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.

7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m⁶A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m⁷GpppN_(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m⁶A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/A_m and m⁶A_m, whereas the lowest was observed for G and G_m. The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the α, β, or γ position of the 5′,5′-triphosphate chain. Three of them were also modified with methyl groups at the 2′-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K_AS) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the β-substituted analog m⁷Gpp_SpG) was characterized by a K_AS that was more than fourfold higher than that of its unmodified counterpart (m⁷GpppG). All analogs modified in the γ position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the α position (i.e., m⁷Gppp_SG and m₂^7,2′-OGppp_SG) were established as S_P and R_P, respectively, using enzymatic digestion and correlation with the S_P and R_P diastereomers of guanosine 5′-O-(1-thiodiphosphate) (GDPαS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.