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
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).
Post-transcriptional Enzymatic mRNA Capping
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).
Co-transcriptional Capping with Dinucleotide Cap Analogs
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.
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-1 Modification and Co-transcriptional Trinucleotide Capping
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.
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.
Methylated Cap Analog for A +1 sites m7G(5′)ppp(5′)A
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.
- A Typical DNase I Reaction Protocol (M0303)
- Capped RNA Synthesis (E2040)
- Capping Protocol (M2080)
- DNA Template Preparation (E2040)
- Evaluation of Reaction Products (E2040)
- High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
- A Typical Tailing Reaction (M0337)
- Purification of Synthesized RNA (E2040)
- RNA Synthesis with Modified Nucleotides (E2040)
- SP6 In Vitro Transcription Reaction Protocol (M0207)
- Standard RNA Synthesis (E2040)
- 2´-O-Methylation of Capped RNA (M0366)
- One-Step Capping and 2´-O-Methylation (M0366)
- Labeling Protocol (M2080)
- Capped RNA Synthesis (E2050)
- Evaluation of Reaction Products (E2050)
- Purification of Synthesized RNA (E2050)
- RNA Synthesis with Modified Nucleotides (E2050)
- Standard RNA Synthesis (E2050)
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- Protocol for Standard RNA Synthesis
- Evaluation of Reaction Products (E2060)
- mRNA Purification (E2060)
- mRNA Purification (E2065)
- mRNA Synthesis with Modified Nucleotides (E2060)
- mRNA Synthesis with Modified Nucleotides (E2065)
- Standard mRNA Synthesis (E2060)
- Standard mRNA Synthesis (E2065)
- Evaluation of Reaction Products (E2065)
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
- sgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050)
- EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (E3322)
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- Purification of Synthesized RNA (E2070)
- RNA Synthesis Protocols (E2070)
- 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
- 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:
- 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:
- 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
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This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene.