DRAQ5 in SARS-CoV-2 COVID-19 Research

Literature Review: DRAQ5™ in SARS-CoV-2, COVID-19 Research

The cell permeant far-red DNA binding dye DRAQ5™ is widely benchmarked as a tool in bench-scale immunofluorescence (IF) and immunohistochemical (IHC) microscopy but also high throughput screening assays for viral infectivity and neutralisation by in-cell western assay™ (ICW), a highly robust semi-quantitative method with a low barrier to entry, and in high content screening (using automated microscopy) as part of the discovery workflow for new or re-purposed anti-viral therapies.

Here we review its deployment in the understanding of SARS-Coronavirus-2 and COVID-19, and in the search for effective vaccination and therapeutics.

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Interrogating COVID-19 cardiac pathology: atypical myocarditis

Fox et al. performed histopathological analysis on autopsy cardiac tissue from COVID-19 patients. Their data suggest that the myocarditis seen is atypical and posit investigation of alternative mechanism for the tissue damage.  DRAQ5 (for nuclei) and Strandbrite Green (RNA; AAT Bioquest) were used for a rapid, non-destructive alternative to H&E staining (after Elfer et al., 2016).

Improving on the plaque reduction neutralisation test (PRNT)

Park et al. proposed a new high throughput assay format to screen for neutralizing antibodies against SARS-CoV-2. They compared the conventional PRNT with a micro-scaled alternative detecting the presence of (de novo) virus in the infected cells rather than plaque reduction.  They did this firstly with an homogeneous assay where signal was developed via a HRP-coupled anti-viral antibody and then ultimately, using the principle of the in-cell western assay™ (ICW), virus was detected with an infra-red fluorophore IRDye™800CW coupled secondary antibody and signals normalised by use of DRAQ5 to correct for cell number variances between wells and measured on LICOR’s Odyssey® imaging system. This fluorescence-based assay also benefited from the use of DRAQ5 since it was possible to estimate cell viability.  Such assays have been previously described for DENV for example (Cox, et al. mAbs. 8.1. (2016): 129-140).

In a further elegant iteration of the ICW assay, Stanifer et al. used it to determine a TCID50 for SARS-CoV-2 infection in Vero cells. From this knowledge, they infected colon carcinoma cells at a MOI of 0.5 as part of their studies into intestinal epithelial cell infection and possible amelioration with type III interferon.

Drug discovery:re-purposing for SARS-CoV-2 infection

To better understand the packaging of nascent virion in infected cells Jack et al. demonstrated that this relied upon the interaction between viral genomes and nucleocapsid (N) protein. Further, they sought to identify compounds that might interfere with this process, where kinase inhibitor nilotinib proved effective.  To further confirm this further, Vero cells were transfected with GFP-tagged N and then allowed to express the protein.  Cells were then exposed to control (DMSO) or doses of nilotinib and thereafter stained with DRAQ5 as counterstain and imaged live for the relative presence of GFP puncta, indicative of the effect of nilotinib to disrupt the phase-separation of N protein. 

Patient-derived anti-SARS-CoV-2 monoclonals:surprising tissue specificities

Kreye et al. generated a number of patient-derived monoclonal antibodies against the S1 subunit of spike protein and tested these for efficacy and non-self reactivity in a mammalian model. The different clones showed tissue structure specificities, for example in frozen, unfixed sections from the hippocampus, where DRAQ5 was used as nuclear counterstain. One clone showed protection from lung pathology.

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References:

Fox, Sharon E., et al. "Unexpected features of cardiac pathology in COVID-19 infection." Circulation (2020) 142:1123–1125

Jack, Amanda, et al. "SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA." bioRxiv (2020). DOI:10.1101/2020.09.14.295824

Kreye, Jakob, et al. "A therapeutic non-self-reactive SARS-CoV-2 antibody protects from lung pathology in a COVID-19 hamster model." Cell (2020). DOI:10.1101/2020.08.15.252320

Park, Jun-Gyu, et al. "Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2." bioRxiv (2020). DOI:10.1101/2020.07.22.216648

Stanifer, Megan L., et al. "Critical role of type III interferon in controlling SARS-CoV-2 infection in human intestinal epithelial cells." Cell reports 32.1 (2020): 107863.

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See the related blog article on DRAQ7's COVID citations and applications

Further reading: 

BioStatus blog article "Anti-virals discovery with DRAQ5" published Sep 8th, 2020

Got questions about DRAQ5™? E-mail us now or visit our website 

Technical datasheet and other key documents can be found on the DRAQ5 product page.

 

Read independent product reviews on DRAQ5, moderated by SelectScience.

 

In-Cell Western Assay™, IRDye™ and Odyssey® are trademarks of LICOR Biosciences.

DRAQ7 in SARS-CoV-2 COVID-19 Research

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