Dataset Information

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

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.

SUBMITTER: Raddaoui N

PROVIDER: S-EPMC7496099 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Raddaoui Nada N Croce Stefano S Geiger Florian F Borodavka Alexander A Möckl Leonhard L Stazzoni Samuele S Viverge Bastien B Bräuchle Christoph C Frischmuth Thomas T Engelke Hanna H Carell Thomas T

Chembiochem : a European journal of chemical biology 20200420 15

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. Covi ...[more]

PMID: 32187837

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Project description:Phages are viruses that infect prokaryotes and can shape microbial communitiesby lysis, thus offering applications in various fields. However, challengesexist in sampling, isolation and accurate prediction of the host specificity ofphages as well as in the identification of newly replicated virions in response toenvironmental challenges. A new workflow using biorthogonal non-canonicalamino acid tagging (BONCAT) and click chemistry (CC) allowed combinedanalysis of phages and their hosts, the identification of newly replicated virions,and the specific tagging of phages with biotin for affinity chromatography.Replication of phage λ in Escherichia coli was selected as a model for workflowdevelopment. Specific labeling of phage λ proteins with the non-canonicalamino acid 4-azido-L-homoalanine (AHA) during phage development in E. coliwas confirmed by LC–MS/MS. Subsequent tagging of AHA with fluorescentdyes via CC allowed the visualization of phages adsorbed to the cell surfaceby fluorescence microscopy. Flow cytometry enabled the automated detectionof these fluorescent phage-host complexes. Alternatively, AHA-labeled phageswere tagged with biotin for purification by affinity chromatography. Despitebiotinylation the tagged phages could be purified and were infectious afterpurification. Applying this approach to environmental samples would enablehost screening without cultivation. A flexible and powerful workflow for thedetection and enrichment of phages and their hosts in pure cultures has beenestablished. The developed method lays the groundwork for future workflowsthat could enable the isolation of phage-host complexes from diverse complexmicrobial communities using fluorescence-activated cell sorting or biotinpurification. The ability to expand and customize the workflow through thegrowing range of compounds for CC offers the potential to develop a versatiletoolbox in phage research. This work provides a starting point for these furtherstudies by providing a comprehensive standard operating procedure.

Dataset Information

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

Publications

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

Similar Datasets

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