady-state techniques to unravel mechanistic details. Furthermore, we describe a cleavage assay to analyze Argonaute 2-mediated slicing using radioactively labeled target strands.

    TitelMethods in Molecular Biology
    Herausgeber (Verlag)Humana Press Inc.
    PublikationsstatusVeröffentlicht - 01.01.2017


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