A: This kit is used for co-transcriptional capping of 5-methyl-cytidine-pseudouridine-containing, in vitro transcribed RNA with T7 RNA polymerase and Anti-Reverse cap analog (ARCA). 5mC- Ψ-mRNAs induce lower innate immune responses in mammalian cells. A reaction following the protocol produces 40 µg RNA from 1 µg DNA in 3 hours. Reaction products are 80% correctly capped and 20% uncapped.
A: mRNAs modified with the incorporation of 5mCTP and ΨTP have demonstrated the ability to reduce RNA-mediated innate immune activation in vivo.
A: Yes, the modified nucleotides 5mCTP and ΨTP are included in the kit as part of the ARCA / GAΨ5mC PreMix.
A: The PreMix contains all four ribonucleotides (GAΨ5mC) and the ARCA. It ensures the optimal concentration of each NTP and ratio of ARCA to GTP (4:1), maximizing transcript capping (~80%) and yield.
A: Addition of DTT is recommended as the RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling. Addition of DTT will not compromise the performance of the reaction.
A: There are no special requirements of your template to cap with ARCA, other than the requirement for a functional T7 promoter where the first base to be transcribed (Base +1) is a “G”.
A: No, you cannot achieve 100% capping using this method. Reaction products are 80% correctly capped and 20% uncapped.
A: Two other drawbacks to this method of capping are: 1) transcriptional yields are limited due to the limiting amount of GTP present in the reactions, and 2) one cannot make “long” transcripts (>4 kb) very efficiently due to the limiting GTP. Both drawbacks can be addressed by supplementing extra GTP into the reaction. The kit contains a separate tube of GTP for this purpose. However, if doing so, one changes the ratio of cap analog to GTP in the reaction which results in a lower capping efficiency.
A: The alternative to co-transcriptional capping is post-transcriptional capping. This method involves enzymatic treatment of the in vitro transcribed (IVT) RNA with capping enzyme as a separate reaction distinct from the transcription reaction. With this method, one can achieve virtually 100% capping efficiency and, because GTP is not limiting in the IVT reaction, IVT yields are maximized, and transcript lengths are not an issue.
CELLSCRIPT™ offers the ScriptCap™ m7G Capping System, ScriptCap™ 2’-O-Methyltransferase Kit, and ScriptCap™ Cap 1 Capping System for post-transcriptional capping.
