Editing Capped mRNA (section)

= 5’AG 3’ for the CleanCap AG reagent, N-7113= =

[https://www.tebu-bio.com/blog/2018/03/13/tips-to-modify-mrna-for-in-vivo-applications-and-delivery-optimisation/ https://www.tebu-bio.com/blog/2018/03/13/tips-to-modify-mrna-for-in-vivo-applications-and-delivery-optimisation/]

CleanCap reagents are easy to use. They are simply compatible with wild-type T7 polymerase. You apply the same quantity for CleanCap and the rNTP (CTP, UTP, ATP, GTP). It could be 5mM of each depending on the usual protocol. Should use ENB's T7-FlashScribe in vitro transcription kit because of its high yield production (180µg from 1µg)!



CleanCap AG is optimized for production of mRNA that initiates with adenosine as the first base, guanosine as the second base, and guanosine as the third base, so AGG.

Unfortunately, most conventional transcripts start with 5’-GGG.

It would be a T7 promoter sequence like that 5’TAATACGACTCACTATAGGGNNNNNNNNNNNN… 3’.

So for optimal results with the CleanCap AG reagent, you would need have the three bases after the TATA box be AGG.

Thus the best T7 promoter sequence would be 5’TAATACGACTCACTATAAGGNNNNNNNNNNNN… 3’

Using that sequence with the CleanCap AG reagent, N-7113, you will get 95-98% capped mRNA with the natural CAP1 structure.

Keeping the benefit of CAP1 structure If you cannot optimize the T7 promoter sequence, you should use the CleanCap GG reagent, N-7133.

The capping efficiency would be similar to the ARCA (it could be 80%), but you will still benefit of a natural CAP1 structure in place of the CAP0 with ARCA. CAP1 boosts translation compare to CAP0 in most cell types.

#2 – Pseudo-UTP and 5-Methyl-CTP to reduce immune response of cells against mRNA

We offer a variety of modified rNTPs suitable for in vitro transcription. This includes the Pseudo-UTP and 5-Methyl-CTP used to reduce cellular response against mRNA

It is now well established that a full substitution with these two modified rNTP reduces the innate immune response of cells. Such modRNA, as called by Pr Chien’s team in their article Nat. Biotechnol. in 2013 (10.1038/nbt.2682), can be used as treatment in vivo with no more need for HPLC purification

They showed that intramyocardial injection of VEGF-A coding modRNA after myocardial infarction (MI) induces vascular regenration (see picture below).

TriLink’s teams and partners have made further investigations and found that the 5-methoxy-UTP fully substitute mRNA can also efficiently reduce the immune response of the cells and the toxicity of the modified mRNA. The use of 5-methoxy-UTP (5moU) substitution is license free.

To track mRNA in vivo and optimise their delivery into cells, Cy5 labelling is possible though translation efficiency correlated inversely with Cyanine 5-UTP substitution.

Transcription with Cyanine 5-UTP:5-Methoxy-UTP at a ratio of 1:3. Substitution in this ratio results in mRNA that is easily visualized and can still be translated in cell culture.