title: Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate.
abstract: A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-mumol scale corresponds to 1 mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100 nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.
authors: Discovery Research Laboratories, Nippon Shinyaku Co. Ltd, 3-14-1 Sakura, Tsukuba City, Ibaraki 305-0003, Japan.
authors: Yoshinobu
authors: Y
authors: Shiba
authors: Hirofumi
authors: H
authors: Masuda
authors: Naoki
authors: N
authors: Watanabe
authors: Takeshi
authors: T
authors: Ego
authors: Kazuchika
authors: K
authors: Takagaki
authors: Kouichi
authors: K
authors: Ishiyama
authors: Tadaaki
authors: T
authors: Ohgi
authors: Junichi
authors: J
authors: Yano
mesh_terms: D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D004987:Ethers / Q000737:chemistry*; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D009570:Nitriles / Q000737:chemistry*; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D009943:Organophosphorus Compounds / Q000737:chemistry; D012322:RNA Precursors / Q000138:chemical synthesis* / Q000737:chemistry; D012263:Ribonucleosides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C520617:2-cyanoethyl methylthiomethyl ether; D004987:Ethers; D035683:MicroRNAs; D009570:Nitriles; D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; D012322:RNA Precursors; D012263:Ribonucleosides; C434331:phosphoramidite