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

Autor: Raddaoui N; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Croce S; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany.; Baseclick GmbH, Floriansbogen 2-4, 82061, Neuried (München, Germany., Geiger F; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Borodavka A; Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.; Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK., Möckl L; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Stazzoni S; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Viverge B; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Bräuchle C; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Frischmuth T; Baseclick GmbH, Floriansbogen 2-4, 82061, Neuried (München, Germany., Engelke H; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany., Carell T; Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2020 Aug 03; Vol. 21 (15), pp. 2214-2218. Date of Electronic Publication: 2020 Apr 20.
DOI: 10.1002/cbic.202000081
Abstrakt: 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 off-target 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.
(© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
Databáze: MEDLINE
Externí odkaz: