mRNA with Pseudouridine and 5-methylcytidine?

Question

I am making mRNA by in vitro transcription for transfection purposes. There is a lot of literature about how protein expression is increased and innate immune activation is decreased by substituting uridine for pseudouridine and cytidine for 5-methylcytidine. I decided to try this, but my yield was lower and the mRNA was shorter than when using natural nucleotides. The papers all say they made the mRNA by simply using the new nucleotides in their in vitro transcription kits, but they never mention any further changes to the protocol. Now I would assume that since pseudouridine and 5-methylcytidine are a little different from the normal substrates for RNA polymerase that they might not be incorporated as quickly and the resulting reaction rate would slow down. It might be possible that the Polyadenylate polymerase that adds the polyA tail doesn't recognize the modified mRNA as well. Both a slower reaction rate and less efficient tailing could make a lower yield of shorter mRNA.

Does anyone here have any experience with IVT using modified nucleotides and can suggest something to improve yields?

Answer 1

After a couple months I got this working. I ordered the psuedouridine and 5 methylcytidine from Trilink. I also switched from the mMessage mMachine kit the the Megascript kit. Megascript has all the nucleotides separated while mMessage mMachine has them pooled. You have to dissolve Trilink nucleotides so that the concentration is the same as the natural nucleotides in your IVT kit. Then you just use the non-natural nucleotides instead of UTP and CTP. Let the IVT reaction run 4 hours. Be careful about RNAse contamination.