Abstract
Bacterial phytochromes are attractive moleculartemplates for engineering fluorescent proteins (FPs) because their near-infrared (NIR) emission significantly extends the spectral coverage of GFP-like FPs. Existing phytochrome-based FPscovalently bind heme-derived tetrapyrrolechromophores and exhibit constitutivefluorescence. Here we introduce Rep-miRFP, an NIRimaging probe derived from bacterialphytochrome, which interacts non-covalently and reversibly with biliverdinchromophore. In Rep-miRFP,the photobleached non-covalent adduct can be replenished with fresh biliverdin, restoring fluorescence. By exploiting this chromophore renewal capability, we demonstrate NIR PAINT nanoscopy in mammalian cells using Rep-miRFP.
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