Novel BRET combination for detection of rapamycin-induced protein dimerization using luciferase from fungus Neonothopanusnambi

• Published in: Heliyon 2024-02, Vol.10 (4), p.e25553-e25553, Article e25553
• Main Authors: Mujawar, Aaiyas, Dimri, Shalini, Palkina, Ksenia A., Markina, Nadezhda M., Sarkisyan, Karen S., Balakireva, Anastasia V., Yampolsky, Ilia V., De, Abhijit
• Format: Article
• Language: English
• Subjects: Bioluminescence resonance energy transfer; Cancer; Cell signaling; Fungal BRET; Luciferase; mTOR signalling; Rapamycin
• ISSN: 2405-8440; 2405-8440
• DOI: 10.1016/j.heliyon.2024.e25553

Bioluminescence resonance energy transfer (BRET) is one of the most promising approaches used for noninvasive imaging of protein-protein interactions in vivo. Recently, our team has discovered a genetically encodable bioluminescent system from the fungus Neonothopanus nambi and identified a novel luciferase that represents an imaging tool orthogonal to other luciferin-luciferase systems. We demonstrated the possibility of using the fungal luciferase as a new BRET donor by creating fused pairs with acceptor red fluorescent proteins, of which tdTomato provided the highest BRET efficiency. Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system. We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture that will provide the unique opportunity to perform multiplexed BRET assessment in the future. •A new BRET reporter was developed by creating a fusion protein of N. nambi luciferase (nnLuz) with tdTomato acceptor protein.•This novel BRET pair of nnLuz-tdTomato was then converted as a genetically encodable sensor to detect FRB-FKBP12 interactions from live cells.•The developed nnLuz-TdTomato BRET pair provides an opportunity to develop multiplexed BRET approach in future.