The analysis of structures of the Nb-antigen complexes shows that a considerable number of contacts with antigen is mediated by the residues in the framework regions 24, which limits this approach to reduce the intracellular level of chromobodies. However, the introduction of mutations at the frameworks may affect the Nb-antigen-binding properties 20, 21, 22, 23. The diversity of Nbs is mainly determined by three complementarity determining regions (CDRs), whereas four framework regions are relatively conserved 19. To control the level of intracellular chromobodies, it was further suggested to use conditionally stable Nbs containing six point mutations in the Nb framework regions 18. These approaches allow to moderate the impact of background signal but do not allow studies of cell populations heterogeneous in antigen expression. Additionally, stable cell lines with a consistent chromobody expression could be generated 16, 17. To reduce the expression level of chromobodies, low or inducible promoters could be selected 14. Accumulation of fluorescent Nbs that remained unbound led to diffuse background signal, affecting the signal of antigen-bound Nbs 13, 14, 15. However, imaging with chromobodies often suffers from their excess inside a cell. It has been shown that genetic fusions of Nbs with FPs of the green fluorescent protein- (GFP-)like family, named chromobodies, enabled visualization of protein dynamics in live cells 13. The advantage of Nbs is their ability to recognize and bind a cognate (specific) antigen intracellularly 12. Despite their small size, Nbs bind antigens with high affinity and specificity 9, 10, 11. Endogenous, not modified, proteins can be visualized by nanobodies (Nbs), which are single-domain 15 kDa antigen-binding fragments derived from camelid heavy-chain-only antibodies 8. While direct tagging of proteins with FPs ensures specificity and allows studying of protein dynamics in live cells, in some cases FP-fusion constructs behave differently from their endogenous analogs, due to altered expression level, turnover and being blocked by FP-tag functional domains 7. Due to the compact single-domain fold with the N and C termini positioned close to each other, unlike BphP-based FPs, miRFP670nano performs well as the internal protein tag 6. 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.

Single domain protein scaffold