期刊
RNA
卷 18, 期 4, 页码 856-868出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.030932.111
关键词
5 '-capped mRNA; RNA chemical synthesis; enzymatic methylation; guanine-N-7-methyl transferase; solid-phase synthesis
资金
- CNRS
- Ministere de l'Education Nationale de la Recherche et Technologie
- European Union [Health-F3-2010-260644]
- Direction Generale de l'Armement [DGA 09co402]
- Fondation pour la Recherche Medicale
- MNRT
- INSERM
The 5' end of eukaryotic mRNA carries a N-7-methylguanosine residue linked by a 5'-5' triphosphate bond. This cap moiety ((7m)GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 5' exonucleases and avoiding its recognition as nonself'' by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 5'-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N-7-methylguanosine, the synthesis of RNAs possessing a cap structure at the 5' end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 2'-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 5'-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN(2'-Om)-RNAs were purified before the N-7-methyl group was added by enzymatic means using the human (guanine-N-7)-methyl transferase to yield (7m)GpppN-RNAs (cap-0) or (7m)GpppN(2'-Om)-RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.
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