title: Synthesis of 5' cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase.
abstract: 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.
authors.affiliation: IBMM, UMR 5247 CNRS-UM1-UM2, Université Montpellier 2, 34095 Montpellier cedex 05, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Thillier
authors.forename: Etienne
authors.initials: E
authors.lastname: Decroly
authors.forename: François
authors.initials: F
authors.lastname: Morvan
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Vasseur
authors.forename: Françoise
authors.initials: F
authors.lastname: Debart
mesh_terms: D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D012315:RNA Caps
chemical_list: D012315:RNA Caps; D008780:Methyltransferases