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Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions
Author(s) -
Holstein Josephin M.,
Muttach Fabian,
Schiefelbein Stephan H. H.,
Rentmeister Andrea
Publication year - 2017
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201604816
Subject(s) - bioorthogonal chemistry , fluorophore , chemistry , click chemistry , fluorescence , methyltransferase , combinatorial chemistry , biomolecule , rna , analyte , cascade reaction , biotinylation , biochemistry , biophysics , biology , catalysis , physics , quantum mechanics , methylation , gene
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.