Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies

miRFP670nano3 offers improved near-infrared imaging and was used to develop fluorescent nanobodies whose stability and fluorescence strongly depend on antigen binding, with broad implications for detecting and manipulating cellular targets. Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. 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. 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. 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. Altogether, NIR-Fbs enable the detection and manipulation of a variety of cellular processes based on the intracellular protein profile.

Automated summary

miRFP670nano3 is a near-infrared fluorescent protein that can be used to develop fluorescent nanobodies, whose stability and fluorescence depend on antigen binding, with broad implications for detecting and manipulating cellular targets, and it has important research significance.