Efficient Suppression of Premature Termination Codons by Engineered Chimeric Pyrrolysine tRNAs

Mutations that introduce premature termination codons (PTCs) within protein-coding genes are associated with incurable and severe genetic diseases. Many PTC-associated disorders are life-threatening and have no approved medical treatment options. Suppressor transfer RNAs (sup-tRNAs) with the capacity to promote translational readthrough of PTCs represent a promising therapeutic strategy to treat these conditions; however, developing novel sup-tRNAs with high efficiency and specificity often requires extensive engineering and screening. Moreover, these efforts are not always successful at producing more efficient sup-tRNAs. Here we show that the pyrrolysine tRNA (tRNAPyl), which naturally translates the UAG stop codon, offers an attractive scaffold for developing potent sup-tRNAs that restore protein synthesis from PTC-containing genes. We created a series of rationally designed Pyrrolysine tRNA Scaffold Suppressor-tRNAs (PASS-tRNAs) that are substrates of bacterial and human alanyl-tRNA synthetase. Using a PTC-containing reporter gene, PASS-tRNAs restore protein synthesis to wild-type levels in bacterial cells. In human cells, PASS-tRNAs display robust and consistent translation activity in two distinct PTC reporter genes with high codon specificity and no observed cytotoxic effects. Collectively, these results unveil a new class of highly efficient sup-tRNAs with great potential for tRNA-based therapeutics.