UC Irvine

The emergence of bright bioluminescent luciferase reporters has enabled versatilebioimaging applications in vitro and in vivo . However, conventional luciferases such as nanoluciferase are sub-optimal reporters for bioluminescence imaging (BLI) in vivo due to their blue-shifted emission which is highly attenuated by tissue, resulting in a dim, less sensitive signal. To overcome this limitation, Nluc and its substrate furimazine have been further engineered to produce red-shifted emission. Additionally, Nluc has been fused to fluorescent proteins, creating reporters with spectrally shifted light emission through bioluminescence resonance energy transfer (BRET). In this study, we employed two BRET-based fusion reporters, CeNLuc and LumiScarlet, as they have previously demonstrated improved imaging properties compared to luciferase-only reporters. We aimed to immobilize CeNLuc and LumiScarlet onto the E2 nanoparticle surface to develop brighter reporters for BLI. We successfully conjugated CeNLuc and LumiScarlet onto E2 using the SpyCatcher-SpyTag conjugation, resulting in CeNLuc-E2 and LumiScarlet-E2 nanoparticles of ~35-nm in size. Furthermore, we were able to control the number of luminescent proteins per particle and demonstrated that CeNLuc-E2 and LumiScarlet-E2 underwent efficient BRET. We also evaluated the luminescence of CeNLuc-E2 and LumiScarlet-E2 using BLI. CeNLuc-E2 particles produced approximately 12-fold greater luminescence than non-immobilized CeNLuc proteins, resulting in a brighter luminescent reporter. In contrast, LumiScarlet-E2 particles exhibited a greater red-shifted emission than CeNLuc-E2, which could be advantageous for more sensitive deep-tissue BLI. These findings demonstrate the potential of CeNLuc-E2 and LumiScarlet-E2 as effective reporters for broad applications in nanomedicine and bioimaging.