Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
Journal article, 2021

Methods for tracking RNA 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, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO 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 tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Author

Tom Baladi

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

AstraZeneca AB

Jesper Nilsson

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

Audrey Gallud

Chalmers, Biology and Biological Engineering, Chemical Biology

Emanuele Celauro

Chalmers, Biology and Biological Engineering, Chemical Biology

Cécile Gasse

Universite d' Evry Val d'Essonne

Fabienne Levi-Acobas

Institut Pasteur, Paris

Ivo Sarac

Institut Pasteur, Paris

Marcel R. Hollenstein

Institut Pasteur, Paris

Anders Dahlén

AstraZeneca AB

Elin Esbjörner Winters

Chalmers, Biology and Biological Engineering, Chemical Biology

Marcus Wilhelmsson

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

Journal of the American Chemical Society

0002-7863 (ISSN) 1520-5126 (eISSN)

Vol. 143 14 5413-5424

Areas of Advance

Nanoscience and Nanotechnology (SO 2010-2017, EI 2018-)

Health Engineering

Subject Categories

Physical Chemistry

Cell Biology

Biochemistry and Molecular Biology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Organic Chemistry

DOI

10.1021/jacs.1c00014

PubMed

33797236

More information

Latest update

7/7/2021 9