Click-based amplification: designed to facilitate various target labelling modes with ultralow background amplification

We describe a signal amplification method termed “Click-based amplification” that can be well integrated with various click-labelling modes, including chemical labelling, genetic incorporation and covalent inhibitor probe mediated target labelling.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

Acridonylalanine (Acd) is photostable, with a high quantum yield and long fluorescence lifetime in water. An evolved tRNA synthetase (RS) enables genetic incorporation of Acd in mammalian cells and its use in fluorescence lifetime imaging microscopy.

Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.