title: Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs.
abstract: Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA(Tyr) mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue-white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA(Tyr).
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Tian
authors.forename: Danni
authors.initials: D
authors.lastname: Deng
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.forename: Dongning
authors.initials: D
authors.lastname: Yao
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Xu
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage / Q000235:genetics; D015723:Gene Library; D016153:Genes, Suppressor; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
chemical_list: D012343:RNA, Transfer