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Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.
Baladi, Tom; Nilsson, Jesper R; Gallud, Audrey; Celauro, Emanuele; Gasse, Cécile; Levi-Acobas, Fabienne; Sarac, Ivo; Hollenstein, Marcel R; Dahlén, Anders; Esbjörner, Elin K; Wilhelmsson, L Marcus.
  • Baladi T; Department of Chemistry and Chemical Engineering, Chemistry and Biochemistry, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
  • Nilsson JR; Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • Gallud A; Department of Chemistry and Chemical Engineering, Chemistry and Biochemistry, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
  • Celauro E; Department of Biology and Biological Engineering, Chemical Biology, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
  • Gasse C; Department of Biology and Biological Engineering, Chemical Biology, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
  • Levi-Acobas F; Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France.
  • Sarac I; Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR3523, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris CEDEX 15, France.
  • Hollenstein MR; Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR3523, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris CEDEX 15, France.
  • Dahlén A; Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR3523, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris CEDEX 15, France.
  • Esbjörner EK; Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • Wilhelmsson LM; Department of Biology and Biological Engineering, Chemical Biology, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1387160
ABSTRACT
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 (H2BGFP). 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 H2BGFP 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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Messenger / Molecular Imaging / Fluorescence / Fluorescent Dyes Type of study: Experimental Studies Topics: Vaccines Limits: Humans Language: English Journal: J Am Chem Soc Year: 2021 Document Type: Article Affiliation country: JACS.1C00014

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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Messenger / Molecular Imaging / Fluorescence / Fluorescent Dyes Type of study: Experimental Studies Topics: Vaccines Limits: Humans Language: English Journal: J Am Chem Soc Year: 2021 Document Type: Article Affiliation country: JACS.1C00014