title: Dual 5' Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions.
abstract: The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5' cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149, Muenster, Germany.
authors.forename: Josephin M
authors.initials: JM
authors.lastname: Holstein
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149, Muenster, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Muttach
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149, Muenster, Germany.
authors.forename: Stephan H H
authors.initials: SHH
authors.lastname: Schiefelbein
authors.affiliation: University of Muenster, Department of Chemistry, Institute of Biochemistry, Wilhelm-Klemm-Straße 2, 48149, Muenster, Germany.|Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Münster, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rentmeister
mesh_terms: D001710:Biotin / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D057930:Click Chemistry; D009670:Encephalitozoon cuniculi / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D016829:Giardia lamblia / Q000201:enzymology; D008780:Methyltransferases / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D012333:RNA, Messenger; C085321:cyanine dye 5; D001710:Biotin; D008780:Methyltransferases
keywords: FRET; RNA; click chemistry; enzyme catalysts; fluorescence