The advent of mRNA-based vaccines and therapeutics has revolutionized modern medicine, as exemplified by the rapid development of COVID-19 vaccines. Unlike traditional biologics, mRNA offers a flexible and rapid production platform capable of encoding virtually any protein of interest. However, manufacturing high-quality mRNA requires stringent control over each step of the production pipeline to ensure efficacy, stability, and safety.

At Creative Enzymes, we provide various enzymes for mRNA production including T7 RNA polymerase, RNase inhibitor, DNase I, vaccinia capping enzyme, and more. Here, we systematically introduce the mRNA production process, emphasizing critical parameters that influence yield, purity, and functionality.

Figure 1. The process of in vitro transcription of mRNA made from a DNA plasmid template that has been linearized. (Knezevic et al., 2021)

DNA Template Preparation

Efficient in vitro transcription (IVT) for synthetic mRNA production begins with the meticulous preparation of a DNA template, most commonly in the form of an engineered plasmid. This template serves as the blueprint for transcription and directly influences the fidelity, stability, and translational efficiency of the resultant mRNA product.

Plasmid Design and Engineering

The plasmid used as a DNA template is a synthetic construct designed to ensure optimal transcription and downstream translational activity. A typical mRNA-encoding plasmid consists of the following core elements:

• Promoter sequence: This is the binding site for a DNA-dependent RNA polymerase, typically derived from bacteriophages such as T7, SP6, or T3. The promoter drives high-yield in vitro transcription and is often positioned immediately upstream of the 5' untranslated region (UTR).
• 5' Untranslated Region (5' UTR): Engineered for optimal ribosomal recruitment, the 5' UTR enhances translation efficiency. Key features include Kozak consensus sequences and minimal secondary structure to facilitate ribosome scanning.
• Open Reading Frame (ORF): This is the coding sequence for the protein of interest. Codon optimization, tailored to the target host's translational machinery, is employed to improve translation efficiency and reduce potential immunogenicity.
• 3' Untranslated Region (3' UTR): The 3' UTR contains sequence elements that regulate mRNA stability and translational control. Common stability motifs include those derived from human β-globin or α-globin genes.
• Polyadenylation signal and poly(A) tail: Either encoded in the plasmid or added post-transcriptionally, a stretch of 70–120 adenosine residues at the 3' end enhances mRNA stability and translational efficacy by mimicking natural eukaryotic transcripts.

Key Design Considerations:

• Codon Optimization: Substituting rare codons with synonymous codons preferred in the target expression system enhances translation without altering the amino acid sequence.
• Avoidance of Immunogenic Motifs: The presence of unmethylated CpG islands, poly(U) sequences, or other pathogen-associated molecular patterns (PAMPs) can stimulate innate immune responses. These sequences are minimized or eliminated during sequence engineering.

Plasmid Propagation and Purification

Following design, the engineered plasmid must be propagated and purified at scale for use in transcription reactions.

• Bacterial Amplification: The plasmid is introduced into a high-copy strain of Escherichia coli (e.g., DH5α) via transformation. Cultures are grown in the presence of selective antibiotics (ampicillin, kanamycin, etc.) to ensure plasmid retention.
• Plasmid Purification: Bacterial cultures are harvested, and plasmids are extracted using alkaline lysis methods followed by purification steps. Commercial silica column-based kits are widely used. For research requiring ultra-pure DNA (e.g., for therapeutic-grade mRNA), cesium chloride (CsCl) gradient ultracentrifugation may be employed.

Quality Control Measures:

• Agarose Gel Electrophoresis: Confirms the integrity of the plasmid and the predominance of the supercoiled form, which is most transcriptionally competent.
• Spectrophotometry (A260/A280 and A260/A230 ratios): Assesses DNA concentration and purity. Acceptable purity ratios are generally ~1.8 for A260/A280 and >2.0 for A260/A230.

Linearization of Plasmid DNA

Plasmid DNA must be linearized prior to IVT to prevent readthrough transcription and yield uniform mRNA transcripts.

• Restriction Enzyme Digestion: A unique restriction site downstream of the poly(A) tail is targeted using enzymes such as BspQI, ApaI, or NotI. The choice of enzyme depends on plasmid design and desired end configuration.
• End Characteristics: To prevent undesirable transcriptional artifacts, enzymes that produce blunt ends or 5' overhangs are preferred over those generating 3' overhangs.
• Template Purification: After digestion, enzymes and small fragments are removed using phenol-chloroform extraction followed by ethanol precipitation or column-based purification (e.g., silica spin columns). The final template should be highly pure, RNase-free, and free from undigested plasmid.

In Vitro Transcription (IVT)

The IVT process synthesizes mRNA in vitro by copying a linear DNA template using phage-derived RNA polymerases. High-yield, high-fidelity mRNA synthesis is critical for applications ranging from functional protein expression to therapeutic RNA manufacturing.

