Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

A wide variety of non-canonical amino acids (ncAAs) can be incorporated into proteins through the coordinated action of a stop codon suppressing tRNA and aminoacyl-tRNA synthetase. However, methods to discover and characterize suppressor tRNAs are generally lacking. In this work, we show that cell-free systems can express functional suppressor tRNAs using endogenous machinery and characterize their activity. This method is compatible with widely used orthogonal tRNAs, such as the Methanocaldococus jannaschii tyrosyl tRNA, the Methanosarcina barkeri pyrrolysyl tRNA, the Methanomethylophilus Alvus pyrrolysyl tRNA, and an engineered Int pyrroysyl tRNA. Modifying the workflow to evaluate TAA suppression revealed that the M. jannaschii and M. alvus are highly functional TAA suppressors in cell-free systems. Finally, we show that we can express two distinct tRNAs simultaneously, enabling the incorporation of multiple, distinct ncAAs. In total, our work shows that cell-free systems are useful platforms to express and characterize tRNAs.