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Short answer: mRNA capping adds a 7-methylguanosine (m7G) nucleotide to the 5′ end of an RNA through an inverted 5′-5′ triphosphate link. It takes three enzymatic steps: remove one phosphate, attach GMP, then methylate the guanine at N7. The cap helps the mRNA resist degradation and be translated efficiently. In the lab, synthetic (in vitro transcribed) mRNA is capped either after transcription with enzymes or during transcription with cap analogs, and that choice decides cap type, yield and template design.
Reader task: researchers and lab staff who need to understand what the 5′ cap is and choose between capping routes for synthetic mRNA. General scientific information only.
Definition and scope
The 5′ cap is the m7GpppN structure found at the start of capped eukaryotic and many viral RNAs. In Shuman’s review in RNA, the m7GpppN cap is described as conferring mRNA stability and efficient translation, and capping as acting on nascent transcripts made by cellular or viral RNA polymerases. Two terms recur in the lab literature: cap0 (the methylated guanosine cap alone) and cap1, a form that mimics mature human mRNA and carries an additional methylation on the first transcribed nucleotide. Cap0 may not be recognised as self by the immune system, while uncapped mRNA with an exposed 5′ phosphate can activate immune responses, according to the NIH-linked article in The Scientist.
How is mRNA capped?
Capping is a three-step enzymatic reaction (Shuman, RNA, 2015):
- An RNA 5′ triphosphatase hydrolyses the 5′ triphosphate end of the transcript to a diphosphate.
- An RNA guanylyltransferase transfers GMP to that diphosphate end, through a covalent enzyme-GMP intermediate, giving a GpppRNA structure.
- An AdoMet:RNA (guanine-N7) methyltransferase methylates the guanine to produce m7GpppRNA.
The vaccinia virus capping enzyme is a well-studied example: a heterodimer of a 97 kDa and a 33 kDa subunit that performs all three reactions. In the cell, this happens on the nascent transcript, which is why capping is usually described as co-transcriptional. In the test tube, the same chemistry can be run after transcription with purified enzymes.
What is the capping process in the lab? Co- versus post-transcriptional capping
For in vitro transcribed (IVT) mRNA there are two routes, as summarised in the Thermo Fisher Behind the Bench article on IVT capping methods:
- Post-transcriptional (enzymatic): transcribe first, then cap with a guanylyltransferase and methyltransferase. Efficiency is high and cap0 or cap1 can be made; the trade-off is multiple purification steps and additional enzymes.
- Co-transcriptional: add a cap analog to the transcription reaction so it is incorporated at the 5′ end. This needs a single purification step and no additional enzymes.
Cap analogs compared (the 5 prime cap in practice)
The table below collects what one source (Thermo Fisher Behind the Bench, read 2026-11-07) states for each route. Vendor-reported efficiency figures are the vendor’s claims, not independent measurements.
| Route | Cap type | Stated characteristics |
|---|---|---|
| Enzymatic, post-transcription | Cap0 or cap1 | High efficiency; multiple purification steps and extra enzymes needed |
| mCap (m7GpppG) | Cap0 | Incorporated in both orientations, so about 50% of capped product is untranslatable; used at about a 4:1 cap:GTP ratio; about 70% capping efficiency |
| ARCA (m7(3′-O-Me)GpppG) | Cap0 | Incorporated in the correct orientation only; still needs a high cap:GTP ratio, which lowers full-length yield |
| CleanCap Reagent AG | Cap1 | No high cap:GTP ratio required; requires an AG initiation sequence in the template instead of the standard GG |
Checklist before choosing a capping route
- Cap type needed: cap0 or cap1. The choice balances efficiency, immunogenicity and manufacturing complexity (The Scientist).
- Template initiation sequence: CleanCap Reagent AG needs an AG initiation sequence instead of the standard GG. A template designed for one route may need redesign for another.
- Purification steps and enzymes: the enzymatic route adds both; co-transcriptional capping avoids them.
