Brighter NIR Bioluminescence System for Mammalian Cell Bioimaging Based on Engineered Railroadworm Luciferase and 6′-Aminoluciferin Analogues

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.