Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

BioxRiv, 2020, 172767 (doi: 10.1101/2020.07.01.172767)

Methods for tracking of RNA molecules inside living cells are critical to probe their dynamics and biological functions, but also to monitor delivery of therapeutic RNA. We here describe a method for fluorescence labeling of RNAs of any length, via the enzymatic incorporation of the minimally perturbing and intrinsically fluorescent tricyclic cytosine analogue tC^O. Using this approach, we demonstrate incorporation of tC^O in up to 100% of all natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). The resulting transcript is fully compatible with both in vitro transcription and subsequent in cell translation. Spectroscopic characterization of the in vitro transcribed mRNA, shows that the incorporation rate of tC^O is on par with cytosine, facilitating efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tC^O-labeled mRNA is efficiently and correctly translated into H2B:GFP upon electroporation as well as lipid-mediated transfection of human Huh-7 cells; correct translation was further confirmed in cell-free systems. Importantly, the spectral properties of the tC^O-modified transcripts and their translation product, in this case H2B:GFP, allow for their straightforward and simultaneous visualization in live cells.