title: Facile synthesis of N-acyl-aminoacyl-pCpA for preparation of mischarged fully ribo tRNA.
abstract: Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minus CA) is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minus CA) transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University , Husargatan 3, Box 596, Uppsala 75124, Sweden.
authors.forename: Marek
authors.initials: M
authors.lastname: Kwiatkowski
authors.forename: Jinfan
authors.initials: J
authors.lastname: Wang
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Forster
mesh_terms: D000215:Acylation; D060326:Chemistry Techniques, Synthetic; D015226:Dinucleoside Phosphates / Q000138:chemical synthesis* / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D012343:RNA, Transfer / Q000737:chemistry*; D012270:Ribosomes
chemical_list: D015226:Dinucleoside Phosphates; D012343:RNA, Transfer