Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions

Holstein J., Muttach F., Schiefelbein S., Rentmeister A.

Forschungsartikel (Zeitschrift) | Peer reviewed

Zusammenfassung

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.

Details zur Publikation

FachzeitschriftChemistry - A European Journal (Chem. Eur. J.)
Jahrgang / Bandnr. / Volume2017
Ausgabe / Heftnr. / Issue25
Seitenbereich6165-6173
StatusVeröffentlicht
Veröffentlichungsjahr2017
Sprache, in der die Publikation verfasst istEnglisch
DOI10.1002/chem.201604816
Link zum Volltexthttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85010384451&origin=inward
StichwörterClick chemistry; Enzyme catalysts; Fluorescence; FRET; RNA

Autor*innen der Universität Münster

Holstein, Josephin
Professur für Biomolecular Label Chemistry (Prof. Rentmeister)
Muttach, Fabian
Professur für Biomolecular Label Chemistry (Prof. Rentmeister)
Rentmeister, Andrea
Professur für Biomolecular Label Chemistry (Prof. Rentmeister)
Schiefelbein, Stephan
Professur für Biomolecular Label Chemistry (Prof. Rentmeister)