mRNA Synthesis with Modified Nucleotides (E2060)

Modified mRNAs containing 5mCTP and Pseudo-UTP have been shown to suppress RNA-mediated innate immune activation in vivo. The HiScribe T7 ARCA mRNA Kit (with tailing) is capable of incorporation of 5mCTP and Pseudo-UTP, into mRNA. Up to 2.5 mM total modified nucleotides can be added into the transcription reaction without impacting the mRNA yield significantly. Other modified UTP or CTP may also be used but RNA yield will vary depending on the properties of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Please note modified nucleosides are not supplied with the kit.

The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP.

  1. Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order:

    Nuclease-free water to 20 µl  
    2X ARCA/NTP Mix 10 µl 1 mM GTP, 4 mM ARCA,
    1.25 mM CTP, 1.25 mM
    UTP, >1.25 mM ATP final
    5mCTP, 10 mM 2.5 µl 1.25 mM 5mCTP final
    Pseudo-UTP, 10 mM 2.5 µl 1.25 mM Pseudo-UTP final
    Template DNA X µl 1 μg
    DTT (0.1)*
    1 µl
    5 mM
    T7 RNA Polymerase Mix 2 µl  
    Total reaction volume 20 µl  

    *Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation.

  2. Follow standard mRNA synthesis protocol.