Novel “anti-reverse” cap analogs with superior translational properties

Abstract

Synthetic analogs of the 5′-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m⁷Gp₃G. Unfortunately, because of the presence of a 3′-OH on both the m⁷Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two “anti-reverse” cap analogs (ARCAs), m⁷3′dGp₃G and m₂^7,3′-OGp₃G, that cannot be incorporated in the reverse orientation because of modifications at the C3′ position of m⁷Guo. In the present study, we have synthesized seven new cap analogs modified in the C2′ and C3′ positions of m⁷Guo and in the number of phosphate residues, m₂^7,2′-OGp₃G, m⁷2′dGp₃G, m⁷2′dGp₄G, m₂^7,2′-OGp₄G, m₂^7,3′-OGp₄G, m⁷Gp₅G, and m₂^7,3′-OGp₅G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2′, like those at C3′, prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.

Keywords

• Cap-dependent translation
• T7 RNA polymerase
• in vitro transcription
• capping efficiency
• dinucleoside tetra- and pentaphosphates
• cap analog inhibition
• translational efficiency
• anti-reverse cap analogs
• eIF4E