Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, *

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA Pyl CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.The site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion, provides new approaches for imaging, controlling and understanding protein function in cells1-3. This approach uses orthogonal aminoacyl tRNA synthetase and tRNA CUA pairs to direct the incorporation of a non-natural amino acid into a target protein in response to an amber stop codon introduced at a desired site in the corresponding gene1,2. The PylRS (encoded by PylS) and tRNA Pyl CUA (encoded by PylT) pair from Methanosarcina species (most commonly M. barkeri or M. mazei, here abbreviated Mb and Mm, respectively) is a particularly useful pair for genetic code expansion2. This pair and its evolved variants have been used to direct the incorporation of numerous non-natural amino acids with diverse structures and functions into proteins produced in Escherichia coli4, Salmonella typhimurium5, Saccharomyces cerevisiae6, mammalian cells7,8 and certain invertebrates and plants, including Caenorhabditis elegans9, Drosophila melanogaster10 and Arabidopsis thaliana11. Genetic code expansion in these model organisms is facilitating new approaches to manipulate and understand the molecular basis of biology1. However there are no reports of developing this pair for genetic code expansion in a live vertebrate.