The most common mRNA design contains a cap structure at the 5'-end. A cap structure fulfills many duties, including maturation, nuclear export, and initiation of translation. However, in terms of vaccines and therapeutics, the most crucial roles are the initiation of translation and the prevention of degradation of mRNA from the 5'-end via exonucleases. Therefore, the cap structure holds a special role in the design of mRNAs.
In the initiation of translation, the affinity of the cap to the eukaryotic initiation factor eIF4E plays a major role. The most straightforward approximation is that for higher translational efficiency, the affinity needs to be greater. In the Cap 0 structure, there are several sites that can be modified to modulate the affinity. One of them is the functionalization of the N2 position of 7-methylguanosine. To this end, scientists from Poland have employed various chemical modifications at the N2 position to elucidate the effect on the performance of the cap structure.
In the inhibition study, all N2-modified m7GpppG (7-methylguanosine-containing Cap 0) analogs were much better inhibitors compared to the standard Cap 0 (m7GpppG). Upon employing the same modification in the ARCA cap, the inhibitory effect reduced, explained by an increase in steric hindrance. In the binding study to the initiation factor eIF4E, all chemically modified m7GpppG analogs bonded more strongly than the standard m7GpppG.
The orientation of a cap in an RNA molecule is extremely crucial, and m7GpppG suffers greatly from such incorrect orientation. The analysis indicated that N2-modification can affect the orientation of the cap molecule, inserting it preferentially into the strand in the correct direction. In general, all modified Cap 0 analogs outperform m7GpppG, indicating the potential of chemical modification in the design of new cap analogs.
Such a unique approach towards cap structures will not only improve our knowledge about their roles and interactions but also push the boundaries for the ideal cap design for various applications.
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