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

Nada Raddaoui,Alexander Borodavka,Florian Geiger,Hanna Engelke,Bastien Viverge,Samuele Stazzoni,Thomas Carell,Stefano Croce,Leonhard Möckl,Christoph Bräuchle,Thomas Frischmuth

doi:10.1002/cbic.202000081

Nada Raddaoui, Alexander Borodavka + Show 9 more

Open Access

https://doi.org/10.1002/cbic.202000081

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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 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.

Highlights

Introduction specificmessenger RNAs (mRNAs) is highly desirable from a diagnostic point of view
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
Basically all malfunctioning processes in cells induce transcriptional changes.[1,2]. These go in hand with altered levels of messenger RNAs

Summary

These multichromophore probes were conveniently prepared

Gene expression varies significantly between individual cells and it is strongly altered in disease states. Viral infections for example lead to early transcription of virus-specific genes that could be exploited for an early diagnosis and characterization of the infection. Basically all malfunctioning processes in cells induce transcriptional changes.[1,2] These go in hand with altered levels of messenger RNAs (mRNAs). In extreme cases disease related mRNA may not be present at all in the normal state. The levels of specific mRNA will be changed, which requires reliable methods to quantify mRNA transcripts. Detection and quantification of a [a] N.

Department of Biochemistry

Results and Discussion

Conclusions