Rapid SARS-CoV-2 Detection Using Amplicon Templated Reporter Enzyme Assembly

ABSTRACT We are proposing to pilot test a new enzyme biosensor technology for the purpose of enhancing isothermal RNA amplification assays for SARS-CoV-2. Our overarching goal is to validate this technology, called DETECT, as biomolecular tool to increase the sensitivity, specificity, and speed of SARS-CoV-2 testing. DETECT is based on a modified split luciferase enzyme complementation assay. Instead of the standard bait and prey fused protein constructs, we connect two non-interacting luciferase fragments to SARS-CoV-2 oligonucleotide probes. Conjugation of the luciferase fragments to the oligonucleotides uses a chemi-enzymatic method developed in the investigator's lab. The oligonucleotides are designed to anneal to adjacent segments in a unique SARS-CoV-2 amplicon. With samples containing the amplicon, the split luciferase fragments are brought together through base pairing of their attached oligonucleotides with the SARS-CoV-2 amplicon. Molecular assembly reconstitutes functional luciferase from the two fragments, enabling robust light output. In preliminary experiments, we validate the central and novel concept of DETECT: protein fragment complementation via nucleic acid base pairing. In controls where base pairing of the oligonucleotides is blocked, either by exonuclease pretreatment or by competitor oligonucleotide, we observe luminescence readings on par with buffer only samples. By contrast, in experimental samples where oligonucleotide base pairing is supported, we observe luciferase signal that is increased 100- fold over background. These preliminary experiments were carried out with the split luciferase- oligonucleotide conjugates at 25 nM. Over the course of this 2-year project, we propose to evaluate the DETECT system quantitatively for specificity, sensitivity and speed, thereby assessing the clinical potential of this biosensor technology. Although our objective here is diagnosing SARS-CoV-2 infection, the DETECT system is easily re-programmed by changing the oligonucleotide probe sequences. Thus DETECT holds promise as a new and innovative diagnostic platform.