Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents
| Autor: | Hirofumi Masuda, Seigo Nagata, Koichi Uetake, Natsuhisa Oka, Tomohiro Hamasaki, Kazuchika Takagaki, Junichi Yano, Takeshi Wada, Tadaaki Ohgi |
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| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
RNA Caps
Peptide CHO Cells Biology Cricetulus Glucagon-Like Peptide 1 Cricetinae Genetics Protein biosynthesis Animals RNA Messenger Phosphorylation Protecting group 3' Untranslated Regions chemistry.chemical_classification Messenger RNA Three prime untranslated region Chinese hamster ovary cell RNA Biological activity Methyltransferases chemistry Biochemistry Synthetic Biology and Chemistry Indicators and Reagents Poly A |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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