Photocaged 5′ cap analogues for optical control of mRNA translation in cells

Abstract

The translation of messenger RNA (mRNA) is a fundamental process in gene expression, and control of translation is important to regulate protein synthesis in cells. The primary hallmark of eukaryotic mRNAs is their 5′ cap, whose molecular contacts to the eukaryotic translation initiation factor eIF4E govern the initiation of translation. Here we report 5′ cap analogues with photo-cleavable groups (FlashCaps) that prohibit binding to eIF4E and resist cleavage by decapping enzymes. These compounds are compatible with the general and efficient production of mRNAs by in vitro transcription. In FlashCap-mRNAs, the single photocaging group abrogates translation in vitro and in mammalian cells without increasing immunogenicity. Irradiation restores the native cap, triggering efficient translation. FlashCaps overcome the problem of remaining sequence or structure changes in mRNA after irradiation that limited previous designs. Together, these results demonstrate that FlashCaps offer a route to regulate the expression of any given mRNA and to dose mRNA therapeutics with spatio-temporal control.

Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures.

“…The most important finding of this experiment was that the presence of Nb protecting group in the cap significantly suppressed mRNA translation, showing only 1.6% of the activity after deprotection. This observation is consistent with a recent report by Klocker et al that the translation of mRNA containing a cap structure with a photocleavage group on the exocyclic amino group of m 7 G could be triggered by light irradiation in cultured cells 30 . They attributed the translation inhibition effect of the photocleavage group to the inhibition of binding to eIF4E 30 .…”

“…This observation is consistent with a recent report by Klocker et al that the translation of mRNA containing a cap structure with a photocleavage group on the exocyclic amino group of m 7 G could be triggered by light irradiation in cultured cells 30 . They attributed the translation inhibition effect of the photocleavage group to the inhibition of binding to eIF4E 30 . Unlike the case above with DiPure/N2, it should be noted that the mRNA prepared with DiPure showed almost the same translation activity before and after light irradiation, indicating that the Nb group at the 2' Oposition does not impair the translational activity.…”

“…The introduction of a benzyl group at the N 2 position of m 7 G or the substitution of a 7-methyl group with an arylmethyl group was reported to enhance the translational activity of mRNA in human cultured cells 54,63,64 . Some cap analogs have been developed for on/off control of the mRNA translation 30,65,66,67 . For example, Ogasawara et al reported the synthesis of cap analogs that contained photo-responsible groups such as phyenylazo or styryl group or at the N 2 or C 8 position of m 7 G and that their cis/trans conformation enabled photo-switchable translational control 65,66,67 .…”

“…The enzymatic treatment is a standard method to remove uncapped mRNA byproducts. For example, the 5'-terminal triphosphate of uncapped RNA will be converted to the monophosphate by RNA 5' polyphosphatase, followed by degrading monophosphorylated RNA using XRN I, a 5'→3' exoribonuclease requiring 5´ monophosphate 23,25,26,30 . However, considering mRNA susceptibility to non-specific degradation, the number of purification processes should be minimized.…”

The Development and Application of Opto-Chemical Tools in the Zebrafish

“…To improve photostability, caged 4-hydroxy-cyclofen (Cyc) [ 89 , 90 , 91 , 92 ] has also been developed to optimize its use in most physiological conditions. Optical control of gene expression with caged small molecules directly against mRNA is scarce, but recently, synthetic 5′ cap analogues with photo-cleavable groups have been developed [ 93 ]. UV irradiation efficiently releases the 5′ cap to interact with the eukaryotic translation initiation factor 4E (eIF4E), prompting the start of translation.…”