Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expres-sion at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant pro-tein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Automated summary

Synthetic biology tools have been developed to control gene expression at the transcription level, but few methods exist for regulating gene expression at the translational level. In this study, split orthogonal aminoacyl-tRNA synthetases (o-aaRS) were designed to control gene translation in bacteria and mammalian cells. These split o-aaRSs function as genetically encoded AND gates, where stop codon suppression is controlled by two distinct molecular inputs. Additionally, they can be used as versatile biosensors to detect protein-protein interactions involved in cancer and SARS-CoV-2 infection.