Triet M. Pham, Morgan G. Howard, Shane M. Carey, Lindsey R. Baker, Edward L. D’Antonio
{"title":"Novel Tetrazolium-Based Colorimetric Assay for Helicase nsp13 in SARS-CoV-2","authors":"Triet M. Pham, Morgan G. Howard, Shane M. Carey, Lindsey R. Baker, Edward L. D’Antonio","doi":"10.3390/biochem4020006","DOIUrl":null,"url":null,"abstract":"Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a human pathogenic virus that encodes for a helicase (SC2Hel) that is essential for viral replication. SC2Hel 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 SC2Hel activity. This SC2Hel assay combines three enzyme-coupled steps involving the ADP-dependent Thermococcus litoralis glucokinase (TlGlcK), Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (LmG6PDH), and Clostridium kluyveri diaphorase (CkDIA). 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.","PeriodicalId":72357,"journal":{"name":"BioChem","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioChem","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/biochem4020006","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a human pathogenic virus that encodes for a helicase (SC2Hel) that is essential for viral replication. SC2Hel 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 SC2Hel activity. This SC2Hel assay combines three enzyme-coupled steps involving the ADP-dependent Thermococcus litoralis glucokinase (TlGlcK), Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (LmG6PDH), and Clostridium kluyveri diaphorase (CkDIA). 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.