Super-sensitive multi-fluorophore RNA-FISH for early virus detection and flow-FISH using click chemistry.

Super-sensitive multi-fluorophore RNA-FISH for early virus detection and flow-FISH using click chemistry.

Raddaoui, Nada;Groce, Stefano;Geiger, Florian;Borodavka, Alexander;Möckl, Leonhard;Stazzoni, Samuele;Viverge, Bastien;Bräuchle, Christoph;Frischmuth, Thomas;Engelke, Hanna;Carell, Thomas;
chembiochem : a european journal of chemical biology 2020
215
raddaoui2020supersensitivechembiochem

Abstract

The reliable detection of transcription events via the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization of the mRNA of a particular gene allows characterizing disease states more directly compared to an analysis on the DNA level. This is particularly needed for the early detection of virus infections as now required for Corona diagnostic. 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 at question. Often a large number of probe strands (often >30) are required to get a reliable signal. The more oligonucleotide probes are however used, the higher are potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne modified DNA oligonucleotides for preparing multiple-fluorophore containing mRNA probes. We found that these multiple-dye probes allow reliable detection and localization of mRNA with only a very small number (5-10) of probe strands. The new method is so sensitive that it even enabled the in situ detection of viral transcripts just 4 hours after infection.

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100544
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10.1002/cbic.202000081
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