Abstract
Red-shifted bioluminescence is highly desirable for diagnostic and imaging applications. Herein, we report a semisynthetic NanoLuc (sNLuc) based on complementation of a split NLuc (LgBiT) with a synthetic peptide (SmBiT) functionalized with a fluorophore for BRET emission. We observed exceptional BRET ratios with diverse fluorophores, notably in the red (I674/I450 > 14), with a brightness that is sufficient for naked eye detection in blood or through tissues. To exemplify its utility, LgBiT was fused to a miniprotein that binds HER2 (affibody, ZHER2), and the selective detection of HER2+ SK-BR-3 cells over HER2- HeLa cells was demonstrated.
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