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 a, b, or g position of the 59,59-triphosphate chain. Three of them were also modified with methyl groups at the 29-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 b-substituted analog m 7 Gpp S pG) was characterized by a K AS that was more than fourfold higher than that of its unmodified counterpart (m 7 GpppG). All analogs modified in the g 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 a position (i.e., m 7 Gppp S G and m 2 7,29-O Gppp S G) were established as S P and R P , respectively, using enzymatic digestion and correlation with the S P and R P diastereomers of guanosine 59-O-(1-thiodiphosphate) (GDPaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.

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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.

The kinetics of binding 7-methyl-GpppG, an analogue of the 5'-mRNA cap, to the cap-binding protein eIF4E, at 20 degrees C, in 50 mM Hepes-KOH buffer, pH 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectrofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inadequate description of the binding mechanism and an additional step is required to accommodate the kinetic data. The rate constants derived from assumed one-step and two-step binding models were determined. The forward rate constants towards the complex formation decrease, and the reverse rate constants increase, with increasing ionic strength. The association rate constants derived from the stopped-flow measurements and the computed diffusional encounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the ligand. Moreover, comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors. The first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionic strengths the ligand must be closer to the binding site to induce the fluorescence quenching.