0000000000285773
AUTHOR
Gemma Estrada Girona
Fluorogenic Tetrazine-Siliconrhodamine Probe for the Labeling of Noncanonical Amino Acid Tagged Proteins
Tetrazine-bearing fluorescent labels enable site-specific tagging of proteins that are genetically manipulated with dienophile modified noncanonical amino acids. The inverse electron demand Diels-Alder reaction between the tetrazine and the dienophile fulfills the criteria of bioorthogonality allowing fluorescent labeling schemes of live cells. Here, we describe the detailed synthetic and labeling protocols of a near infrared emitting siliconrhodamine-tetrazine probe suitable for super-resolution imaging of residue-specifically engineered proteins in mammalian cells.
Inducible Genetic Code Expansion in Eukaryotes
Abstract Genetic code expansion (GCE) is a versatile tool to site‐specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl‐tRNA‐synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression whe…
Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes.
How to make an organelle in eukaryotes A key step in the evolution of complex organisms like eukaryotes was the organization of specific tasks into organelles. Reinkemeier et al. designed an artificial, membraneless organelle into mammalian cells to perform orthogonal translation. In response to a specific codon in a selected messenger RNA, ribosomes confined to this organelle were able to introduce chemical functionalities site-specifically, expanding the canonical set of amino acids. This approach opens possibilities in synthetic cell engineering and biomedical research. Science , this issue p. eaaw2644