Enzymatic Modification of the 5' Cap with Photocleavable ONB-Derivatives Using GlaTgs V34A

The 5' cap of mRNA plays a critical role in mRNA processing, quality control and turnover. Enzymatic availability of the 5' cap governs translation and could be a tool to investigate cell fate decisions and protein functions or develop protein replacement therapies. We have previously reported on the chemical synthesis of 5' cap analogues with photocleavable groups for this purpose. However, the synthesis is complex and post-synthetic enzymatic installation may make the technique more applicable to biological researchers. Common 5' cap analogues, like the cap 0, are commercially available and routinely used for in vitro transcription. Here, we report a facile enzymatic approach to attach photocleavable groups site-specifically to the N² position of m⁷ G of the 5' cap. By expanding the substrate scope of the methyltransferase variant GlaTgs V34A and using synthetic co-substrate analogues, we could enzymatically photocage the 5' cap and recover it after irradiation.