title: Site-Specific Fluorescent Labeling of Argonaute for FRET-Based Bio-Assays.
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.
authors.affiliation: Department of Microbiology, Single Molecule Biochemistry Lab, Universität Regensburg, Universitätstraße 31, 93053, Regensburg, Germany.|Institute of Molecular Medicine, Universitätsklinikum Schleswig-HolsteinUniversity of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Willkomm
authors.affiliation: Department of Microbiology, Single Molecule Biochemistry Lab, Universität Regensburg, Universitätstraße 31, 93053, Regensburg, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Zander
authors.affiliation: Department of Microbiology, Single Molecule Biochemistry Lab, Universität Regensburg, Universitätstraße 31, 93053, Regensburg, Germany. Dina.Grohmann@ur.de.
authors.forename: Dina
authors.initials: D
authors.lastname: Grohmann
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000302:isolation & purification*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D020868:Gene Silencing; D063747:Methanocaldococcus / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D060565:Argonaute Proteins; D005456:Fluorescent Dyes
keywords: Archaea; Argonaute; Gene silencing; Methanocaldococcus jannaschii; Unnatural amino acid incorporation