title: Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents.
abstract: Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2'-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5'-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5'-cap and a 3'-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications.
authors.affiliation: Discovery Research Laboratories, Nippon Shinyaku Co, Ltd, 3-14-1 Sakura, Tsukuba, Ibaraki 305-0003, Japan. s.nagata@po.nippon-shinyaku.co.jp
authors.forename: Seigo
authors.initials: S
authors.lastname: Nagata
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hamasaki
authors.forename: Koichi
authors.initials: K
authors.lastname: Uetake
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Masuda
authors.forename: Kazuchika
authors.initials: K
authors.lastname: Takagaki
authors.forename: Natsuhisa
authors.initials: N
authors.lastname: Oka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Wada
authors.forename: Tadaaki
authors.initials: T
authors.lastname: Ohgi
authors.forename: Junichi
authors.initials: J
authors.lastname: Yano
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis / Q000235:genetics*; D007202:Indicators and Reagents; D008780:Methyltransferases / Q000378:metabolism; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D007202:Indicators and Reagents; D012315:RNA Caps; D012333:RNA, Messenger; D011061:Poly A; D052216:Glucagon-Like Peptide 1; D008780:Methyltransferases; C055099:mRNA (nucleoside-O(2'))-methyltransferase