Molehills over Mountains: Exploring a Novel Mechanism for Direct Detection of SARS-CoV-2

Abstract

Over the course of the COVID-19 pandemic, certain methods of testing became more heavily preferred than others, indicating shifting priorities in the face of a prolonged and pervasive public health crisis. From studying changing preferences it is clear there is a need for a more agile platform for pathogen detection that is decentralized, direct, simple to produce, simple to use, and can easily adaptable to other emerging pathogens. Taking this into account, several mechanisms for direct detection were explored that target the interaction between the receptor binding domain (RBD) on the SARS-CoV-2 spike protein and the peptidase domain on the human ACE2 receptor, the latter of which serving as the primary entry point for the virus into human cells. The most promising mechanism was a weak mutant RBD “lock” that is bound to a recombinant-hACE2 peptidase domain with a lower binding affinity than that of wild type RBD. Both the lock and the recombinant hACE2 would be linked to separate split-GFP parts that are constitutively bound and fluorescing. In the presence of virus, the lock would be displaced, tearing the intact split-GFP fragments apart and disrupting the fluorescent signal, indicating infection. Experimental validation was pursued for this idea, and transfection and expression of component parts was demonstrated. Future steps are described that involve working towards a more rigorous proof of concept, including tuning of the mutant lock via site-directed mutagenesis, and investigating binding dynamics of the mutant lock against SARS-CoV-2 RBD. Commercialization of this novel platform is explored, as well as an examination of the feasibility of the idea as a truly pathogen-agnostic platform through the context of a measles sensor case-study.