Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

Abstract

Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering**

“…[32] Furthermore, photo-caging of eNB using ONBY or NPY resulted in blocking of target binding when nanobody and target were coexpressed in cultured HeLa cells. [36,37] While ONBY was not sufficient to abolish binding in our intracellular assay, we found that uncaging by illuminating animals using a 365 nm LED (640 seconds illumination, 10 mW/…”

“…This observation was unexpected since previously published in vitro binding assays, using photocaged eNB purified from E. coli , showed that Y37 substitution for ONBY reduces binding affinity 10,000 fold and blocks binding in vitro , and NPY reduces binding affinity 420 fold [32] . Furthermore, photo‐caging of eNB using ONBY or NPY resulted in blocking of target binding when nanobody and target were co‐expressed in cultured HeLa cells [36,37] …”

“…The resulting improved efficiency of uncaging at 365 nm makes NPY preferable for in vivo applications [41] . ONBY and NPY have been used to optically control nanobody binding in human cell lines [36,37] …”

“…[32,34] Intracellular use of photo-caged nanobodies was first shown by delivering purified photocaged nanobodies synthesised in E. coli to HeLa cells expressing the antigen, [35] and subsequently by directly expressing a photo-caged nanobody in a cultured human cell line. [36,37] A number of factors influence nanobody/antigen interaction, including pH, ionic concentration, incubation time, and concentration of nanobody and antigen. [38] The disruption of binding by the photocaged amino acid observed in one system may therefore not correctly predict binding when applying photocaged nanobodies in other systems or at different concentrations.…”

“…Intracellular use of photo‐caged nanobodies was first shown by delivering purified photocaged nanobodies synthesised in E. coli to HeLa cells expressing the antigen, [35] and subsequently by directly expressing a photo‐caged nanobody in a cultured human cell line [36,37] …”