Reaction Components

A standard IVT reaction includes:

• RNA Polymerase: T7, SP6, or T3 RNA polymerases are used based on the corresponding promoter in the template. T7 polymerase is most common due to its high transcriptional efficiency.
• Nucleotide Triphosphates (NTPs): Equimolar concentrations of ATP, GTP, CTP, and UTP, typically in the range of 4–8 mM, provide the building blocks for RNA synthesis.
• Reaction Buffer: Contains essential cofactors such as magnesium ions (Mg²⁺), dithiothreitol (DTT) to maintain redox balance, and spermidine to stabilize nucleic acids.
• RNase Inhibitors: RNase Inhibitors are added to protect nascent RNA from degradation during the transcription process.

Figure 2. Saccharomyces cerevisiae RNAP II CTD. (Pal, 2020)

Optimizing IVT Conditions

• NTP Concentration: Excessively high NTP concentrations can lead to premature termination or truncated transcripts, while insufficient levels may limit yield. A balanced concentration (~4–8 mM) is optimal.
• Magnesium Ion Concentration: Mg²⁺ is crucial for polymerase activity and fidelity. Optimal concentrations range between 20–30 mM, and may require titration based on template and scale.
• Incubation Parameters: Standard IVT reactions are conducted at 37°C for 2–4 hours. Prolonged incubation may increase yield but also elevates the risk of RNA degradation or impurity formation.

Co-Transcriptional Modifications

To improve the translational performance and reduce immunogenicity of synthetic mRNA, several modifications can be incorporated directly during transcription.

5' Capping Strategies:

• Cap 0 (m⁷GpppG): The simplest form of eukaryotic mRNA cap. Often added in molar excess to GTP to ensure effective incorporation.
• ARCA (Anti-Reverse Cap Analog): Prevents reverse cap incorporation and ensures correct 5'-end orientation, enhancing ribosome recognition.

Nucleotide Modifications:

• Pseudouridine (Ψ) and N1-methylpseudouridine (m¹Ψ): These analogs reduce innate immune recognition (e.g., by TLR7/8) and enhance translational efficiency.
• 5-Methylcytidine (m⁵C): Improves mRNA stability by resisting nuclease degradation and contributes to translation optimization.
• Other modifications (e.g., 2-thiouridine, N6-methyladenosine) are being explored for therapeutic mRNA engineering to improve pharmacokinetics and reduce inflammatory signaling.

Post-Transcriptional Processing

Following in vitro transcription (IVT), the resulting RNA must undergo a series of post-transcriptional modifications and purification steps to yield a mature, stable, and translation-competent messenger RNA (mRNA). These processes ensure that the synthetic transcript mimics natural eukaryotic mRNA in both structure and function, thereby maximizing its efficacy in downstream applications such as protein expression and mRNA-based therapeutics.

Enzymatic Capping (If Not Performed Co-Transcriptionally)

While co-transcriptional capping strategies or the use of Anti-Reverse Cap Analogs (ARCAs) are increasingly preferred, enzymatic capping remains a robust alternative, particularly for research-grade and therapeutic-grade mRNA production where precise control over capping efficiency and fidelity is required.

• Vaccinia Capping Enzyme (VCE): This multifunctional enzyme complex catalyzes the sequential enzymatic addition of a 5' cap structure known as Cap 0 (m⁷GpppN), where "m⁷G" denotes a 7-methylguanosine linked via a 5'–5' triphosphate bridge to the first transcribed nucleotide. VCE activity involves RNA triphosphatase, guanylyltransferase, and (guanine-N7)-methyltransferase functions. The cap plays a critical role in transcript stability, efficient translation initiation, and resistance to exonucleolytic degradation.
• 2'-O-Methyltransferase: Converts Cap 0 to Cap 1 (m⁷GpppNm) by methylating the ribose 2'-hydroxyl group of the first transcribed nucleotide. Cap 1 structures are recognized as "self" by host immune systems, thereby significantly reducing innate immune responses mediated by Toll-like receptors (TLRs), RIG-I, and MDA5.

The enzymatic capping method allows near-quantitative conversion of uncapped RNA to the fully capped Cap 1 structure, especially useful for sensitive applications such as vaccine development and gene replacement therapies.

Figure 3. Sequential enzymatic activities leading to RNA 5'-end cap. (Pal, 2020)

Poly(A) Tailing

In the absence of an encoded polyadenylation tract within the plasmid DNA, the 3' poly(A) tail must be enzymatically added post-transcriptionally.

• Poly(A) Polymerase (PAP): Catalyzes the template-independent addition of adenosine monophosphates to the 3' hydroxyl terminus of the RNA transcript. This enzymatic reaction typically utilizes ATP as a substrate and is carried out in optimized buffer systems to ensure homogeneity and appropriate tail length.
• Optimal Poly(A) Tail Length: Empirical studies suggest that an approximately 100–150 nucleotide poly(A) tail offers the best compromise between transcript stability, translational efficiency, and resistance to cytoplasmic degradation. Over-elongation can affect transcript folding and translation, and shorter tails can compromise stability.

