Chemical synthesis of a biologically active natural tRNA with its minor bases

• Published in: Nucleic acids research Vol. 20; no. 19; pp. 5159 - 5166
• Main Authors: Gasparutto, Didier, Livache, Thierry, Bazin, Hervé, Duplaa, Anne-Marie, Guy, André, Khorlin, Alexander, Molko, Didier, Roget, André, Téoule, Robert
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11.10.1992
• Subjects: Alanine-tRNA Ligase - metabolism; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Electrophoresis, Polyacrylamide Gel; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Indicators and Reagents; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic acids; Nucleosides - chemical synthesis; Oligodeoxyribonucleotides; Polymerase Chain Reaction - methods; Rna, ribonucleoproteins; RNA, Transfer, Ala - chemical synthesis; RNA, Transfer, Ala - isolation & purification; RNA, Transfer, Ala - metabolism; RNA-Directed DNA Polymerase; Transcription, Genetic; Alanine; Aminoacylation; Complementary DNA; t-RNA; Nucleotide sequence; Escherichia coli; Reverse transcription; Bacteria; Chemical synthesis; In vitro; Enterobacteriaceae
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/20.19.5159

The complete chemical synthesis of an E.coli tRNAAla with its specific minor nucleosides, dlhydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2′-O-tertiobutyldi-methylsilyl-ribonucleoside-3′-O-(2-cyanoethyl-N-ethyl-N-methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxy-acetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. TriethylamJne trls-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNAAla has been transcribed into cDNA by reverse transcriptase and sequenced. With E.coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A290 and 6×104M-1s-1, respectively.