Over the last years, mRNA-based experimental approaches have been successfully used in a broad range of research applications (genome editing, gene replacement, vaccine, immunotherapy, cellular reprogramming…) without risk of integration into the genome of the host cell.
mRNA molecules, mimicking fully processed, capped and polyadenylated mRNA, can now be reliably produced via in vitro transcription methods while reducing innate immune stimulation. In this post, 4 tips & Tricks are presented to help produce and optimally use your mRNAs in your research and drug development projects.
#1 – mRNA capping
In vitro production of mRNA has made significant progress. We can note a new capping method providing a natural CAP1 structure at high efficiency.
How to use CleanCap AG?
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.
By the way I would suggest the T7-FlashScribe in vitro transcription kit because of its high yield production (180µg from 1µg)!
The secret of the CleanCap AG
Each CleanCap reagent is to be used with a specific initiating sequence.
I recommend the CleanCap AG reagent N-7113, because it provides the best capping efficiency.
A reagent for a specific T7 promoter sequence
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
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.
#3 – Simple codon optimization
Furthermore, U-depletion, playing with the genetic code, can improve translation and thus increase protein function in the cells as illustrated below with the endonuclease CAS9 protein that generates indels.
#4 – Tracking the mRNA with Cy5
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.
I would recommend 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.
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