Literature Review: DRAQ7™ in SARS-CoV-2, COVID-19 Research
The far-red DNA binding dye DRAQ7™ is now widely adopted as a state-of-the-art viability dye for flow cytometry/cell sorting including exclusion of dead cells, in assays for cell death mechanisms (e.g. apoptosis) and single cell analysis (e.g. RNA-seq, ATAC-seq). It has become the reagent of choice to enable real-time cell health monitoring to disclose desired / undesired cellular toxicity in drug discovery and in the study of disease mechanisms by both 2D cell culture and 3D microtissues (e.g. spheroids, organoids).
Here we review its deployment in the understanding of the SARS-Coronavirus-2 and COVID-19, and in the search for effective vaccination.
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Drug Discovery: re-purposing for acute lung injury (ALI)
Malimova et al. performed a high content screen to identify candidate compounds that reduce expression of Muc-1, implicated in ALI and ARDS (acute respiratory distress syndrome). A 7-day time-lapse cell viability high content imaging screen on the Perkin Elmer Opera Phenix was included that utilised DRAQ7 and CellEvent Caspase 3/7 Green (ThermoFisher) to identify cytotoxicity across dilution series of each compound tested. Fostamatinib a SYK-inhibitor proved to have promising efficacy.
Cell Therapy: SARS-CoV-2-specific allogeneic T-cells
Cooper et al. have demonstrated the expansion of T cells specific for SARS-CoV-2 with retained central memory phenotype from convalescent donors for therapeutic use. The phenotypic assessment of T cell lineage used a 7-colour (VioBlue, VioGreen, FITC, PE, PerCp-Vio700, PE-Vio770, APC) multi-parameter antibody panel with dead cells excluded using DRAQ7, on the Miltenyi Biotec MACSQuant Analyzer.
Inflammation in COVID-19: impaired cytotoxic function, expansion of MDSC
An international consortium led by Giuseppe Ippolito (Bordoni et al. & Agrati et al.) has described striking features of COVID-19. MDSC expansion went to 90% of MNCs and cytotoxic cell function was significantly impaired in severe COVID-19 patients. For MDSC characterization DRAQ7 was used to exclude dead cells from a 7-colour (Pacific Blue, Krome Orange, FITC, ECD, PC5.5, PC7, APC-A750) multi-parameter antibody panel analyzed on the Beckman Coulter Cytoflex further demonstrating DRAQ7's excellent spectral compatibility.
snRNA-seq & snATAC-seq profiling: age-related gene regulation in COVID-19
Wang et al. isolated nuclei from COVID-19 diseased and healthy lung tissue and sorted these from debris and as singleton events into wells for downstream snATAC-seq and in bulk for droplet-based snRNA-seq. Nuclei sorting was facilitated by DRAQ7 staining on the SH800 Sorter (Sony Biotechnology). Gene expression of ACE2 and TMPRSS2 in alveolar epithelial cells was significantly higher in the adult lung compared to two age-cohorts of childhood lung.
Vaccine development targeting APC: spike protein subunit S1 fused to Fc
Herrmann et al. demonstrated the potential for a vaccine immunogen based on fusing SARS-CoV-2 spike protein subunit S1 to a human immunoglobulin Fc moiety thereby taking advantage of this direct targeting of antigen-presenting cells. To verify the successful electroporation of S1-Fc dsDNA into target cells Cy3-tagged dsDNA was visualised by fluorescence microscopy. Subsequent production of S1-Fc protein in the electroporated cells was determined by indirect immunofluoresence as was its presence in frozen fixed tissue sections from S1-Fc immunized mice. In all cases DRAQ7 was used as the nuclear counterstain. Note: we would recommend DRAQfx FIX & GO™ as a benchtop, dropper bottle-convenience nuclear counterstain for fixed cells and tissue sections.
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References:
Agrati, Chiara, et al. "Expansion of myeloid-derived suppressor cells in patients with severe coronavirus disease (COVID-19)." Cell Death & Differentiation (2020): DOI:10.1038/s41418-020-0572-6.
Bordoni, Veronica, et al. "An inflammatory profile correlates with decreased frequency of cytotoxic cells in COVID-19." Clinical Infectious Diseases (2020). DOI:10.1093/cid/ciaa577.
Cooper, Rachel S., et al. "Rapid GMP-compliant expansion of SARS-CoV-2-specific T cells from convalescent donors for use as an allogeneic cell therapy for COVID-19." bioRxiv (2020). DOI:10.1101/2020.08.05.237867.
Herrmann, Andreas, et al. "A Targeted Vaccine against COVID-19: S1-Fc Vaccine Targeting the Antigen-Presenting Cell Compartment Elicits Protection against SARS-CoV-2 Infection." bioRxiv (2020). DOI: 10.1101/2020.06.29.178616.
Malimova, Maria, et al. "A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic." bioRxiv (2020). DOI:10.1101/2020.06.30.180380.
Wang, Allen, et al. "Single Nucleus Multiomic Profiling Reveals Age-Dynamic Regulation of Host Genes Associated with SARS-CoV-2 Infection." bioRxiv (2020). DOI:10.1101/2020.04.12.037580.
See the related blog article on DRAQ5's COVID citations and applications
Further reading:
BioStatus blog article "Anti-virals discovery with DRAQ5" published Sep 8th, 2020
Technical datasheet and other key documents can be found on the DRAQ7 product page.
Read independent product reviews on DRAQ7, moderated by SelectScience.
Got questions about DRAQ7™? E-mail us now or visit our website
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