Messenger RNA (mRNA) production, particularly in vitro synthesis, is a cornerstone of modern biotechnology, playing a central role in applications ranging from vaccine development to protein replacement therapies. The process involves the concerted action of multiple enzymes that ensure efficient, high-fidelity synthesis, maturation, and purification of mRNA. This article provides a comprehensive overview of the essential enzymes utilized during in vitro mRNA production, detailing their specific functions and relevance at each stage of the workflow.

Creative Enzymes offers enzymes for mRNA production that are critical for each stage of this process, from plasmid linearization to mRNA capping and polyadenylation.

Overview of mRNA Production

mRNA serves as the intermediary between genetic information encoded in DNA and the synthesis of proteins. In vitro transcription (IVT) of mRNA has emerged as a critical platform for therapeutic development. The generation of functional and translationally competent mRNA requires not only the synthesis of the RNA strand but also post-transcriptional modifications such as 5' capping, 3' polyadenylation, and rigorous purification. These steps are mediated by a set of highly specialized enzymes, each with distinct mechanistic roles that contribute to the integrity and translational efficiency of the final mRNA product.

Figure 1. (a) Overview of essential structural elements of in vitro transcribed (IVT) mRNA. (b) Initiation of cap-dependent translation. The initiation of cap-dependent translation is started by binding the eukaryotic translation initiation factor 4E (eIF4E) to the 5' cap. The eIF4E bound to the 5' cap interacts with a variety of translation initiation factors, and the poly A binding protein (PABP) is also incorporated into the translation initiation complex, circularizing the mRNA. During this initiation process, the 40s ribosome is recruited to the initiation complex, subsequently scanning start codon for the initiation of translation. In addition, the 40s ribosome can also be recruited into the IRES located in the 5' UTR, initiating cap-independent translation. (Kwon et al., 2018)

DNA Template Preparation in Vitro

Before transcription begins, a high-quality linear DNA template containing the gene of interest under the control of a promoter (usually T7, SP6, or T3) is needed. Enzymes involved at this stage include:

Restriction Endonucleases

Restriction enzymes like EcoRI, BamHI, and BsaI are commonly used to linearize plasmid DNA. Linearization is crucial to prevent read-through transcription and ensure defined mRNA ends.

DNA Polymerases

High-fidelity DNA polymerases such as Phusion or Q5 are employed during PCR amplification to create or amplify the transcription template. These enzymes offer proofreading activity to reduce mutations that could compromise the mRNA's function or stability.

Enzymatic Components of the mRNA Production Process in Vitro

RNA Polymerase: Origin and Regulation

In vitro systems predominantly utilize bacteriophage-derived RNA polymerases such as T7, SP6, or T3 RNA polymerase. These enzymes are single-subunit, promoter-specific, and highly processive. They do not require accessory proteins or chromatin remodeling and operate under defined buffer conditions, enabling rapid and efficient transcription from plasmid or linear DNA templates containing the appropriate phage promoter.

5' Capping Mechanisms

In vitro capping can be achieved either:

• Post-transcriptionally, using vaccinia virus capping enzyme and 2'-O-methyltransferase.
• Co-transcriptionally, via incorporation of synthetic cap analogs such as Anti-Reverse Cap Analog (ARCA) directly into the transcription reaction.

These methods are designed to mimic natural capping structures and reduce innate immune activation, although they lack the regulatory context of the endogenous capping complex.

3' Polyadenylation

In vitro, polyadenylation is accomplished either:

• Enzymatically using E. coli or yeast poly(A) polymerase, which adds adenine residues to the 3' end without a template.
• Template-encoded, by incorporating a poly(A) tail sequence directly into the DNA template used for transcription.

The enzymatic approach offers more control over tail length but is not regulated by RNA-binding proteins or cleavage sites as in vivo.

Figure 2. Basic workflow for mRNA synthesis. The in vitro synthesis of mRNA starts with the preparation of the DNA template containing the gene of interest (depicted in orange), which can be linearized plasmid DNA, a PCR product, or a cDNA template. These DNA templates will be used for the in vitro transcription of mRNA using an RNA polymerase, followed by mRNA capping at the 5' untranslated region, addition of a poly(A) tail at the 3' untranslated region (optional in cases were a poly(A) is included in the DNA template), and purification of the final mRNA. UTR, untranslated regions. (Campillo-Davo et al., 2021)

RNA Processing Enzymes and Complexes

In vitro mRNA production bypasses nuclear RNA processing pathways. Introns are typically omitted from synthetic mRNA constructs, and splicing is unnecessary. No RNA editing or nuclear export machinery is involved. Instead, chemical modifications and optimized UTRs (untranslated regions) are incorporated during design to enhance translational efficiency and stability in the cytoplasm.

Nuclease and Contaminant Management

To prevent degradation during in vitro synthesis, RNase inhibitors are added throughout the reaction and purification processes. Additionally, DNase I is used to degrade the DNA template post-transcription, ensuring a pure mRNA preparation devoid of template DNA contamination.

Summary of Enzymatic Roles

Recommendation Products

Creative Enzymes offers a comprehensive range of high-quality enzymes for research & diagnostic use, including enzymes for mRNA production. By providing high quality enzymes and expert support, Creative Enzymes plays a vital role in advancing the production of mRNA vaccines and contributing to global health solutions. Contact us for any questions or requests!