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mRNA Capping

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Capping and tailing are key steps in producing active synthetic mRNA for use in functional studies; these modifications prevent degradation and facilitate translation in eukaryotic cells. 

RNA Capping 

Most eukaryotic cellular mRNAs are modified at their 5 ́ends by the addition of a 7-methyl guanosine (m7G) residue in a 5 ́ → 5 ́ triphosphate linkage to the first encoded nucleotide of the transcript. The mRNA cap structure engages critical translation factors to recruit ribosomes to mRNAs, promoting translation. 

Cap structures can be added to in vitro transcripts in two ways:

  • After transcription by using capping enzymes, GTP and S-adenosyl methionine (SAM)
  • During transcription by including cap analogs 
Figure 1: 5’ Cap Structure


 

Schematic representation of mRNA 5′ cap structure indicating the 7-methylguanosine, shown in yellow, and the 5′ end of the mRNA, shown in blue. The 2′-O-methyl group present in Cap-1 structures is shown in red.

Figure 2: GLuc Expression
 



Purified Cap-0 and uncapped GLuc mRNA were transfected into HeLa cells and incubated overnight (16 hrs.) at 37°C. Cell culture supernatants from each well were assayed for GLuc and CLuc activity and luminescence values were recorded. The GLuc luminescence values were normalized to the luminescence values of Cap-0 CLuc RNA.

Post-transcriptional Enzymatic mRNA Capping 

Highest efficiency mRNA capping is achieved using the Faustovirus Capping Enzyme (NEB #M2081). This system has three enzymatic activities (RNA triphosphatase, guanylyltransferase, guanine methyltransferase); all are necessary for the addition of a complete Cap-0 structure, m7Gppp5 ìN. In vitro transcripts can be capped in less than one hour in the presence of the capping enzyme, reaction buffer, GTP and the methyl donor, SAM. All capped structures are added in the proper orientation for recognition by the translational machinery, unlike co-transcriptional addition of some cap analogs (1). 

Co-transcriptional Capping with Dinucleotide Cap Analogs

Anti-Reverse Cap Analog (ARCA) [3′-0-Me-m7G(5′)ppp(5′)G RNA Cap Structure Analog, (NEB #S1411)] is the preferred cap analog for co-transcriptional capping. Transcription with ARCA produces 100% translatable capped transcripts, because it can only incorporate in the ‘correct’ orientation, where the N7-methylguanosine is at the terminus [m7G(5 ́)pppG-RNA] (2,3). 


In contrast, the standard cap analog [m7G(5 ́)ppp(5 ́)G RNA Cap Structure Analog (NEB #S1404)] can be incorporated in either orientation [m7G(5′) pppG-RNA] or [G(5′)pppm7G-RNA], resulting in a mixture of transcripts (2,4). mRNAs with cap analog incorporated in the incorrect orientation are not efficiently translated, resulting in lower protein yields (1). The RNA products are a mixture of 5′-capped and 5′-triphosphorylated transcripts. This may necessitate purification or treatment with a phosphatase in order to avoid unintended immune stimulation by 5 ́-triphosphorylated RNA. 

Figure 3: Schematic representation of alternative mRNA synthesis workflows


Enzyme-based capping (top) is performed after in vitro transcription using 5′-triphosphate RNA, GTP, and S-adenosyl- methionine (SAM). Cap-0 mRNA can be converted to Cap-1 mRNA using mRNA cap 2 ́′O-methyltransferase (MTase) and SAM in a subsequent or concurrent reaction. The methyl group transferred by the MTase to the 2′-O of the first nucleotide of the transcript is indicated in red. Conversion of ~100% of 5′-triphosphorylated transcripts to capped mRNA is routinely achievable using enzyme-based capping.

Co-transcriptional capping (bottom) uses an mRNA cap analog, shown in yellow, in the transcription reaction. For ARCA (anti reverse cap analog) (left), the cap analog is incorporated as the first nucleotide of the transcript. ARCA contains an additional 3′-O-methyl group on the 7-methylguanosine to ensure incorporation in the correct orientation. The 3′-O-methyl modification does not occur in natural mRNA caps. Compared to reactions not containing cap analog, transcription yields are lower. ARCA-capped mRNA can be converted to Cap-1 mRNA using mRNA cap 2′-O-MTase and SAM in a subsequent reaction. CleanCap Reagent AG (right) uses a trinucleotide cap analog that requires a modified template initiation sequence. A natural Cap-1 structure is accomplished in a single reaction.

