Co-transcriptional capping is when cap analogs are directly added to the in vitro transcription reaction mix and incorporated simultaneously into the 5’ end of mRNA as it is synthesized. Both of the main co-transcriptional capping technologies, CleanCap® (TriLink BioTechnologies) and ARCA (Anti-reverse cap analog), can be used in the Takara IVTpro workflow.
The CleanCap and ARCA approaches each have their own set of pros and cons. The advantages of each approach vary depending on the specific needs of the scientists.
CleanCap is the latest generation of cap analogs and enables synthesis of cap-1 mRNA in a single step, but can be costly and often has patent/licensing requirements. ARCA synthesizes cap-0 mRNA first during the IVT reaction. An additional reaction, performed by an enzyme such as mRNA cap 2'-O-methyltransferase, is required to produce cap-1 mRNAs. The capping efficiency of ARCA is slightly low (50–80%) compared to CleanCap (>95%). Regardless, the lack of licensing requirements for ARCA attracts some users.
CleanCap approach
The linearized template vector in Cloning Kit for mRNA Template (Cat. # 6143) or Takara IVTpro mRNA Synthesis System (Cat. # 6141) has AGG as the transcription start site (Figure 1). This makes the vector compatible with use of CleanCap Reagent AG (No. N-7113 or N-7413).
For the vector information and a detailed protocol of in vitro transcription along with CleanCap reagents, please see the Takara IVTpro product overview or the User Manuals for Takara IVTpro mRNA Synthesis System (Cat. # 6141) or Takara IVTpro T7 mRNA Synthesis Kit (Cat. # 6144).
Figure 1. Transcription start site in Linearized Template Vector. The start site, AGG, is shown in red. This vector is included in Cloning Kit for mRNA Template (Cat. # 6143) or Takara IVTpro mRNA Synthesis System (Cat. # 6141) and is compatible with CleanCap Reagent AG (No. N-7113 or N-7413).
ARCA approach
If you use ARCA for co-transcriptionally capping the mRNA using Takara IVTpro mRNA Synthesis System (Cat. # 6141), it is necessary to modify the transcription start site. In this case, the Linearized Template Vector’s start site (+1 position) must be changed from A to G (Figure 2).
Figure 2. Modification of the transcription start site. Some cap analogs, such as ARCA, may require a different transcription site for the cap analog to be incorporated co-transcriptionally. Here, the Linearized Template Vector, included in the Cloning Kit for mRNA Template (Cat. # 6143) or Takara IVTpro mRNA Synthesis System (Cat. # 6141) is changed from AGG to GGG (shown in red).
