Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging

Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular shows superior sensitivity over other commonly used bioluminescence reporters in vitro and in vivo. This pair was adapted to develop a bioluminescence resonance energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.