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.
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
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.
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