tetano
Editor, Senior Moderator
Sci Rep
. 2023 Jan 31;13(1):1753.
doi: 10.1038/s41598-023-28984-4.
A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection
Masashi Arakawa[SUP] 1 2 [/SUP], Akiho Yoshida[SUP] 3 4 [/SUP], Shinya Okamura[SUP] 3 4 [/SUP], Hirotaka Ebina[SUP] 3 5 4 [/SUP], Eiji Morita[SUP] 6 7 [/SUP]
Affiliations
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.
. 2023 Jan 31;13(1):1753.
doi: 10.1038/s41598-023-28984-4.
A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection
Masashi Arakawa[SUP] 1 2 [/SUP], Akiho Yoshida[SUP] 3 4 [/SUP], Shinya Okamura[SUP] 3 4 [/SUP], Hirotaka Ebina[SUP] 3 5 4 [/SUP], Eiji Morita[SUP] 6 7 [/SUP]
Affiliations
- PMID: 36720982
- DOI: 10.1038/s41598-023-28984-4
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.