Compared to BphP-derived NIR FPs, miRFP670nano is twofold smaller, naturally monomeric, tolerant to an acidic environment, denaturation conditions, cell fixation and has notably greater stability in mammalian cells 6. We recently reported a miRFP670nano, the first single-domain NIR FP, developed from CBCR 6.
In contrast, cyanobacteriochromes (CBCRs) can bind chromophores via the GAF domain only and allow engineering of small 17 kDa NIR FPs. However, for BV attachment BphP-based FPs require two PAS and GAF domains tightly interlinked by a complex ‘knot’ structure and have a relatively high molecular weight of 35 kDa. Most available NIR FPs were engineered from bacterial phytochrome photoreceptors (BphPs) 3 that use as a chromophore biliverdin IVα (BV), available in mammalian cells 4, 5. NIR FPs allow labeling of whole organisms, specific cell populations, organelles or individual proteins, and enable spectral multiplexing with FPs, biosensors and optogenetic tools active in the visible range 1. Optical imaging with near-infrared (NIR) fluorescent proteins (FPs) provides increased tissue penetration depths and a better signal‐to‐noise ratio due to reduced light-scattering, tissue absorption and autofluorescence in the NIR region (650–900 nm) 1, 2. Altogether, NIR-Fbs enable the detection and manipulation of a variety of cellular processes based on the intracellular protein profile. Applying NIR-Fbs as destabilizing fusion partners, we developed molecular tools for directed degradation of targeted proteins, controllable protein expression and modulation of enzymatic activities.
NIR-Fbs allowed background-free visualization of endogenous proteins, detection of viral antigens, labeling of cells expressing target molecules and identification of double-positive cell populations with bispecific NIR-Fbs against two antigens. By exploring miRFP670nano3 as an internal tag, we engineered 32 kDa NIR fluorescent nanobodies, termed NIR-Fbs, whose stability and fluorescence strongly depend on the presence of specific intracellular antigens. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications.