Stabilized 5' Cap Analogue for Optochemical Activation of mRNA Translation

The 5' cap is a distinguishing feature of transcripts made by polymerase II and characterized by an N7-methylated guanosine (m⁷G) linked to the first transcribed nucleotide by a 5'-5' triphosphate bridge. It stabilizes eukaryotic mRNAs and plays a crucial role in translation initiation. Its importance in mRNA processing, translation, and turnover makes the 5' cap a privileged structure for engineering by non-natural modifications. A photocleavable group at the 5' cap of guanosine was recently used to mute translation of exogenous mRNAs. Its removal by light enabled direct control of protein production at the posttranscriptional level. Modifications in the triphosphate bridge impede degradation by specific decapping enzymes and maintain translation. Here, we combined 5' cap modifications at different positions and investigated how they impact 5' cap-dependent processes in distinct manners. We synthesized 5' cap analogues with a photocleavable group at the N²-position of m⁷G in addition to a medronate in the triphosphate bridge to obtain a photoactivatable 5' cap analogue featuring a methylene group between the β and γ phosphates. The resulting Medronate-FlashCap transiently or permanently impeded distinct crucial interactions of the 5' cap required for translation and degradation. We show that the Medronate-FlashCap is compatible with in vitro transcription to generate muted mRNA and that light can be used to activate translation in cells. After light-induced removal of the photocleavable group, the Medronate-FlashCap remained stable against degradation by the decapping enzyme DcpS. The additional methylene group renders the 5' cap resistant to DcpS, while maintaining the interaction with cap-binding proteins.