title: A Genetically Encodable System for Sequence-Specific Detection of RNAs in Two Colors.
abstract: Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non-fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths.
authors.affiliation: Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Strasse 2, 48149, Münster, Germany.|Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146, Hamburg, Germany.
authors.forename: Stefanie J
authors.initials: SJ
authors.lastname: Kellermann
authors.affiliation: Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Strasse 2, 48149, Münster, Germany. a.rentmeister@uni-muenster.de.|Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Wilhelm-Klemm-Strasse 2, 48149, Münster, Germany. a.rentmeister@uni-muenster.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rentmeister
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015202:Protein Engineering; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017423:Sequence Analysis, RNA / Q000379:methods*; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C465505:pumilio protein, human; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D012313:RNA
keywords: GFP; Pumilio homology domain; RNA recognition; fluorescent probes; protein engineering