Red-shifted luciferase–luciferin pairs for enhanced bioluminescence imaging

Abstract

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.

Advances in bioluminescence imaging: new probes from old recipes

“…The optimized BRET construct (termed Antares2) exhibited enhanced red-shifted light emission and more robust bioluminescence in deep tissues. [24] Related BRET probes with Rluc have similarly provided bioluminescent probes that emit in the near-infrared regime. [25]…”

“…Light emission was observed upon furimazine or DTZ administration (3.3 μmol i.p.). Mouse images were reproduced with permission from ref 24.…”

Identification of Factors Complicating Bioluminescence Imaging

“…Similar to our previous observation for i.p. injection of DTZ, 15 the background signal from i.v. injection was also low (Figure 3).…”

“…14 Recently, our lab engineered NanoLuc into teLuc, which emits teal photos in the presence of a synthetic diphenylterazine (DTZ) substrate ( Figure 2B). 15 Moreover, further red-shifted emission has been achieved by fusing NanoLuc or teLuc with fluorescent proteins for bioluminescence resonance energy transfer (BRET). [15][16][17][18] These studies have partially fulfilled the increasing demand of using the same reporters for in vitro and in vivo, fluorescence-and bioluminescence-based assays.…”

“…15 Moreover, further red-shifted emission has been achieved by fusing NanoLuc or teLuc with fluorescent proteins for bioluminescence resonance energy transfer (BRET). [15][16][17][18] These studies have partially fulfilled the increasing demand of using the same reporters for in vitro and in vivo, fluorescence-and bioluminescence-based assays. In particular, the Antares2-DTZ pair was able to produce ~10-fold more detectable bioluminescence than FLuc at the standard D-luciferin concentration in a deep-tissue transfection mouse model.…”