A mature mRNA ready for efficient translation by the ribosome contains two major modifications: a 5′ cap structure and a poly(A) tail. The m7G cap structure makes up of a 7-methylguansine triphosphate linked to the 5′ end of the mRNA via a 5′ → 5′ triphosphate linkage (m7G cap). The m7G cap, also known as cap 0 structure, is essential for the majority of protein translation in vivo. The m7G cap also protects the mature mRNA from degradation, allows for a regulated degradation mechanism, enhances pre-RNA splicing and directs nuclear export. In vivo, the cap 0 structure can be further modified to cap 1 structure by adding a methyl group to the 2’O position of the initiating nucleotide of the mRNA. Recently, the literature suggests that the 2′O methylation in the cap 1 structure helps the mRNA evade innate immune response in vivo.
NEB offers RNA capping options that includes (1) post-transcriptional capping by using the Vaccinia Capping System (NEB# M2080) on on in vitro transcripts and (2) co-transcriptional capping by using cap analogs during in vitro transcription. Post-transcriptional capping offers the advantage of the capability of 100% capping on large quantity of in vitro transcripts. On the other hand, co-transcriptional capping using cap analog allows for a single-step workflow and the flexibility to incorporation non-canonical cap structures to the RNA, with a tradeoff of reduced transcription yield (due to the lower GTP concentration used in transcription) and incomplete capping (due to the competition between the cap analog and GTP for the initiation nucleotide position.)
For in vivo applications such as expression of protein via transfection of exogenous mRNA, (1) incorporation of cap 1 structure via the use of Vaccinia virus cap 2’O methyltransferase (NEB# M0366) and (2) incorporation of a poly(A) tail via a poly(T) track in the template or using Poly(A) Polymerase (NEB# M0276) are recommended.
Vaccinia Capping System
Based on the Vaccinia Virus Capping Enzyme, the Vaccinia Capping System (NEB# M2080) provides the necessary components to add 7-methylguanylate cap structures (Cap 0) to the 5´ end of RNA. In eukaryotes, these terminal cap structures are involved in stabilization, transport and translation of mRNAs. Enzymatic production of capped RNA is an easy way to improve the stability and translational competence of RNA used for in vitro translation, transfection and microinjection. Alternatively, use of labeled GTP in a reaction provides a convenient way to label any RNA containing a 5′ terminal triphosphate.
Advantages of the Vaccinia Capping System
- Capping mRNA prior to in vivo or in vitro translation
- Labeling 5′ end of mRNA
RNA Cap Analogs
The 5′ terminal m7G cap present on most eukaryotic mRNAs promotes in vitro and in vivo translation, at the initiation level. For most RNAs, the cap structure increases stability, decreases susceptibility to exonuclease degradation, and promotes the formation of mRNA initiation complexes. Certain prokaryotic mRNAs with 5′ terminal cap structures are translated as efficiently as eukaryotic mRNAs in a eukaryotic cell-free protein synthesizing system. Splicing of certain eukaryotic substrate RNAs has also been observed to require a cap structure.