New Far‐Red and Near‐Infrared Fluorescent Phycobiliproteins with Excellent Brightness and Photostability

Abstract Far‐red and near‐infrared fluorescent proteins can be used as fluorescence biomarkers in the region of maximal transmission of most tissues and facilitate multiplexing. Recently, we reported the generation and properties of far‐red and near‐infrared fluorescent phycobiliproteins, termed BeiDou Fluorescent Proteins (BDFPs), which can covalently bind the more readily accessible biliverdin. Far‐red BDFPs maximally fluoresce at ∼670 nm, while near‐infrared BDFPs fluoresce at ∼710 nm. In this work, we molecularly evolved BDFPs as follows: (a) mutations L58Q, S68R and M81K of BDFPs, which can maximally enhance the effective brightness in vivo by 350 %; (b) minimization and monomerization of far‐red BDFPs 2.1, 2.2, 2.3, and near‐infrared BDFPs 2.4, 2.5 and 2.6. These newly developed BDFPs are remarkably brighter than the formerly reported far‐red and near‐infrared fluorescent proteins. Their advantages are demonstrated by biolabeling in mammalian cells using super‐resolution microscopy.