Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5′ end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization.

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Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3′→5′ mRNA decay. Some previous studies suggested that both m7GpppG and m7GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m7GDP (7-methylguanosine diphosphate) and m32,2,7GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5′→3′ mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode and yeast). Furthermore, whereas mononucleoside diphosphates (m7GDP and m32,2,7GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m7GTP and m32,2,7GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m7GTP and m32,2,7GTP are cleaved at a slower rate than their corresponding dinucleotides (m7GpppG and m32,2,7GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m7GDP, they have a high binding affinity for m7GDP and m7GDP potently inhibits DcpS hydrolysis of m7GpppG, suggesting that m7GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m7GDP in mRNA metabolic pathways, due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.