A Modular Platform for Quantitative Two-Color Bioluminescence Combining NanoLuc and Red-Shifted NanoPrism Luciferases

Bioluminescent reporters are widely used to monitor and image biological processes. Among these, NanoLuc luciferase and its complementation variants (LgBiT/SmBiT and LgBiT/HiBiT) are commonly used due to their brightness, sensitivity, and compatibility with prolonged kinetic measurements. However, the single-channel emission of these NanoLuc-based systems (460 nm peak) limits their use in multiplexed assays. Prior efforts to shift NanoLuc’s emission employed bioluminescence resonance energy transfer (BRET) to a proximal fluorescent protein or organic fluorophore. Building on this concept, we engineered high-efficiency BRET reporters, termed NanoPrism luciferases, by inserting circularly permuted NanoLuc or LgBiT into a surface loop of the self-labeling HaloTag protein. These NanoPrisms achieve a ∼90% BRET efficiency by optimally positioning NanoLuc variants near a fluorophore covalently bound to HaloTag. The binary design further supports high- and low-affinity complementation, allowing applications in HiBiT knock-in cells and tracking protein–protein interactions, respectively. Pairing red-shifted NanoPrisms with unmodified NanoLuc or its complementation variants, we created a two-color bioluminescent reporter platform featuring bright signals of similar intensity and >100 nm spectral separation, allowing quantitative, simultaneous measurement of two molecular readouts within the same sample. Here, we demonstrate the platform’s utility for monitoring a degradation target alongside a control protein and for tracking two distinct events within a biological pathway, using plate-based detection and bioluminescence imaging. By enabling concurrent measurements within the same sample, the system provides insights into cellular dynamics while reducing variability and complexity associated with parallel single-channel assays.