Site-Specific Fluorescent Labeling, Single-Step Immunocytochemistry, and Delivery of Nanobodies into Living Cells

The smallest natural antibody fragments currently available are single-domain antibodies obtained from camelid species and sharks (variable new antigen receptors). These molecules consist of a single amino acid chain of ~120 amino acids that adopts a typical immunoglobulin fold. Single-domain antibodies (nanobodies) are monovalent and can be isolated from immunized animals, from naïve libraries, or from synthetic libraries. Importantly, their complete DNA sequences are readily obtained by default, which greatly facilitates their rapid manipulation for various applications. Here, a PCR-based protocol for inserting a sortase A recognition sequence at the carboxy-terminus of a nanobody is described. Subsequently, a sortase A-catalyzed biochemical reaction results in tagging of the nanobody with a short carboxy-terminal amino acid sequence that carries a non-canonical residue (propargyl glycine). This allows click chemistry to be performed with an azido-derivatized fluorophore, with the ensuing fluorescent nanobody being covalently and site-specifically labeled. The labeled nanobody can be used directly for immunocytochemistry, omitting the classical secondary antibody step. Also described are methods for delivery of fluorescent nanobodies into the cytoplasm of mammalian cells by photoporation, a very low-toxicity approach involving laser light and graphene quantum dots. The combined protocol embodies a novel route for studying protein function in living cells at high resolution.