Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry

Raddaoui, Nada; Croze, Stefano; Geiger, Florian; Borodavka, Alexander; Möckl, Leonhard; Stazzoni, Samuele; Viverge, Bastian; Bräuchle, Christoph; Frischmuth, Thomas; Engelke, Hanna and Carell, Thomas (18. March 2020): Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry. In: ChemBioChem

Abstract

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential offtarget binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5–10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection.

• Item Type: Journal article
• EU Funded Grant Agreement Number: 741912
• EU Projects: Horizon 2020 > ERC Grants > ERC Advanced Grant > ERC Grant 741912: EPiR - The Chemical Basis of RNA Epigenetics
• Form of publication: Publisher's Version
• Keywords: click chemistry; fluorescence probes; mRNA detection; RNA-FISH; viral infection
• Faculties: Chemistry and Pharmacy > Department of Chemistry
• Subjects: 500 Science > 540 Chemistry
• ISSN: 1439-7633
• Language: English

References

[1] A. Raj, A. Van Oudenaarden, Cell 2008, 135, 216–226. [2] A. Eldar, M. B. Elowitz, Nature 2010, 467, 167–173. [3] S. Itzkovitz, A. van Oudenaarden, Nat. Methods 2011, 8, 12–19. [4] A. Raj, P. van den Bogaard, S. A. Rifkin, A. van Oudenaarden, S. Tyagi, Nat. Methods 2008, 5, 877–879. [5] A. Raj, S.Tyagi. Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. In: Single molecule Tools: Chapter 17, Fluorescence based approaches, Part A; Walter, N. G. B. T.- M. in E., Ed.; Academic Press, 2010, 472, 365–368. [6] S. Semrau, N. Crosetto, M. Bienko, M. Boni, P. Bernasconi, Cell Rep. 2011, 6, 18–23. [7] T. Muramoto, D. Cannon, M. Gierliński, A. Corrigan, G. J. Barton, J. R. Chubb, Proc. Mont. Acad. Sci. 2012, 109, 7350–7355. [8] A. M. Femino, F. S. Fay, K. Fogarty, R. H. Singer, Science 1998, 280, 585– 590. [9] C. Larsson, I. Grundberg, O. Söderberg, M. Nilsson, Nat. Methods 2010, 7, 395–397. [10] T. Trcek, T. Lionnet, H. Shroff, R. Lehmann, Nat. Protoc. 2017, 12, 1326– 1348. [11] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004–2021; Angew. Chem. 2001, 113, 2056–2075. [12] K. V. Gothelf, K. A. Jørgensen, Chem. Rev. 1998, 98, 863–910. [13] S. Bräse, C. Gil, K. Knepper, V. Zimmermann, Angew. Chem. Int. Ed. 2005, 44, 5188–5240. [14] C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–3064. [15] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41, 2596–2599. [16] K. Gutsmiedl, D. Fazio, T. Carell, Chem. Eur. J. 2010, 16, 6877–6883. [17] J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T. Carell, Org. Lett. 2006, 8, 3639–3642. [18] P. M. E. Gramlich, S. Warncke, J. Gierlich, T. Carell, Angew. Chem. Int. Ed. 2008, 47, 8350–8358. [19] P. M. E. Gramlich, C. T. Wirges, A. Manetto, T. Carell, Angew. Chem. Int. Ed. 2008, 47, 8350–8358. [20] S. Hesse, A. Manetto, V. Cassinelli, J. Fuchs, L. Ma, N. Raddaoui, A. Houben, Chromosom. Res. 2016, 24, 299–307. [21] J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T. Carell, Org. Lett. 2006, 8, 3639–3642. [22] R. Arrigucci, Y. Bushkin, F. Radford, K. Lakehal, P. Vir, R. Pine, D. Martin, J. Sugarman, Y. Zhao, G. S. Yap, Nat. Protoc. 2017, 12, 1245–1260. [23] A. Borodavka, E. C. Dykeman, W. Schrimpf, D. C. Lamb, elife 2017, 6, e27453. [24] F. Rodriguez, O. R. Burrone, C. Eichwald, J. Gen. Virol. 2004, 85, 625–634. [25] E. N. Salgado, S. Upadhyayula, S. C. Harrison, J. Virol. 2017, 91, e00651–17. [26] A. Borodavka, U. Desselberger, J. T. Patton, Curr. Opin. Virol. 2018, 33, 106–112.