Chemical synthesis of a biologically active natural tRNA with its minor bases
- 1 January 1992
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 20 (19) , 5159-5166
- https://doi.org/10.1093/nar/20.19.5159
Abstract
The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl 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. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) 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/A260 and 6 x 10(4)M-1s-1, respectively.Keywords
This publication has 15 references indexed in Scilit:
- Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramiditesNucleic Acids Research, 1990
- Aminoacylation of RNA minihelices with alanineNature, 1989
- Solid phase synthesis of the 5′-half of the initiator t-RNA fromB.subtilisNucleic Acids Research, 1989
- Prevention of chain cleavage in the chemical synthesis of 2′-silylated oligoribonucleotidesNucleic Acids Research, 1989
- Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templatesNucleic Acids Research, 1987
- The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groupsNucleic Acids Research, 1987
- Solid phase synthesis of oligoribonucleotides using o-nitrobenzyl protection of 2′-hydroxyl via a phosphite triester approachNucleic Acids Research, 1986
- Isolation and nucleotide sequence analysis of tRNAAlaGGC from Escherichia coli K-12Journal of Bacteriology, 1985
- Polymer support oligonucleotide synthesis XVIII1.2): use ofβ-cyanoethyi-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final productNucleic Acids Research, 1984
- Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.Proceedings of the National Academy of Sciences, 1981