- Yield versus capping: ARCA-type analogs trade full-length RNA yield for correct orientation because of the high cap:GTP ratio.
- Vendor figures: treat capping-efficiency percentages as vendor-reported unless you have measured them in your own system.
What are the three steps of mRNA processing?
In eukaryotic cells, pre-mRNA processing consists of 5′ capping, splicing (removal of introns) and 3′ polyadenylation (addition of the poly(A) tail). Capping is the 5′ end step described above. Splicing and polyadenylation have their own mechanisms and are not covered in detail here; a hybridisation method for checking RNA size and abundance is described in the northern blot guide.
Why is capping and tailing done? Poly(A) tails and poly(A) polymerase
The cap and the poly(A) tail both support stability and translation. A Nature Reviews Molecular Cell Biology review (Passmore and Coller, 2022) notes that poly(A) tails are present on almost every eukaryotic mRNA, were first proposed to promote translation and prevent degradation, and have a more complex role than first thought: poly(A)-binding protein can stimulate tail removal (deadenylation), and the interplay between tails, binding protein, translation and decay regulates gene expression.
For synthetic mRNA, the tail can be added in two ways. It can be encoded in the DNA template, or added afterwards with a poly(A) polymerase. A 2026 Nucleic Acids Research paper (Spiewla et al.) reports that long poly(A) stretches can recombine during plasmid amplification in bacteria, which impairs production of IVT mRNA with long tails, and that segmenting the tail with heteronucleotide spacers stabilised the sequence and increased translation in their experiments (up to six-fold compared with an A90 tail for the best segmented design). That result comes from the authors’ own experimental systems.
Common misunderstandings
- “Capping is only a stability tag.” Sources describe both stability and efficient translation as functions of the cap.
- “All cap analogs give correctly oriented caps.” Standard mCap can be incorporated in either orientation, which is the reason anti-reverse analogs such as ARCA exist.
- “Cap0 and cap1 are interchangeable.” They differ in how closely they resemble mature human mRNA, which is why immunogenicity is part of the selection criteria.
- “The tail is an afterthought.” Tail length and design affect stability and translation, and template design can affect plasmid stability.
Where it matters for labs
Capping choices are made when designing the DNA template, selecting transcription reagents and planning purification. Because the initiation sequence and the cap:GTP ratio differ between methods, the decision belongs at the design stage rather than after a transcription run. Related reading on the site: the Kozak sequence guide for translation initiation context, viral vector types for an alternative delivery route to nucleic-acid constructs, and the RNA extraction kit guide for upstream RNA handling.
Frequently asked questions
How is mRNA capped?
By three sequential enzymatic reactions: triphosphatase, guanylyltransferase and N7-methyltransferase, giving an m7GpppRNA 5′ end (Shuman, 2015).
What are the three steps of mRNA processing?
5′ capping, splicing and 3′ polyadenylation.
What is the capping process?
The enzymatic addition of an inverted, methylated guanosine to the 5′ end of a transcript. For synthetic mRNA it is done after transcription with enzymes or during transcription with a cap analog.
Why is capping and tailing done?
The cap supports stability and efficient translation; the poly(A) tail is part of the regulation of translation and mRNA decay in the cytoplasm.
Sources
- Shuman S. RNA capping: progress and prospects. RNA. 2015. pmc.ncbi.nlm.nih.gov/articles/PMC4371356 (read 2026-11-07).
- Thermo Fisher Scientific, Behind the Bench: Methods of IVT mRNA capping. thermofisher.com (read 2026-11-07; vendor source).
- The Scientist: 5 things to know about mRNA capping for next-generation vaccines and therapies. scientist.com (read 2026-11-07).
- Passmore LA, Coller J. Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nat Rev Mol Cell Biol. 2022. PMC7614307 (abstract read 2026-11-07).
- Spiewla T et al. PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA. Nucleic Acids Res. 2026. PMC12805897 (abstract read 2026-11-07).
