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Stealth fluorescence labeling for live microscopy imaging of mRNA delivery
Stealth fluorescence labeling for live microscopy imaging of mRNA delivery
International audience; 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.
- French National Centre for Scientific Research France
- Université Paris Diderot France
- AstraZeneca (United Kingdom) United Kingdom
- University of Paris-Saclay France
- Institut Pasteur France
Microsoft Academic Graph classification: Green fluorescent protein law.invention chemistry.chemical_compound law medicine.diagnostic_test Chemistry Electroporation Translation (biology) Transfection Cell biology Cytosine Flow cytometry Confocal microscopy medicine Messenger RNA RNA Nucleic acid
MESH: Cytosine, translation, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, fluorescence labeling, Biochemistry, Histones, Colloid and Surface Chemistry, MESH: COVID-19, nucleobase triphosphate, MESH: Histones, Molecular Structure, [CHIM.ORGA]Chemical Sciences/Organic chemistry, MESH: Fluorescent Dyes, Molecular Imaging, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, MESH: Spectrometry, Fluorescence, MESH: Cell Line, Tumor, mRNA, MESH: Molecular Structure, Green Fluorescent Proteins, Catalysis, Article, Fluorescence, Cytosine, MESH: Green Fluorescent Proteins, Cell Line, Tumor, [CHIM]Chemical Sciences, Humans, RNA, Messenger, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: RNA, Messenger, Fluorescent Dyes, MESH: Humans, MESH: Molecular Imaging, MESH: Fluorescence, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, COVID-19 Drug Treatment, live cell imaging, Spectrometry, Fluorescence, drug delivery
MESH: Cytosine, translation, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, fluorescence labeling, Biochemistry, Histones, Colloid and Surface Chemistry, MESH: COVID-19, nucleobase triphosphate, MESH: Histones, Molecular Structure, [CHIM.ORGA]Chemical Sciences/Organic chemistry, MESH: Fluorescent Dyes, Molecular Imaging, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, MESH: Spectrometry, Fluorescence, MESH: Cell Line, Tumor, mRNA, MESH: Molecular Structure, Green Fluorescent Proteins, Catalysis, Article, Fluorescence, Cytosine, MESH: Green Fluorescent Proteins, Cell Line, Tumor, [CHIM]Chemical Sciences, Humans, RNA, Messenger, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: RNA, Messenger, Fluorescent Dyes, MESH: Humans, MESH: Molecular Imaging, MESH: Fluorescence, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, COVID-19 Drug Treatment, live cell imaging, Spectrometry, Fluorescence, drug delivery
Microsoft Academic Graph classification: Green fluorescent protein law.invention chemistry.chemical_compound law medicine.diagnostic_test Chemistry Electroporation Translation (biology) Transfection Cell biology Cytosine Flow cytometry Confocal microscopy medicine Messenger RNA RNA Nucleic acid
59 references, page 1 of 6
1. Crooke, S. T.; Witztum, J. L.; Bennett, C. F.; Baker, B. F., RNA-Targeted Therapeutics. Cell Metabolism 2018, 27 (4), 714- 739.
2. Dowdy, S. F., Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 2017, 35 (3), 222-229.
3. Crooke, S. T.; Wang, S.; Vickers, T. A.; Shen, W.; Liang, X.- h., Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol. 2017, 35, 230.
4. Pei, D.; Buyanova, M., Overcoming Endosomal Entrapment in Drug Delivery. Bioconjugate Chem. 2018.
5. Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stöter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M., Image-based analysis of lipid nanoparticlemediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31 (7), 638-646. [OpenAIRE]
6. Armitage, B. A., Cyanine Dye-Nucleic Acid Interactions. In Heterocyclic Polymethine Dyes: Synthesis, Properties and Applications, Strekowski, L., Ed. Springer Berlin Heidelberg: Berlin, 2008; pp 11-29.
7. Zhang, Y.; Kleiner, R. E., A Metabolic Engineering Approach to Incorporate Modified Pyrimidine Nucleosides into Cellular RNA. J. Am. Chem. Soc. 2019, 141, 3347-3351.
8. Custer, T. C.; Walter, N. G., In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines. Protein. Sci. 2017, 26 (7), 1363-1379.
9. Shimomura, O.; Johnson, F. H.; Saiga, Y., Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. J. Cell. Compar. Physl. 1962, 59 (3), 223-239.
10. Jung, G., Fluorescent Proteins: The Show Must go on! In Fluorescent Analogues of Biomolecular Building Blocks: Design and Applications, Wilhelmsson, L. M.; Tor, Y., Eds. Wiley: 2016; pp 55- 90.
citations This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).21 popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.Top 10% influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).Top 10% impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.Top 10% citations This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).21 popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.Top 10% influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).Top 10% impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.Top 10% Powered byBIP!