Polyadenylation is a critical determinant of mRNA half-life and recruitment to the ribosomal complex in eukaryotic cells.

DNase Treatment

Residual DNA templates from IVT reactions must be rigorously removed to comply with regulatory guidelines, prevent innate immune activation, and eliminate background in functional assays.

• Turbo DNase (Recombinant RNase-free DNase I): Efficiently hydrolyzes single- and double-stranded DNA under conditions that preserve RNA integrity. This treatment is typically performed post-IVT and post-polyadenylation, followed by heat inactivation or purification to remove the enzyme.

DNase treatment is essential not only for therapeutic applications but also for analytical workflows such as RT-qPCR, where DNA contamination can confound quantification.

mRNA Purification

Purification is arguably the most critical step in the production of therapeutic-grade mRNA. This stage aims to remove impurities such as proteins, residual DNA, truncated transcripts, nucleotides, enzymes, and endotoxins. A variety of purification strategies are employed, often in combination, to achieve the required quality and regulatory standards.

Precipitation Methods

Precipitation remains a widely used primary purification method due to its simplicity, scalability, and cost-effectiveness.

• Lithium Chloride (LiCl) Precipitation: LiCl selectively precipitates long RNA species while leaving behind most proteins, short RNA fragments, free nucleotides, and salts in the supernatant. The standard protocol involves incubation at 4°C followed by high-speed centrifugation.
• Ethanol Precipitation: Typically used in conjunction with salts (e.g., sodium acetate), ethanol precipitation efficiently recovers RNA from dilute solutions. This step is especially useful for buffer exchange and desalting prior to enzymatic processing or chromatographic purification.

Although precipitation does not provide high-resolution separation, it is often integrated into multi-step purification protocols to reduce bulk impurities.

Chromatographic Purification

Chromatography-based techniques offer superior resolution and specificity for the isolation of full-length, high-purity mRNA transcripts.

• Oligo(dT) Affinity Chromatography: Exploits the affinity between poly(A) tails and oligo(dT)-conjugated resins (e.g., sepharose or magnetic beads). This approach selectively captures polyadenylated transcripts and removes truncated RNAs and non-polyadenylated contaminants. It is particularly effective for purifying mRNA from total IVT mixtures.
• Size-Exclusion Chromatography (SEC): Separates molecules based on hydrodynamic radius. SEC can effectively remove short transcripts, degraded RNA, and unincorporated nucleotides while preserving the native conformation of full-length mRNA.
• Ion-Exchange Chromatography (IEX): Separates nucleic acids based on net charge. Anion-exchange resins (e.g., DEAE or Q-sepharose) are commonly used under low-salt conditions. Fine-tuning of salt gradients allows separation of capped vs. uncapped mRNA, as well as full-length vs. truncated products.

Chromatographic techniques are especially useful when producing clinical-grade mRNA requiring high batch-to-batch consistency.

High-Performance Liquid Chromatography (HPLC)

HPLC is a powerful analytical and preparative tool in mRNA purification, enabling separation of mRNA isoforms, impurities, and structurally similar species.

• Reverse-Phase HPLC (RP-HPLC): Based on hydrophobic interactions between RNA and the column matrix. Typically uses a C18 column and ion-pairing reagents (e.g., triethylammonium acetate) to enhance resolution. RP-HPLC excels at resolving modified and unmodified mRNA, truncated species, and residual enzymatic proteins.
• Hydrophobic Interaction Chromatography (HIC): Useful for distinguishing capped versus uncapped RNA based on differences in hydrophobicity. HIC is often employed as a quality control tool or as a secondary purification step to enhance final purity.

These methods are valuable in quality assurance (QA) settings and are frequently integrated into good manufacturing practice (GMP) pipelines for mRNA-based therapeutics.

Filtration-Based Methods

Tangential Flow Filtration (TFF) is A scalable and efficient method for buffer exchange, RNA concentration, and impurity removal. TFF systems utilize hollow fiber or flat-sheet membranes with defined molecular weight cutoffs to retain RNA while allowing the passage of small molecules and salts.

TFF is frequently used in both upstream and downstream processing stages, particularly in large-scale mRNA production. It is compatible with continuous manufacturing strategies and is well-suited for high-throughput operations.

Figure 4. Summary of production of functional mRNA. (Papaioannou et al., 2023)

The mRNA production pipeline is a meticulously controlled process encompassing DNA template preparation, IVT, capping, purification, and rigorous quality control. Advances in enzymatic modifications, chromatographic purification, and analytical techniques have significantly enhanced mRNA yield, stability, and translational efficiency. As regulatory frameworks evolve and novel technologies emerge, mRNA manufacturing will continue to improve, enabling broader therapeutic applications.

At Creative Enzymes, we provide the high-performance enzymes required at every step of the mRNA production pipeline—including plasmid linearization, in vitro transcription, capping, and polyadenylation. Contact us today with your questions and inquiries!