RNA Synthesis

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

Post-transcriptional Enzymatic mRNA Capping

Highest efficiency mRNA capping is achieved using the Vaccinia Capping System (NEB #M2080). This system has three enzymatic activities (RNA triphosphatase, guanylyltransferase, guanine methyltransferase); all are necessary for the addition of a complete Cap-0 structure, m7Gppp5′N(1,2). 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. Capping is ~100% efficient and all capped structures are added in the proper orientation for recognition by the translational machinery, unlike co-transcriptional addition of some cap analogs(3).

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.

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-transcrip- tional 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](4,5).

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(4,6). mRNAs with cap analog incorporated in the incorrect orientation are not efficiently translated, resulting in lower protein yields (3). The RNA products are a mixture of 5′-capped and 5′-triphosphorylated transcripts. This may necessi- tate 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(7) 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. Molecular Structure of CleanCap Reagent AG

Figure 5. Comparison of RNA Yields from In Vitro Reagent AG Transcription Reactions with no cap analog, ARCA, or 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.



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.Guo, P.X. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA, 87, 4023–4027.
2. Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472–24479.
3. Grudzien, E., et al. (2004) RNA, 10, 1479–1487.
4. Stepinski, J., et al. (2001) RNA, 7, 1486–1495.
5. Peng, Z.-H., et al. (2002) Org. Lett. 4, 161–164.
6. Pasquinelli, A. E., Dahlberg, J. E. and Lund, E. (1995) RNA, 1, 957–967.
8. Kuge, H., et al. (1998) Nucleic Acids Res, 26, 3208–3214.

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Protocols for RNA Synthesis
    Publications related to RNA Synthesis
  1. Eyler, D.E., Franco, M.K., Batool, Z., Wu, M.Z., Dubuke, M.L., Dobosz-Bartoszek, M., Jones, J.D., Polikanov, Y.S., Roy, B., Koutmou, K.S 2019. Pseudouridinylatio of mRNA coding sequences alters translation Proc Natl Acad Sci U S A. 116(46), PubMedID: 31672910, DOI: 10.1073/pnas.1821754116
  2. Wu, M.Z., Asahara, H., Tzertzinis, G., Roy, B. 2020. Synthesis of low immunogenicity RNA with high-temperature in vitro transcription RNA. , PubMedID: 31900329, DOI:
  3. Potapov, V., Fu, X., Dai, N., Correa, I.R., Jr., Tanner, N.A., Ong, J.L 2018. Base modificatons affecting RNA polymerase and reverse transcriptase fidelity Nucleic Acids Res. 46(11): 5753-5763, PubMedID: 29750267, DOI:
  4. Wulf, Madalee; Buswell, John; Chan, Siuhong; Dai, Nan; Marks, Katherine; Tzertzinis, George; Whipple, Joe; Correa, Ivan; Schildkraut, Ira; 2019. The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping Sci Rep. 9 (1), PubMedID: 31197197, DOI: 10.1038/s41598-019-45083-5
RNA Reagents Overview
Recommended HiScribe RNA Synthesis Kits by Application
RNA Polymerase Selection Chart
NEB offers RNA polymerases that can be used for in vitro synthesis of RNA for a wide variety of downstream applications. Additional polymerases are offered for the generation of untemplated homoribopolymeric tails for reverse transcription or labeling.
RNA Ligase Selection Chart
NEB offers a variety of ligases for RNA research with unique specificities.
Cap Analog Selection Chart
NEB offers a number of cap analogs that increase stability, decrease susceptibility to exonuclease degradation, and promote the formation of mRNA initiation complexes.
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