Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging

While conventional antibodies have been an instrument of choice in immunocytochemistry for some time, their small counterparts known as nanobodies have been much less frequently used for this purpose. In this study we took advantage of the availability of nanobody cDNAs to site-specifically introduce a non-standard amino acid carrying an azide/alkyne moiety, allowing subsequent Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC). This generated a fluorescently labelled nanobody that can be used in single step immunocytochemistry as compared to conventional two step immunocytochemistry. Two strategies were explored to label nanobodies with Alexa Fluor 488. The first involved enzymatic addition of an alkyne-containing peptide to nanobodies using sortase A, while the second consisted of incorporating para-azido phenylalanine at the nanobody C-terminus. Through these approaches, the fluorophore was covalently and site-specifically attached. It was demonstrated that cortactin and beta-catenin, cytoskeletal and adherens junction proteins respectively, can be imaged in cells in this manner through single step immunocytochemistry. However, fixation and permeabilization of cells can alter native protein structure and form a dense cross-linked protein network, encumbering antibody binding. It was shown that photoporation prior to fixation not only allowed delivery of nanobodies into living cells, but also facilitated j3-catenin nanobody Nb86 imaging of its target, which was not possible in fixed cells. Pharmacological inhibitors are lacking for many non-enzymatic proteins, and it is therefore expected that new biological information will be obtained through photoporation of fluorescent nanobodies, which allows the study of short term effects, independent of gene-dependent (intrabody) expression.