Fast In Vitro Synthesis and Direct Labeling of Nanobodies for Prototyping in Microscopy Applications.

Small antigen binders, such as nanobodies, have become widely used in biomedical research and pharmaceutical development. However, the pipeline for the generation of functional conjugated probes and drugs from identified binders remains a major time-consuming bottleneck. Here, we developed a method for fast nanobody production and conjugation based on an in vitro synthesis platform. Our system allows for small batch synthesis of nanobodies with the inclusion of a noncanonical amino acid (NCAA). This NCAA can then be used for direct conjugation of molecules to the synthesized nanobody using click-chemistry, reducing the time from binder-encoding DNA to a conjugated probe tremendously. In this study, we conjugated a fluorescent dye to an anti-Green fluorescent protein (GFP) nanobody and attained a fully functional probe suitable for advanced super-resolution microscopy within a short time frame of 2 days. Our work illustrates that an in vitro synthesis platform in combination with click-chemistry can be successfully employed to produce conjugated small antigen binding probes. The fast production and conjugation, combined with the possibility for parallelization as well as precise analysis by microscopy, forms an excellent platform for nanobody prototyping. The here-illustrated method can be used for quick selection and benchmarking of obtained nanobody sequences/clones, e. g., from a phage-display, for use as conjugated small-molecule carriers. This procedure can accelerate the bioengineering of nanobodies for research and pharmaceutical applications.