Novel Tetrazolium-Based Colorimetric Assay for Helicase nsp13 in SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a human pathogenic virus that encodes for a helicase (<i>SC2</i>Hel) that is essential for viral replication. <i>SC2</i>Hel has the ability to unravel dsRNA or dsDNA in an NTP-dependent manner from the 5′ to 3′ directionality. The standard helicase assay from studies involving SARS-CoV and SARS-CoV-2 have relied on the concept of fluorescence resonance energy transfer. Adding to the collection of helicase assays, herein, we have developed a novel tetrazolium-based colorimetric assay system for the detection of ADP that is produced via <i>SC2</i>Hel activity. This <i>SC2</i>Hel assay combines three enzyme-coupled steps involving the ADP-dependent <i>Thermococcus litoralis</i> glucokinase (<i>Tl</i>GlcK), <i>Leuconostoc mesenteroides</i> glucose-6-phosphate dehydrogenase (<i>Lm</i>G6PDH), and <i>Clostridium kluyveri</i> diaphorase (<i>Ck</i>DIA). Iodonitrotetrazolium chloride (INT), a colorimetric tetrazolium reagent, was used in the final step of the assay that converted into INT-formazan during reduction. INT-formazan in the assay’s buffered solution at pH 7.6 exhibited an intense colorimetric response at a wavelength maximum of 505 nm. The assay exhibited excellent performance characteristics as it revealed a Z’ factor of 0.87 and it has the potential to be further adopted into high-throughput screening studies for therapeutic drug discovery research.