Enzymes for mRNA Production

Steps of In Vitro mRNA Synthesis

Figure 1. Creative Enzymes offers a full range of enzymes for in vitro mRNA synthesis.

The synthesis of mRNA in vitro involves a multi-step process, each requiring specific enzymes to ensure high yield and quality of the final product. Below are the essential steps, along with the roles of the required enzymes.

1. Plasmid Linearization

The first step in mRNA synthesis involves the linearization of the plasmid DNA template. This is crucial because linear DNA templates are more efficient for in vitro transcription compared to their circular counterparts.

Enzymes Used:

• BsaI, BspQI, XbaI: These are restriction enzymes used to linearize the plasmid DNA. By cutting the plasmid at specific sequences, they create linear templates that serve as efficient starting points for in vitro transcription.

2. In Vitro Transcription

Once the plasmid is linearized, the next step is the transcription of DNA into mRNA. This is achieved using RNA polymerase, which synthesizes RNA from the DNA template.

Enzymes Used:

• T7 RNA Polymerase: This enzyme catalyzes the synthesis of RNA from a DNA template, incorporating the appropriate nucleotides to form mRNA. Creative Enzymes provides both GMP-grade T7 RNA Polymerase and Premium T7 RNA Polymerase to ensure high transcription efficiency and fidelity.
• Inorganic Pyrophosphatase: This enzyme removes inorganic pyrophosphate produced during the synthesis of RNA, driving the reaction forward and increasing the yield of mRNA.
• RNase Inhibitor from Mouse: RNA molecules are susceptible to degradation by ribonucleases (RNases). Murine RNase Inhibitor binds to and inhibits RNases, protecting the synthesized mRNA from degradation.
• DNase I: This enzyme is used to remove the DNA template after transcription, ensuring that the final mRNA product is free of DNA contaminants.

3. Capping and Polyadenylation

To make the mRNA functional and stable, it undergoes post-transcriptional modifications, such as capping and polyadenylation. The 5' cap and 3' poly(A) tail are essential for mRNA stability and translation efficiency.

Enzymes Used:

• Vaccinia Capping Enzyme: This enzyme adds a 5' cap to the mRNA. The cap structure protects mRNA from degradation and is recognized by the cellular machinery for translation.
• mRNA Cap 2'-O-Methyltransferase: This enzyme further modifies the 5' cap by adding a methyl group at the 2'-O position of the ribose sugar. This modification enhances the stability and translational efficiency of the mRNA.
• E.coli Poly(A) Polymerase: This enzyme adds a poly(A) tail to the 3' end of the mRNA. The poly(A) tail improves mRNA stability and aids in its export from the nucleus to the cytoplasm.

Role of Creative Enzymes in mRNA Synthesis

Creative Enzymes is committed to providing high-quality enzyme products and services, essential for the successful in vitro synthesis of mRNA. With a comprehensive product line that includes GMP and non-GMP enzymes, Creative Enzymes supports the efficient and reliable production of mRNA vaccines.

Advantages of Creative Enzymes

• High-Quality Products: Creative Enzymes offers enzymes that meet rigorous quality standards, ensuring high purity, activity, and reliability.
• Comprehensive Product Range: The availability of a wide range of enzymes, including T7 RNA Polymerase, restriction enzymes, and modifying enzymes, allows for streamlined mRNA synthesis workflows.
• GMP-Grade Enzymes: For applications requiring the highest quality standards, Creative Enzymes provides GMP-grade enzymes, which are crucial for clinical and commercial mRNA vaccine production.
• Expert Technical Support: With a team of highly trained scientists, Creative Enzymes offers expert technical support to help customers optimize their mRNA synthesis processes.

Conclusion

The in vitro synthesis of mRNA is a complex process involving multiple steps and specialized enzymes. The enzymes provided by Creative Enzymes are critical for each stage of this process, from plasmid linearization to mRNA capping and polyadenylation. By offering high-quality enzymes and expert support, Creative Enzymes plays a vital role in advancing the production of mRNA vaccines, contributing to global health solutions.