Introduction

mRNA capping refers to the addition of a protective cap structure to the 5' end of a precursor mRNA. While mRNA capping occurs primarily in eukaryotes, it is also one of the essential steps for in vitro mRNA transcription, where synthetic mRNA is generated from custom DNA templates in highly regulated laboratory conditions. This step is necessary to improve mRNA stability and ensure efficient nuclear transport and translation.

The purpose of synthetic mRNA capping is to replicate the 5'-cap structures connected to the first nucleotides of newly transcribed eukaryotic mRNA. Cap-0 is the preliminary structure composed of 7-methylguanosine. Cap-0 is 2'-O-methylated in higher eukaryotes, including humans, to form cap 1 and 2¹.

The Process of mRNA Capping

Types of mRNA Capping:

Co-transcriptional Capping:

In co-transcriptional capping, a cap analog is added to the transcription reaction mixture. Thus, it is automatically incorporated into the nascent mRNA transcript emerging from the mixture. Cap-1 and -2 analogs offer the best advantage during co-transcriptional capping because they make the emerging mRNA transcript stable, safe, and translationally efficient. In contrast, capping with cap-0 may require an additional enzymatic step to generate cap-1.

Co-transcriptional capping is more straightforward than post-transcriptional capping, as it eliminates the additional enzymatic capping and subsequent purification steps. However, the cap analog must compete with guanine triphosphate (GTP) for the 5' end of the mRNA, which may impact efficiency.

Post-transcriptional Capping:

Post-transcriptional capping involves an initial mRNA synthesis followed by an additional enzymatic reaction that adds the 5' cap to the mRNA. Common viral capping enzymes used include: 1) Vaccinia virus capping enzyme (VCE) for synthesizing Cap 1 mRNA, and 2) Faustovirus capping enzyme (FCE) due to its broader temperature range and higher enzyme activity than VCE.

Post-transcriptional capping occurs in a stepwise manner. First, a series of enzymatic reactions and the co-substrate S-adenosyl-L-methionine (SAM) are conducted to synthesize the cap-0 structure and add it to the nascent uncapped mRNA. Next, the mRNA Cap 2′-O-Methyltransferase (2′-O-MTase) converts cap-0 to a cap-1 structure². This method provides more robust control over the cap structure than co-transcriptional capping. However, it also requires not only additional enzymatic reaction steps but also more purification, potentially elongating in vitro transcription processes.

Cap Analogues in Co-transcriptional Capping

• The standard cap analog (mCap) is a cap-0 mimic that was initially used in IVT. It consists of 7-methylguanosine triphosphate linked to guanosine³. A key disadvantage of mCap is that it can be incorporated in both forward and reverse orientations, with the latter form unable to participate in translation. As a result, only a portion of the mCap-capped mRNA is translated.
• Anti-reverse Cap Analogs (ARCA) contain modifications to the 7-methylguanosine end of cap-0 to prevent reverse transcription⁴. Thus, higher efficiency is achieved when translating the capped transcripts.
• Trinucleotide caps are of Cap-1 structure derived from the 2′-O-methylation on Cap-0 analogs. Compared to dinucleotide cap analogs like ARCA, trinucleotide caps reduce the risk of innate immune response while overseeing pre-mRNA splicing more efficiently⁵.
• Tetranucleotide analogs (cap-2) were synthesized in a 2022 study through further methylation. Although translation efficiency did not change significantly, the cap analog contributed to immune evasion⁶.

Importance and Applications of mRNA capping

In eukaryotes, 5'-cap prevents premature mRNA degradation by exonucleases while recruiting the eukaryotic translation initiation factor eIF4E for efficient translation⁷. The advanced cap-1 and cap-2 structures play a significant role in mammalian development⁸. Furthermore, the mRNA cap differentiates between endogenous and foreign mRNA, prompting innate immune response against the latter. The threat of immunogenicity highlights the need for appropriate capping of IVT mRNA for therapeutic applications.

The clinical significance of mRNA capping was brought to light with the advent of COVID-19 vaccines, where the key challenge was to retain the integrity of the mRNA fragment. Many SARS-CoV-2 candidates used capping methods to overcome the challenge. Post-transcriptional capping with VCE was employed for the Moderna vaccine mRNA-1273⁹. In the Pfizer-BioNTech vaccines for SARS-CoV-2, the mRNA was co-transcriptionally capped with a trinucleotide analog. It was reported that the protocol used in the vaccine development resulted in 94% capping efficiency¹⁰.

Advancements in mRNA capping have implications for mRNA vaccines and therapeutics for many other conditions, from cancer to cardiovascular diseases. In a January 2025 study, a therapeutic mRNA harboring a post-transcriptionally-added cap-1 analog was developed for treating heart failure, exhibiting increased stability, half-life, and efficacy in mouse models¹¹.

Methods for Detecting mRNA Capping

Detection of capping is crucial in mRNA characterization during IVT workflows. Main detection methods include:

• LC-MS/MS - It can help identify and quantify Cap 0, Cap 1, and Cap 2 structures.
• Cap-Specific Enzymatic Digestion & Gel Analysis - It can be used to differentiate between capped and uncapped RNA.
• Thin-layer chromatography (TLC) - The presence of different cap analogs can be confirmed by radiolabeling nucleotides on the cap.
• ELISA - Cap-specific antibodies can help quantify cap-0 and cap-1.
• RT-qPCR - 5' cap efficiency is quantified through reverse transcriptase (RT) efficiency, as capped 5' ends are necessary for RT priming.

References

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3. Hata T, Nakagawa I, Shimotohno K, Miura K-i. The synthesis of α, γ-dinucleoside triphosphates. The confronted nucleotide structure found at the 5′-terminus of eukaryote messenger ribonucleic acid. Chem Lett 1976;5(9):987-990.
4. STEPINSKI J, WADDELL C, STOLARSKI R, DARZYNKIEWICZ E, RHOADS RE. Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl (3′-O-methyl) GpppG and 7-methyl (3′-deoxy) GpppG. RNA 2001;7(10):1486-1495.
5. Henderson JM, Ujita A, Hill E, Yousif‐Rosales S, Smith C, Ko N, et al. Cap 1 messenger RNA synthesis with co‐transcriptional cleancap® analog by in vitro transcription. Current protocols 2021;1(2):e39.
6. Drazkowska K, Tomecki R, Warminski M, Baran N, Cysewski D, Depaix A, et al. 2′-O-Methylation of the second transcribed nucleotide within the mRNA 5′ cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion. Nucleic Acids Res 2022;50(16):9051-9071.
7. Gentry RC, Ide NA, Comunale VM, Hartwick EW, Kinz-Thompson CD, Gonzalez RL. The mechanism of mRNA cap recognition. Nature 2025;637(8046):736-743.
8. Dohnalkova M, Krasnykov K, Mendel M, Li L, Panasenko O, Fleury-Olela F, et al. Essential roles of RNA cap-proximal ribose methylation in mammalian embryonic development and fertility. Cell Rep 2023;42(7).
9. Corbett KS, Edwards DK, Leist SR, Abiona OM, Boyoglu-Barnum S, Gillespie RA, et al. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature 2020;586(7830):567-571.
10. Sahin U, Muik A, Derhovanessian E, Vogler I, Kranz LM, Vormehr M, et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020;586(7830):594-599.
11. Guo Y, Sun T, Li M, Chen Z, Liu Y, Luo X, et al. Revolutionizing Heart Failure Therapy: Harnessing IVT mRNA and Fusion Protein Technology to Prolong rhBNP Half-Life. Pharm Res 2025:1-13.

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