PrismaLuc luciferase enables tunable near-infrared bioluminescence for

Bioluminescence imaging (BLI) enables non-invasive monitoring of biological processes in living animals, yet most bioluminescent reporters emit at fixed wavelengths with limited spectral flexibility, constraining their use in multiplexed and deep-tissue applications. Bioluminescence resonance energy transfer (BRET) offers a solution by coupling a luciferase to a proximal fluorescent protein or synthetic fluorophore that absorbs the emitted light and re-emits it at a longer wavelength. However, existing BRET systems built from luciferase-fluorescent protein fusions are limited by suboptimal energy transfer efficiency and the fixed photophysical properties of their genetically encoded chromophores. We recently introduced PrismaLuc, a high-efficiency BRET reporter, in which a circularly permuted NanoLuc is inserted into HaloTag, enabling efficient energy transfer to a covalently bound synthetic fluorophore whose emission wavelength is selected by choice of ligand. In our initial report, PrismaLuc was paired with JF549 HaloTag ligand for two-colour cell-based assays. Here, we extend PrismaLuc to