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A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

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

Karilyn Porter¹, Mike Killoran¹, Robin Hurst¹

¹Promega Corporation, 2800 Woods Hollow, Fitchburg, Wisconsin 53711, United States.

ACS Chemical Biology

|March 20, 2026

Summary

Researchers developed NanoPrism luciferases, a two-color bioluminescent reporter system. This platform enables simultaneous measurement of two molecular readouts in the same sample, enhancing cellular dynamics studies.

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Bioluminescent reporters like NanoLuc luciferase are valuable for biological process monitoring.
Current NanoLuc systems emit a single channel (460 nm peak), limiting multiplexed assays.
Previous attempts to alter emission involved bioluminescence resonance energy transfer (BRET).

Purpose of the Study:

To engineer novel, high-efficiency BRET reporters for multiplexed bioluminescence.
To develop a two-color bioluminescent reporter platform for simultaneous molecular readout.
To demonstrate the platform's utility in complex biological pathway analysis.

Main Methods:

Engineered NanoPrism luciferases by inserting circularly permuted NanoLuc variants into HaloTag protein loops.
Achieved high BRET efficiency (~90%) by optimizing NanoLuc placement near a covalently bound fluorophore.
Created a two-color system by pairing red-shifted NanoPrisms with unmodified NanoLuc or its complementation variants.

Main Results:

NanoPrisms demonstrated high BRET efficiency and binary design for varied complementation affinities.
Developed a two-color reporter platform with bright signals, similar intensity, and >100 nm spectral separation.
Successfully applied the platform for monitoring protein degradation and tracking distinct biological pathway events.

Conclusions:

The NanoPrism platform enables quantitative, simultaneous measurement of two molecular readouts in a single sample.
This system offers enhanced insights into cellular dynamics by reducing variability and complexity.
The developed reporter system expands capabilities for multiplexed bioluminescent assays.