Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein-Protein Interaction Analysis, and Protein Function Exploration

Abstract

Over the last two decades, nanobodies or single-domain antibodies have found their way in research, diagnostics, and therapy. These antigen-binding fragments, derived from Camelid heavy chain only antibodies, possess remarkable characteristics that favor their use over conventional antibodies or fragments thereof, in selected areas of research. In this review, we assess the current status of nanobodies as research tools in diverse aspects of fundamental research. We discuss the use of nanobodies as detection reagents in fluorescence microscopy and focus on recent advances in super-resolution microscopy. Second, application of nanobody technology in investigating protein-protein interactions is reviewed, with emphasis on possible uses in mass spectrometry. Finally, we discuss the potential value of nanobodies in studying protein function, and we focus on their recently reported application in targeted protein degradation. Throughout the review, we highlight state-of-the-art engineering strategies that could expand nanobody versatility and we suggest future applications of the technology in the selected areas of fundamental research.

Keywords: VHH; engineering; fundamental research; nanobody; protein–protein interactions; single-domain antibody; super-resolution microscopy; targeted protein degradation.

Figure 1

Crystal structure of a gelsolin nanobody. A nanobody is typically composed of three complementarity-determining regions (CDRs), alternated with four framework regions (FRs). CDR1 is depicted in yellow, CDR2 in magenta, CDR3 in red, and the FRs are depicted in green. Image of PDB ID 2X1O (5) created with PyMOL.

Figure 2

Mechanisms of different nanobody-labeling strategies for super-resolution microscopy. N-hydroxysuccinimide ester-labeling 1 (top) randomly labels primary amines 2 in the nanobody. The other techniques mentioned (cysteine-maleimide, Sortase A, and furan technology) site-specifically label introduced tags (respectively, cysteine 5, sortag or LPETG 8, and furylalanine 11).

Figure 3

Overview of different reported approaches to investigate protein–protein interactions using a GFP-targeting nanobody. In the fluorescent-three-hybrid (F3H) strategy (top), the GFP nanobody delocalizes GFP-tagged bait toward a defined subcellular location. The nanobody can also be fused with bait protein and as such, relocalize bait toward GFP-tagged membrane protein (middle). Finally, the GFP nanobody can be used to deliver upconversion nanoparticles (UCNPs) toward GFP-tagged bait (bottom). Binding between GFP-bait and labeled prey is then validated by colocalization, Förster or lanthanide-based resonance energy transfer (FRET or LRET, respectively).

Figure 4

Schematic representation of reported strategies that combine the ubiquitin proteasome pathway with GFP nanobody for targeted degradation of GFP-fusion protein. In the deGradFP and Protein interference approach, the substrate recognition domain of the cullin-RING E3 ubiquitin ligase (CRL) complex is replaced by a high-affinity GFP nanobody. The affinity-directed protein missile (AdPROM) technique on the other hand, implies fusing the GFP nanobody with the substrate recognition domain of the complex.