Updated : Friday, 31 January, 2025 @ 9:10 AM CT
Just as enzymes are the workhorses of our cells, they continue to play a vital role on a much larger scale in manufacturing mRNA therapeutics. In vitro transcription has already enabled society to vaccinate the world against COVID-19 and there’s much more yet to come.
There are dozens of clinical trials testing mRNA vaccines in diseases from acute gastroenteritis to Zika. Delivering these new therapeutics on a mass scale requires specialist manufacturing processes far removed from the chemical synthesis of small molecule drugs.
In vitro transcription recreates the processes happening inside each of us just by using a few key enzymes. Then further specialist enzymes can also help with characterising and purifying the reaction products to ensure high quality. In this blog post we take a quick look at the steps to make this happen and the enzymes involved.
Figure 1. An overview of steps involved in mRNA IVT. Precise workflows may vary and some steps, like capping and tailing, may be completed together during transcription.
Manufacturing the mRNA
T7 RNA polymerase is the most commonly used enzyme for in vitro transcription. The NxGen T7 RNA Polymerase is a reliable and robust enzyme that recognises the T7 promoter and terminator sequences in the DNA template with high specificity. The transcription process takes only a few hours with a plentiful supply of nucleoside triphosphates.
Throughout the transcription process, it’s vital to prevent the mRNA product being degraded. An RNase inhibitor, like RiboGuard™ protects against several common RNases. Riboguard doesn’t interfere with the enzymes used to analyse the RNA and is less prone to oxidation than older RNase inhibitors.
A poly(A) tail is a vital element of mRNA that protects it from nuclease degradation and
promotes protein translation. If the tail isn’t already encoded in the DNA template, it can be added post-transcriptionally with poly(A) polymerase. The Poly(A) Polymerase Tailing Kit provides the enzyme and adenosine monophosphate residues to add a poly(A) tail to the 3′ end of any RNA.
The mRNA also requires a 5′ cap for effective translation in vivo. Although many developers use capping agents during transcription, it’s also possible to add the cap post-transcription using enzymes.
A variety of capping enzymes are available, which partially hydrolyse the triphosphate group, create a 5′-5′ linkage to a guanidine and methylate the guanidine at the N7 position. This can then be converted to the eukaryotic Cap-1 structure with a 2′‐O‐methyltransferase enzyme.
To learn about generating the DNA template and getting the best performance for transcription, download our in-depth ebook.
Purification and characterisation
After transcribing the mRNA and adding the necessary elements, it is essential to purify and characterise the reaction product. This helps to ensure that it meets clinical standards and matches the desired structure.
The DNA transcription template needs to be removed, which can be achieved with DNase I. This enzyme degrades both double and single-stranded DNA, leaving just short oligonucleotides and mononucleotides that can be easily separated with a column.
Uncapped RNA molecules can be removed from the mix using two enzymes together. RNA 5′ Polyphosphatase removes two phosphate groups at the 5′ end of uncapped RNA transcripts, leaving one remaining 5′ phosphate group. This leaves them unprotected against degradation by Terminator™ 5′-Phosphate-Dependent Exonuclease, a 5′→3′ exonuclease.
To characterise the purified mRNA product, it can be broken down into fragments and assessed using LC-MS. One approach for this is to use RNase H, which degrades RNA in a DNA:RNA hybrid. Developers can design a cleavage probe that hybridises with the mRNA at the required cleavage site to generate the desired fragment. Hybridase is a high-performance RNase H that maintains activity as high as 95 °C and will not digest free RNA or DNA.
Another important enzyme is RNase A, which degrades single-stranded RNA at C and U residues. This digests most of the mRNA, leaving the poly-A tail ready for characterisation.
To make it easier to analyse mRNA fragments with LC-MS, T4 Polynucleotide Kinase (PNK) can standardise the phosphate groups at the termini. It dephosphorylates 3′ ends and introduces a monophosphate group to 5′ hydroxyl groups.
Our ebook ‘Enzymes for mRNA in vitro transcription’ contains full details about using enzymes for purification and characterisation.
Circular RNA
A similar process is necessary for manufacturing circular RNA molecules. However, there is no need for capping and tailing – instead enzymes can be used for ligating the 5'-phosphate and 3'-hydroxyl ends. For example, T4 DNA ligase can join two RNA ends and circularise mRNA. T4 
DNA ligase only acts on double-stranded regions, so a complementary oligo is needed as a splint to bring the ends together in a double helix at the ligation site.
However, there are many other circularisation methods, which are further detailed in our ebook.
After circularisation, the precursor linear RNA can be removed using RNase R, a 3′ to 5′ exoribonuclease that digests most linear RNAs. RNase R leaves the circRNA untouched, helping to purify the product and avoid potential safety risks from impurities.
Choosing the right enzymes
Partnering with preferred suppliers that guarantee the products are produced in facilities certified to ISO-13485 quality system standards can ensure that enzymes for IVT are high-quality and reliable. Potential consequences for using sub-optimal raw materials include:
Enhanced risk of workflow delays from lot-to-lot variation in enzyme performance
- Additional purification increases production costs
- Potential for nonconformance and supplier qualification issues during regulatory review by Competent Authorities
- Difficulty scaling production for commercialisation
- Wasted time and resources on repeat work due to variability in raw material performance
When developing and scaling IVT manufacturing, it might become important to customise the formulation of IVT enzymes. Popular enzyme customisations for IVT mRNA synthesis include:
- Different concentrations
- Triton free or glycerol free
- Custom QC based on your requirements
- Batch reservation
- Lot retention
- Custom storage or reaction buffers
- Bulk dispense
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For more information about enzymes for mRNA in vitro transcription, check out our ebook full of tips and insights to optimise your enzyme performance. |
