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

Abstract

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.

New near-infrared fluorescent probes and tools

“…If a transcriptional activator is fused to this NIR-fluorescent nanobody, transcription will become dependent on the presence of a specific protein. This is nicely shown for a GAL4 promotor 4 . One can also fuse an inhibitory protein domain to the NIR-fluorescent nanobody -for instance, to modulate cAMP-dependent kinase signaling 4 .…”

“…This is nicely shown for a GAL4 promotor 4 . One can also fuse an inhibitory protein domain to the NIR-fluorescent nanobody -for instance, to modulate cAMP-dependent kinase signaling 4 .…”

“…Using a single domain from a cyanobacteriochrome, a much smaller variant, miRFP670nano, has been generated 3 . This issue of Nature Methods describes 4 the engineering of a twofold brighter single-domain miRFP670nano3 variant with a quantum yield of 0.19 and extinction coefficient of 129,000 M −1 cm −1 . Introducing an additional covalent bond between the apo-protein and the biliverdin chromophore of miRFP670nano3 further enhances maturation, leading to a stunning fourfold increased brightness as detected in cells as compared to miRFP670nano.…”

“…As a result, miRFP670nano3 is by far the best NIR-FP available to date. Oliinyk et al 4 show the versatility of this new probe for NIR-FP tagging of a variety of proteins and possibilities for deep tissue imaging.…”

“…The technique can also be used to remove a protein of interest in a manner dependent on the absence of, for instance, GFP by fusing an anti-GFP nanobody to a nanobody against a protein of interest. Subsequently, the latter protein will be degraded in cells that do not ectopically express GFP 4 . Using cell-type-specific promotors, this allows cell-type-specific protein depletion, with many applications in developmental biology or medical biology.…”