Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.
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.
Scaringe
S A
SA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Francklyn
C
C
Usman
N
N
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009570:Nitriles / Q000138:chemical synthesis* / Q000737:chemistry; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000138:chemical synthesis; D014320:Triphenylmethyl Compounds / Q000138:chemical synthesis* / Q000737:chemistry
D009570:Nitriles; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D014320:Triphenylmethyl Compounds