In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid.
tRNA-specific chemical aminoacylation was achieved with nonnatural amino acids. A nonnatural amino acid was activated as a thioester derivative, and the latter was linked through a spacer to the N-terminal of a 9-mer peptide nucleic acid that is complementary to the 3'-terminal region of yeast phenylalanine tRNA. Efficient aminoacylation was observed when the amino acid thioester-spacer-PNA conjugate was mixed with the tRNA. The PNA-assisted aminoacylation was also successful in an Escherichia coli in vitro protein synthesizing system that contained an orthogonalized tRNA. The in situ aminoacylation/in vitro translation gave a mutant protein in which the nonnatural amino acid was incorporated into the position directed by a CGGG 4-base codon/anticodon pair.
Ninomiya
Keiko
K
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Minohata
Toshikazu
T
Nishimura
Masaki
M
Sisido
Masahiko
M
D000215:Acylation; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D017076:Computer-Aided Design; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000096:biosynthesis / Q000737:chemistry*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D004952:Esters; D020135:Peptide Nucleic Acids; D012346:RNA, Transfer, Amino Acyl; D013438:Sulfhydryl Compounds