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Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

Abstract : Methods for tracking RNA molecules inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts of any length, through enzymatic incorporation of the triphosphate of tC O-a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tC O in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tC O is similar to cytosine, which not only indicates tC O 's excellent nucleotide analogue properties, but also the possibility for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tC Olabeled mRNA is efficiently and correctly translated into H2B:GFP inside human cells. Hence, we not only pioneer the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript can be correctly translated. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that transfected tC O-labeled RNA, unlike the corresponding state-of-the-art fluorescently labeled RNA, translates equally efficient as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics as well as in vaccines, like the ones currently under development against SARS-CoV-2.
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https://hal-pasteur.archives-ouvertes.fr/pasteur-03228730
Contributor : Marcel Hollenstein <>
Submitted on : Tuesday, May 18, 2021 - 12:53:23 PM
Last modification on : Thursday, June 10, 2021 - 12:37:40 PM

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Distributed under a Creative Commons Attribution - NonCommercial 4.0 International License

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Tom Baladi, Jesper Nilsson, Audrey Gallud, Emanuele Celauro, Cécile Gasse, et al.. Stealth fluorescence labeling for live microscopy imaging of mRNA delivery. Journal of the American Chemical Society, American Chemical Society, 2021, 143 (14), pp.5413-5424. ⟨10.1021/jacs.1c00014⟩. ⟨pasteur-03228730⟩

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