Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells

Abstract

Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the α-, β-, or γ-phosphate moieties, m₂ ^7,2′-OGppp_SG, m₂ ^7,2′-OGpp_SpG, and m₂ ^7,2′-OGp_SppG, respectively. The new compounds were also modified at the 2′-O position of the m⁷Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the α- and β-phosphates. Oligonucleotides capped with m₂ ^7,2′-OGpp_SpG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m₂ ^7,2′-OGpp_SpG (D1) and both isomers of m₂ ^7,2′-OGppp_SG were partially resistant. Luciferase mRNA capped with m₂ ^7,2′-OGpp_SpG (D2) had a t _1/2 of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m⁷Gp₃G-capped mRNA. Luciferase mRNAs capped with m₂ ^7,2′-OGpp_SpG (D1) and m₂ ^7,2′-OGpp_SpG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m⁷Gp₃G. The greater yield of protein due to combining higher translational efficiency with longer t _1/2 of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.

Keywords

• ARCA
• S-ARCA
• phosphorothioate cap analogs
• hDcp2
• translational efficiency
• mRNA stability
• in vitro transcription