A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection
Abstract Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we develope...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-01-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-28984-4 |
| _version_ | 1828041658039533568 |
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| author | Masashi Arakawa Akiho Yoshida Shinya Okamura Hirotaka Ebina Eiji Morita |
| author_facet | Masashi Arakawa Akiho Yoshida Shinya Okamura Hirotaka Ebina Eiji Morita |
| author_sort | Masashi Arakawa |
| collection | DOAJ |
| description | Abstract Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped β-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown. |
| first_indexed | 2024-04-10T17:19:36Z |
| format | Article |
| id | doaj.art-9600a9a4fb65416e9fc5e6378d223f3d |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-10T17:19:36Z |
| publishDate | 2023-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-9600a9a4fb65416e9fc5e6378d223f3d2023-02-05T12:12:36ZengNature PortfolioScientific Reports2045-23222023-01-0113111110.1038/s41598-023-28984-4A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infectionMasashi Arakawa0Akiho Yoshida1Shinya Okamura2Hirotaka Ebina3Eiji Morita4Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki UniversityVirus Vaccine Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Institute for Open and Transdisciplinary Research Initiatives, Osaka UniversityVirus Vaccine Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Institute for Open and Transdisciplinary Research Initiatives, Osaka UniversityVirus Vaccine Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Institute for Open and Transdisciplinary Research Initiatives, Osaka UniversityDepartment of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki UniversityAbstract Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped β-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown.https://doi.org/10.1038/s41598-023-28984-4 |
| spellingShingle | Masashi Arakawa Akiho Yoshida Shinya Okamura Hirotaka Ebina Eiji Morita A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection Scientific Reports |
| title | A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection |
| title_full | A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection |
| title_fullStr | A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection |
| title_full_unstemmed | A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection |
| title_short | A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection |
| title_sort | highly sensitive nanoluc based protease biosensor for detecting apoptosis and sars cov 2 infection |
| url | https://doi.org/10.1038/s41598-023-28984-4 |
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