Abstract
Deciphering the molecular mechanisms of eukaryotic Argonaute proteins is crucial for the understanding of RNA interference (RNAi), a posttranscriptional gene silencing process. Fluorescence-based single-molecule studies like single-molecule Förster resonance energy transfer (FRET) between a donor and acceptor dye represent a versatile tool to gain a mechanistic understanding of the structural dynamics of a biomolecular complex. Until today it was not possible to site-specifically introduce fluorophores into eukaryotic Argonaute. Using an archaeal Argonaute variant from Methanocaldococcus jannaschii that closely resembles its eukaryotic counterpart, we site-specifically incorporated fluorescent probes into Argonaute. In this chapter, we first describe how to express archaeal Argonaute with the site-specifically engineered unnatural amino acid para-azido-l-phenylalanine (pAzF) and subsequently describe the coupling of a fluorophore exploiting the unique chemistry of the azide group of pAzF. In the second part of the chapter, we present a methodological approach that probes complex formation between acceptor-labeled archaeal Argonaute and guide and target nucleic acids equipped with a donor fluorophore which ultimately allows single-molecule FRET measurements. Furthermore we describe binding and cleavage assays that report on the functionality of Argonaute–nucleic acid complexes.
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Willkomm, S., Zander, A., Grohmann, D. (2017). Site-Specific Fluorescent Labeling of Argonaute for FRET-Based Bio-Assays. In: Schmidt, M. (eds) Drug Target miRNA. Methods in Molecular Biology, vol 1517. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6563-2_20
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DOI: https://doi.org/10.1007/978-1-4939-6563-2_20
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