Towards mRNA with superior translational activity: synthesis and properties of ARCA tetraphosphates with single phosphorothioate modifications??

New Journal of Chemistry Pub Date: 2010-03-10 DOI: 10.1039/B9NJ00644C

Abstract

We describe the chemical synthesis and preliminary biophysical and biochemical characterization of a series of mRNA 5′-end (cap) analogs designed as reagents for obtaining mRNA molecules with augmented translation efficiency and stability in vivo and as useful tools to study mRNA metabolism. The analogs share three structural features: (i) the 5′,5′-bridge is elongated to a tetraphosphate to increase their affinity to translation initiation factor eIF4E; (ii) a single phosphorothioate modification at either the α, β, γ or δ-position of the tetraphosphate has been made to decrease their susceptibility to enzymatic degradation and/or to modulate their interaction with specific proteins; and (iii) a 2′-O-methyl group has been added to the ribose of 7-methylguanosine (a characteristic of Anti-Reverse Cap Analogs (ARCAs), which are incorporated into mRNA during in vitro transcription exclusively in the correct orientation). The dinucleotides bearing the modified tetraphosphate bridge were synthesized by ZnCl2-mediated coupling between two mononucleotide subunits with isolated yields of 30–65%. The preliminary biochemical results show that mRNAs capped with new analogs are 2.5–4.5 times more efficiently translated in a cell-free system than m7GpppG-capped mRNAs, which makes them promising candidates for RNA-based therapeutic applications such as gene therapy and anticancer vaccines.

Graphical abstract: Towards mRNA with superior translational activity: synthesis and properties of ARCA tetraphosphates with single phosphorothioate modifications
Recommended Literature