iNClusive: a database collecting useful information on non-canonical amino acids and their incorporation into proteins for easier genetic code expansion implementation

Abstract

The incorporation of non-canonical amino acids (ncAAs) into proteins is a powerful technique used in various research fields. Genetic code expansion (GCE) is the most common way to achieve this: a specific codon is selected to be decoded by a dedicated tRNA orthogonal to the endogenous ones. In the past 30 years, great progress has been made to obtain novel tRNA synthetases (aaRSs) accepting a variety of ncAAs with distinct physicochemical properties, to develop robust in vitro assays or approaches for codon reassignment. This sparked the use of the technique, leading to the accumulation of publications, from which gathering all relevant information can appear daunting. Here we present iNClusive (https://non-canonical-aas.biologie.uni-freiburg.de/), a manually curated, extensive repository using standardized nomenclature that provides organized information on ncAAs successfully incorporated into target proteins as verified by mass spectrometry. Since we focused on tRNA synthetase-based tRNA loading, we provide the sequence of the tRNA and aaRS used for the incorporation. Derived from more than 687 peer-reviewed publications, it currently contains 2432 entries about 466 ncAAs, 569 protein targets, 500 aaRSs and 144 tRNAs. We foresee iNClusive will encourage more researchers to experiment with ncAA incorporation thus contributing to the further development of this exciting technique.

Graphical Abstract

Figure 1.

Scheme of the orthogonal translation system for the incorporation of ncAAs into target proteins. The heterologous aaRS and its cognate tRNA (blue) do not interact with the endogenous aaRSs and tRNAs (grey). The orthogonal aaRS recognizes the ncAA and loads it onto its cognate tRNA. The ncAA-loaded tRNA then interacts with endogenous ribosomes to allow for the translation of the target mRNA. In this example, the amber STOP codon (UAG) codes for the ncAA. aaRS, aminoacyl tRNA synthetase. ncAA, non-canonical amino acid. For simplicity only one endogenous aaRS–tRNA pair is shown (grey box).

Figure 2.

Scheme depicting the workflow used to generate the iNClusive database. The hits were generated using the ‘Publish or Perish’ software (31) upstream of Google Scholar. The publications were then manually reviewed and inserted into a preliminary database. The SMILES of the ncAAs were generated with the Optical Structure Recognition (OSRA) software (32) and compared with those given in the PubChem database (33). The retrieved information was confirmed manually by a different team member and only then added into the iNClusive database. A second review process done by a third distinct person was additionally carried out on 10% of randomly picked entries.

Figure 3.

Pie charts showing the number of entries in the database for the indicated entities. For instance, the number 396 for sfGFP indicates that this protein has been modified 396 times. Two entries might differ only for the organism used for the experiment or for the aminoacyl tRNA synthetase employed. Similarly, the number 191 for 4-azido-l-phenylalanine does not mean this ncAA has been incorporated in 191 different target proteins. Entries are distinct if any of the other categories in the database are different, such as tRNA, protein, organism in which the incorporation was tested, etc.