A: The MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2 produces capped RNA by in vitro transcription (IVT) using T7 RNA polymerase and the Anti-Reverse Cap Analog (ARCA).
A: ARCA contains a 3′-O-methyl group on the m7G nucleotide and because of that it can only be incorporated in the correct orientation at the 5′-end of the RNA during an in vitro transcription/capping reaction. This is not true for the standard cap analog (m7G[5′]ppp[5′]G). Thus, ARCA incorporation results in the synthesis of capped RNA that is more efficiently translated in vivo than standard cap analog.
A: The optimal templates for in vitro transcription reaction are linear double-stranded DNA molecules with 5′-protruding ends. DNA templates with blunt ends are less preferable and templates with 3′-protruding ends should not be used.
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: The ARCA/NTP PreMix contains all four ribonucleotides and the ARCA. The PreMix ensures the optimal concentration of each NTP and ratio of ARCA to GTP (4:1), maximizing transcript capping (~80%) and yield. Because the concentration of GTP in the reaction is limiting, the ARCA is preferentially incorporated as the first or 5′-terminal G of the transcript.
A: The reactions for synthesizing ARCA-capped RNA are very similar to setting up a standard IVT reaction. A mix of NTPs and ARCA cap analog are incorporated into the IVT reaction, mixed well, and incubated at 37°C for 30 minutes to 2 hours. The RNA is then purified using any standard RNA purification method.
A: Yes, please follow the recommendations listed.
- Optimizing Yields for Long Templates: Synthesis of transcripts ≥5 kb may require the addition of 1-2 µl of 20 mM GTP to the standard protocol. While this may decrease the percentage of capped transcript to 50-60%, it will increase the yield of full-length transcript.
- Optimizing Yields for Short Templates: Synthesis of transcripts <500 b requires reaction times of 2 hours to maximize yields.
A: Yes, the reaction yields can be increased by modifying the protocol for some templates.
- Extend the incubation to 4 hours. Do not extend the reaction time beyond 4 hours.
- Increase the template concentration.
- Increase the reaction temperature to 42°C.
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.
