Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites

作者：Stephen A. Scaringe, Christopher S. Francklyn, Nassim Usman · 发表于：Nucleic Acids Research · 年份：1990 · DOI：10.1093/nar/18.18.5433 · 被引用次数：264 · 研究领域：RNA and protein synthesis mechanisms、DNA and Nucleic Acid Chemistry、Advanced biosensing and bioanalysis techniques

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.