Abstract
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.
Highlights
Coronavirus disease (COVID-19) emerged by the end of 2019 in Wuhan, China, and led to more than 240 million infections and over 5 million deaths as of 1 November 2021 (Johns Hopkins University Dashboard)
Our RNA-FISH approach uses two types of bioinformatically designed DNA oligonucleotides (Tsanov et al, 2016): (i) unlabeled primary oligos consisting of two parts: a specific sequence complementary to a selected subregion of the target RNA and a readout sequence that is identical among all primary oligos (FLAP sequence), (ii) a fluorescently labeled secondary oligo complementary to the FLAP sequence, allowing visualization by light microscopy
We presented CoronaFISH, an approach based on single molecule inexpensive FISH (smiFISH) (Tsanov et al, 2016) permitting visualization of the positive and negative RNA strands of SARS-CoV-2
Summary
Coronavirus disease (COVID-19) emerged by the end of 2019 in Wuhan, China, and led to more than 240 million infections and over 5 million deaths as of 1 November 2021 (Johns Hopkins University Dashboard). Viral replication occurs in the host cell’s cytoplasm, which is massively reorganized (V’kovski et al, 2020). The genomic positive-strand viral RNA (vRNA) serves as a template for transcription and replication. The virus synthesizes its own RNA-dependent RNA polymerase (RdRP) to generate negativesense RNA replication intermediates. This negative strand acts as template for replication of new full-length positive-stranded RNA genomes and for transcription of several smaller, subgenomic positive-stranded RNAs (sgRNAs). These sgRNAs are used to synthesize all other viral proteins in spatially confined replication complexes. Many aspects of the SARS-CoV-2 viral replication cycle, including the subcellular location of vRNA synthesis, are still not fully understood and under active investigation (V’kovski et al, 2020)
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