Zbigniew M. Darzynkiewicz

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

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.

Interaction of human decapping scavenger with 5' mRNA cap analogues : structural requirements for catalytic activity

The cap structure is a specific feature of the 5 � end of mRNA which plays an important role in the post-transcriptional control in gene expression. A major step of gene regulation occurs at the level of mRNA turnover. Degradation of most eukaryotic mRNAs entails the removal of the cap structure in the various pathways. A human scavenger decapping enzyme (hDcpS) catalyses the cleavage of the residual cap structure m 7 GpppN and/or short oligonucleotides after the 3 � → 5 � exosom mediated digestion. In this paper we report a fluorescence study of association process of hDcpS with m 7 GMP, m 7 GDP and selected dinucleotide cap analogues resistant to enzymatic hydrolysis. The calculated values of association constants (Kas) and corresponding Gibbs free energies (� G ◦ ) depend on the type of substituents and their positions in the cap molecule, indicating which structural modifications are crucial for the catalysis.

Affinity of dinucleotide cap analogues for human decapping scavenger (hDcpS).

Eukaryotic cells utilize scavenger decapping enzymes to degrade cap structure following 3′-5′ mRNA decay. Human DcpS recently has been described as a highly specific hydrolase (a member of the HIT family) that catalyses the cleavage of m 7 GpppG and short capped oligoribonucleotides. We have demonstrated here that cap-1 (m 7 GpppGm) is a preferred substrate among several investigated dinucleotide cap analogues m 7 Gp n N (n = 3–5, N is a purine or pyrimidine base) and m 7 GMP is always one of the reaction product. Cap analogues containing pyrimidine base instead of guanine or diphosphate chain are resistant to hydrolysis catalyzed by human scavenger. Contrary to the other enzymes of HIT family, hDcpS activity is not stimulated by Mg 2+.