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Stealth Fluorescenc...
Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
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- Baladi, Tom, 1991 (author)
- Chalmers tekniska högskola,Chalmers University of Technology,AstraZeneca AB
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- Nilsson, Jesper, 1984 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Gallud, Audrey, 1988 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Celauro, Emanuele, 1980 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Gasse, Cécile (author)
- Université d'Évry Val-d'Essonne,University of Évry Val d'Essonne
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- Levi-Acobas, Fabienne (author)
- Institut Pasteur, Paris
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- Sarac, Ivo (author)
- Institut Pasteur, Paris
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- Hollenstein, Marcel R. (author)
- Institut Pasteur, Paris
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- Dahlén, Anders (author)
- AstraZeneca AB
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- Esbjörner Winters, Elin, 1978 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Wilhelmsson, Marcus, 1974 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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(creator_code:org_t)
- 2021-04-02
- 2021
- English.
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 143:14, s. 5413-5424
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Abstract
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- 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.
Subject headings
- NATURVETENSKAP -- Kemi -- Fysikalisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Physical Chemistry (hsv//eng)
- NATURVETENSKAP -- Biologi -- Cellbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Cell Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
- NATURVETENSKAP -- Kemi -- Organisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Organic Chemistry (hsv//eng)
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Baladi, Tom, 199 ...
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Nilsson, Jesper, ...
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Gallud, Audrey, ...
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Celauro, Emanuel ...
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Gasse, Cécile
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Levi-Acobas, Fab ...
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Sarac, Ivo
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Hollenstein, Mar ...
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Dahlén, Anders
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Esbjörner Winter ...
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Wilhelmsson, Mar ...
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- About the subject
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Chemical Science ...
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and Physical Chemist ...
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Cell Biology
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Biochemistry and ...
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- MEDICAL AND HEALTH SCIENCES
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MEDICAL AND HEAL ...
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and Medical Biotechn ...
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and Medical Biotechn ...
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Chemical Science ...
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and Organic Chemistr ...
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Chalmers University of Technology