Nowadays gene manipulation techniques ("DNA therapy") undergo progressive development and become widely used in industry and medicine. Since new advances in mRNA technologies are capable for obtaining particles with increased stability and translational efficiency, RNA become an attractive alternative for advancement of DNA therapy. For the past years studies have been conducted to explore different modification in mRNA cap structure and its effect on RNA properties. Recently we have shown that modification of the cap structure at the N2 position of 7-methylguanosine leads to an enhancement in translation inhibition. Currently, we have decided to exploit translational properties of mRNA capped with the ARCA (anti-reversed cap) analogs modified within N2 position of purine moiety s. We designed and synthesized three new dinucleotide cap analogs and investigated them in the rabbit reticulocyte lysate (RRL) and the human embryonic kidney derived HEK293 cell line, in vitro translational model systems. The obtained data indicate that, in both translational assays, the cap analogs synthesized by us when incorporated into mRNA improved its translational properties compared to the ARCA capped transcripts. Furthermore, the introduced modifications enhanced stability of the capped transcripts in HEK293 cells, which become higher compared to that of the transcripts capped with regular cap or with ARCA. Additionally one of the synthesized cap analogs revealed strong translation inhibition potency in RRL system, with IC value 1.7 µM.
Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m2,2,7G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m2,2,7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m2,2,7G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m2,2,7G-cap. Nematode and mammalian eIF4E both have a low affinity for m2,2,7G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m2,2,7G-cap and the m2,2,7G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m2,2,7G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m2,2,7G-cap is present. These data have implications for the contribution of 5′-UTRs in mRNA translation and the function of different eIF4E isoforms.
Synthetic analogs of the 5′ end of mRNA (cap structure) are widely used in molecular studies on mechanisms of cellular processes such as translation, intracellular transport, splicing, and turnover. The best-characterized cap binding protein is translation initiation factor 4E (eIF4E). Recognition of the mRNA cap by eIF4E is a critical, rate-limiting step for efficient translation initiation and is considered a major target for anticancer therapy. Here, we report a facile methodology for the preparation of N2-triazole-containing monophosphate cap analogs and present their biological evaluation as inhibitors of protein synthesis. Five analogs possessing this unique hetero-cyclic ring spaced from the m7-guanine of the cap structure at a distance of one or three carbon atoms and/or additionally substituted by various groups containing the benzene ring were synthesized. All obtained compounds turned out to be effective translation inhibitors with IC 50 similar to dinucleotide triphosphate m 7 GpppG. As these compounds possess a reduced number of phosphate groups and, thereby, a negative charge, which may support their cell penetration, this type of cap analog might be promising in terms of designing new potential therapeutic molecules. In addition, an exemplary dinucleotide from a corresponding mononucleotide containing benzyl substituted 1,2,3-triazole was prepared and examined. The superior inhibitory properties of this analog (10-fold vs. m 7 GpppG) suggest the usefulness of such compounds for the preparation of mRNA transcripts with high translational activity.
Preparative scale synthesis of fourteen new N2-modified mononucleotide 5’ mRNA cap analogues was achieved. The key step involved use of an SNAr reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC50 ~ 0.9 μM was a N2-p-metoxybenzyl-7-methylguanosine-5’-monophosphate 35.
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