De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs.

The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by aminoacyl-tRNA synthetase/tRNA pairs, which direct the unique coupling of specific amino acids with specific anticodons. The evolutionary record suggests that a primitive genetic code expanded into the current genetic code, over billions of years, through duplication and specialization (neofunctionalization) of aminoacyl-tRNA synthetases and tRNAs from common ancestral synthetase/tRNA pairs. This process produced the current set of mutually orthogonal aminoacyl-tRNA synthetases and tRNAs that direct natural protein synthesis. Here we demonstrate the creation of new orthogonal pairs, which are mutually orthogonal with existing orthogonal pairs, de novo, by a logical series of steps implemented in the laboratory, via the de novo generation of orthogonality in RNA-RNA interactions, protein-RNA interactions, and small molecule substrate selection by protein catalysts. Our laboratory evolution experiments provide experimental evidence for duplication and specialization as a plausible route to the current set of synthetases and tRNAs via natural evolution. Moreover our experiments extend billions of years of natural evolution and demonstrate that the small number of naturally occurring orthogonal aminoacyl-tRNA synthetase/tRNA pairs do not place an intrinsic limitation on the scope of synthetic genetic code expansion for the incorporation of multiple distinct unnatural amino acids into proteins or the synthesis and evolution of unnatural polymers in cells.