Abstracts

The SARS-CoV-2 virus infects human cells using a spike (S) protein, an antigen that decorates the surface of the virus particles. In this study, we have revealed a novel aspect of the SARS-CoV-2 S protein’s bioluminescent enzyme (luciferase)-like ability to catalyze the oxidative luminescent reaction of Cypridina luciferin, which is a substrate of the marine luminous sea firefly (Vargula hilgendorfii). Moreover, we have successfully demonstrated that the pseudo-luciferase activity of the S protein itself offers a new method to detect the SARS-CoV2 S protein. From fireflies to lantern fish, most of the animals produce light emission through an enzymatic reaction between the substrate (luciferin) and the enzyme (luciferase). Although a luciferin typically emits light only in the presence of the corresponding luciferase, imidazopyrazinone-type (IPT) luciferins, which are widely present in luminous marine organisms, can emit light when encountering other proteins including serum albumins that aren’t considered enzymes. Then, we first investigated 36 different IPT luciferins’ abilities to react with a single unit of the SARS-CoV-2 S protein. As a result, only Cypridina luciferin emitted light. An adequate amount of light could also be detected with the spike protein in its natural state, as three units folded together. Additional experiments indicated that the Cypridina luciferin was selective because it was recognized from only the interfaces between the units of the S protein and did not glow when exposed to six proteins that occur in human saliva. We defined this specific luminescence reaction by non-luciferase biomolecules as “biomolecule-catalyzing chemiluminescence (BCL)”. Finally, we found that the luciferin could detect the amount of the S protein in human saliva with the same accuracy as an ELISA (Enzyme-linked Immunosorbent Assay). The BCL-based assay system delivered results in one minute — significantly faster than the current rapid point-of-care tests. Our developed approach could serve as the basis for a simple “mix and read” test in which Cypridina luciferin is added to untreated saliva from someone suspected of having COVID-19. This finding opens the door to develop a novel platform to detect virus antigens simply and rapidly without antibodies and genetic manipulation. Furthermore, we have succeeded in identifying the functional group in Cypridina luciferin that selectively recognizes enzymes or enables efficient luminescence reactions in S-protein-catalyzed luminescence systems. The function of each functional group in Cypridina luciferin has not been clarified in the natural Cypridina luciferase system. Therefore, this finding represents a significant contribution to the elucidation of the unknown reaction mechanism in bioluminescence (BL) and to the extension of BL applications in molecular biology and diagnostic measurement studies.