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RNA synthesis monitoring

In addition to BrU, a common approach for RNA synthesis monitoring relies on incorporation of ethynyl-labeled uridine (5-EU[1,2]) instead of their natural counterpart uracil. This traditional method however, is hampered by cytotoxicity of Cu(I) which is generated during copper-catalyzed labeling of 5-EU with Azide-functionalized detection molecules (fluorescent Azides, Azides of (Desthio)Biotin or FLAG Azide)(Fig. 1).

5-Vinyl-uridine (5-VU) can potentially be used as a replacement for the copper-catalyst requiring 5-EU (5-Ethynyluridine)to measure de novo RNA synthesis in proliferating cells. This assumption is based on the previously demonstrated suitability of 5-Vinyl-2'-deoxyuridine (5-VdU) for DNA synthesis monitoring[3]. Labeling of the resulting Vinyl-functionalized RNA is achieved via Copper-free Alkene-Tetrazine Ligation that allows to introduce a Biotin group for subsequent purification (via Tetrazine-containing Biotinylation Reagents) or a fluorescent group for subsequent microscopic imaging (via Tetrazine-containing Fluorescent Dyes).

 

Figure 1: Workflow of RNA synthesis monitoring with 5-EU.

Selected References

[1] Jao et al. (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. USA 105 (41):15779.
[2] Darzynkiewicz et al. (2011) Cytometry of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on "Click Chemistry". Cytometry A 79A:328.
[3] Rieder et al. (2014) Alkene-Tetrazine Ligation for Imaging Cellular DNA. Angew. Chem. Int. Ed. 53:9168.