Cap-1 Modification and Co-transcriptional Trinucleotide Capping

The Cap-1 structure has been reported to enhance mRNA translation efficiency (5) and hence may help improve expression in mRNA transfection and in microinjection experiments. 

Cap-0 transcripts can be enzymatically converted to cap-1 in vitro. mRNA Cap 2′-O-Methyltransferase (NEB #M0366) adds a methyl group at the 2′-O position of the first nucleotide adjacent to the cap structure at the 5′ end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap-0) resulting in a Cap-1 structure. Alternatively, Cap-1 mRNA can be synthesized co-transcriptionally with a trinucleotide cap analog such as CleanCap Reagent AG. The use of CleanCap Reagent AG results in significant advantages over traditional dinucleotide co-transcriptional capping. CleanCap Reagent AG is a trinucleotide with a 5′-m7G joined by a 5′-5′ triphosphate linkage to an AG sequence. The adenine has a methyl group on the 2′-O position. The incorporation of this trinucleotide in the beginning of a transcript results in a Cap-1 structure.

Figure 4. Comparison of RNA Yields from In Vitro Transcription Reactions with no cap analog, ARCA, or CleanCap Reagent AG





Figure 5. Molecular Structure of CleanCap Reagent AG



 

RNA Cap Analog Selection Chart

The 5′ terminal m7G cap present on most eukaryotic mRNAs promotes translation, in vitro, 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 mRNA in a eukaryotic cell-free protein synthesizing system. Splicing of certain eukaryotic substrate RNAs has also been observed to require a cap structure.

PRODUCT

APPLICATION

HiScribe T7 mRNA Kit with CleanCap Reagent AG*

*kit only
  • High yield
  • Natural Cap-1 structure
  • Produces 100% translatable transcripts
  • Highest efficiency capping

Anti-Reverse Cap Analog 3′-O-Me-m7G(5′) ppp(5′)G
  • Produces 100% translatable capped transcripts
  • Co-transcriptional capping with T7 (NEB #M0251), Hi-T7 (NEB #M0658), SP6 (NEB #M0207), and T3 RNA polymerases (NEB #M0378)
  • Synthesis of m7G capped RNA for in vitro splicing assays
  • Synthesis of m7G capped RNA for transfection or microinjection

Standard Cap Analog m7G(5′)ppp(5′)G
  • Co-transcriptional capping with T7, Hi-T7, SP6 and T3 RNA polymerases
  • Synthesis of m7G capped RNA for in vitro splicing assays
  • Synthesis of m7G capped RNA for transfection or microinjection

Unmethylated Cap Analog G (5′)ppp(5′)G
  • Co-transcriptional capping with T7, Hi-T7, SP6 and T3 RNA polymerases
  • Synthesis of unmethylated G capped RNA

Methylated Cap Analog for A +1 sites m7G(5′)ppp(5′)A
  • Co-transcriptional capping with T7 RNA polymerase from the phi2.5 promoter that contains an A at the transcription initiation site
  • Synthesis of m7G capped RNA for in vitro splicing assays
  • Synthesis of m7G capped RNA for transfection or microinjection

Unmethylated Cap Analog for A +1 sites G(5′)ppp(5′)A
  • Co-transcriptional capping with T7 RNA polymerase from the phi2.5 promoter that contains an A at the transcription initiation site
  • Synthesis of unmethylated G capped RNA
  • Synthesis of A capped RNA
 

1. Grudzien, E., et al. (2004) RNA, 10, 1479–1487.
2. Stepinski, J., et al. (2001) RNA, 7, 1486–1495.
3. Peng, Z.-H., et al. (2002) Org. Lett. 4, 161–164. 
4. Pasquinelli, A. E., Dahlberg, J. E. and Lund, E. (1995) RNA, 1, 957–967. 
5. Kuge, H., et al. (1998) Nucleic Acids Res, 26, 3208–3214.