Since their discovery just over 25 years ago, the single variable domain from heavy-chain-only antibodies plays a role in an increasing number of antibody-based applications. Structural and biophysical studies have revealed that the small, ∼15 kDa, single variable domain found in camelids displays versatility in target recognition. Such insight has served as the foundation to develop and engineer VHH domains with enhanced properties capable of targeting a range of therapeutically relevant protein antigens or low-molecular weight haptens. Furthermore, the modular nature of VHH domains allows them to be introduced into constructs that are simply not possible with conventional antibodies. Here, we review the structural and biophysical properties of VHH domains, highlight recent VHH-based therapeutics and diagnostics, and provide insight into VHH engineering that may pave the way to next-generation single domain antibody applications. Impact statement The development of novel antibody formats, beyond conventional antibodies, opens new possibilities in medical therapies, diagnostics, and general life science applications requiring affinity reagents. The camelid VHH domain, from heavy-chain-only antibodies, has emerged as a jack-of-all-trades module for novel affinity reagents. Applications include targeted cancer therapies, novel antimicrobial agents, conformation specific reagents, and tailor-made molecular switch entities. The breadth of unique uses for the VHH will continue to grow, opening new opportunities to treat and understand disease.

Coumarin moieties react with thymine and cytosine in DNA by photoinduced [2+2] cycloaddition, which allows quantitative DNA interstrand crosslink (ICL) formation. Here, we report the application of coumarin analogues for DNA photoligation and the rearrangement of coumarin-induced ligation to ICL products. Both DNA sequences and the linker units at position 4 of the coumarin moieties affected coumarin-induced DNA photoligation. A flexible linker unit favored DNA ICL formation but led to inefficient photoligation, whereas coumarins without linker units greatly increased DNA photoligation efficiency. DNA photoligation induced by the coumarin moiety was photoswitchable. Ligation products were formed between coumarin and dT or dC upon 350 nm irradiation but reverted to the original single-stranded oligodeoxyribonucleotides (ODNs) upon 254 nm irradiation. Rearrangement of ligated ODNs into ICL products occurred during the switchable (350 nm/254 nm) processes. Additionally, photoinduced cleavage of coumarin 3 occurred with dC-3 cycloadducts upon 254 nm irradiation, which was confirmed by mass spectrometry analysis.