Brighter NIR Bioluminescence System for Mammalian Cell Bioimaging Based on Engineered Railroadworm Luciferase and...

Luciferases have been extensively used as reporter genes for bioimaging purposes. For bioimaging in live animals, however, far-red- and NIR-emitting systems are better suited. Despite many efforts to develop such NIR-emitting systems, currently only a few systems have been commercialized, including AkaLuc/akalumine, which emits at ∼650 nm. Departing from the natural red-emitting luciferase of Phrixotrix hirtus railroadworm in combination with 6'-(1-pyrrolidinyl)-luciferin (N5), we previously reported the development of a first efficient far-red-emitting system (650 nm). Here, we further improved this system, developing a luciferase that emits far-red bioluminescence with the natural substrate d-luciferin (630 nm), and a more efficient NIR bioluminescence with 6'-amino analogues (650-664 nm). The combination of this luciferase with N5 displayed superior properties compared with the commercial akalumine/AkaLuc system (645 nm), with high affinity for N5, higher catalytic efficiency and thermostability, more sustained luminescence, and more red-shifted spectrum (660 nm). COS-1 fibroblasts expressing this luciferase/N5 combination also displayed a brighter BL signal and more red-shifted spectrum when compared with the commercial akalumine/AkaLuc system, providing a promising NIR system for mammalian BL imaging.