Home Research Labs Chan Lab Chan Lab Enzymatic co-transcriptional capping technologies

Enzymatic co-transcriptional capping technologies

The 5′ cap is an integral part of functional mRNA molecules. To overcome the hurdle of a multistep workflow of capped RNA synthesis, we created a fusion protein consisting of FCE and T7 RNA polymerase that can efficiently install the Cap-0 structure to synthetic RNA molecules co-transcriptionally in vitro.

Diagram showing the creation of a fusion protein consisting of FCE and T7 RNA polymerase

 

In this experiment, a FLuc transcript was transcribed and modified by the FCE::T7RNAP fusion protein and vaccinia cap 2′-O-methyltransferase or by the T7RNAP in the presence of FCE and vaccinia cap 2′-O-methyltransferase over a 90-minute period. While the level of Cap-1 incorporation into the synthesized RNA increased over time for co-transcriptional capping using separate enzymes (left panel), the level of Cap-1 incorporation achieve >95% in the first 10 minutes and after using the FCE::T7RNAP fusion (right panel). This suggests that the FCE::T7RNAP fusion is able to incorporate the cap structure into the RNA co-transcriptionally.

 

Two bar graphs showing FLuc transcribed and modified by fusion protein and capping enzyme

Reference:
Chan et al (2023), bioRxiv, DOI: 10.1101/2023.10.28.564488