Genetically encoding non-canonical amino acids with chemical functionalities not present in the set of 20 natural residues is a powerful tool for introducing modifications into proteins. Novel amino acids with bioorthogonally reactive side chains can be employed to specifically modify, label, link, and control proteins as tools for understanding protein function and in applied areas as specifically modified components of diagnostics and therapeutics. The ability to encode multiple different, position-specific handles for protein modification has been limited by the fact that all 64 triplet codons have assigned functions. We have developed a fluorescence-based screen to evaluate the ability to reassign the meaning of sense codons in E. coli that monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. Using this system we have evaluated the reassignment of multiple sense codons typically read through wobble interactions using the Methanocaldococcus jannaschii (M. jannaschii) orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. We describe the efficiency and specificity of sense codon reassignment at multiple codons and our ability to improve these qualities through the application of our fluorescence-based screen in a high throughput mode to select improved variants from large libraries. We describe the application of optimized aminoacyl tRNA synthetase and tRNA variants to incorporate non-canonical amino acids in response to reassigned sense codons. Our measurements contribute to understanding the space of interactions that determines the fidelity of the genetic code and provide the infrastructure for expanding the ability to use the already existing set of non-canonical amino acids in the genetically directed construction of proteins produced from expanded genetic codes.

Exploring the space of sense codon reassignment in E. coli

251st National Meeting (2016)

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