Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons

Abstract

Genetic incorporation of noncanonical amino acids has emerged as a powerful tool for the study of protein structure and function. While the three triplet nonsense codons have been widely explored, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acid mutagenesis. Here we demonstrated for the first time that two orthogonal quadruplet codons could be used to simultaneously encode two different noncanonical amino acids within a single protein in bacterial cells. To achieve this, we fine-tuned the interaction between aminoacyl-tRNA synthetase and tRNA, which afforded up to 21-fold improvement in quadruplet codon decoding efficiency. This work represents a significant step toward the use of multiple quadruplet codons for noncanonical amino acid mutagenesis. Simultaneous incorporation of two or more noncanonical amino acids is of significant importance for biological applications that can benefit from multiple unique functional groups, such as fluorescence resonance energy transfer and nuclear magnetic resonance studies, and ultimately for the synthesis of completely unnatural biopolymers as new biomaterials.

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

“…9 Indeed, four-base codons have been used alone, and in combination with nonsense codons, to encode two and three distinct ncAAs in the same protein gene. 1,5,10 However, translation of four-base codons is very inefficient on wildtype ribosomes. 3,11,12 It is likely for this reason that four-base codons have not been widely adopted.…”

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

“…To do this, we cloned quadruplet versions of tyrosine synthetase cassettes. These contained a tRNA with a quadruplet anticodon and synthetases modified with the F261S and D286E mutations, which have been shown to encourage UCUA-tRNA aminoacylation for paceytlphenylalanine 38 . This quadruplet system was able to successfully incorporate at UAGA codons with low efficiency but also incorporated into the UAG codon with similar efficiency (Supp.…”

Intersecting Xenobiology and Neometabolism To Bring Novel Chemistries to Life

“…To overcome the disadvantage, more blank codons are necessary and new strategies have been developed based on quadruplet codons [119] -that, despite of being functional, still require considerable improvement in their efficiency. [120] The first FAA subjected to site-specific incorporation was p-fluorophenylalanine by expressing a tRNA Phe (Amber)/phenylalanyl-tRNA synthetase pair from yeast in E. coli, as described in the pioneer work of Furter. [121] Improved orthogonality was later achieved by Young and co-workers [122] by applying an evolved Methanococcus jannaschii aaRS-tRNA pair that suppresses the amber stop codon, and enables the incorporation of p-fluorophenylalanine (among other unnatural amino acids) into peptides.…”