Why polyA tail stability matters for mRNA therapeutic development? Current challenges in the plasmid DNA manufacturing process
An increasing number of mRNA-based therapeutics are now reaching the clinic, spanning applications in infectious diseases, oncology, and rare genetic disorders. Recent successes, such as the personalized base editor therapy used to treat Baby KJ or Moderna’s individualized neoantigen vaccine, demonstrated the growing clinical validation of mRNA-based therapies (1). Despite this clinical success, strategies to reduce the dosage of mRNA drug substance while prolonging protein expression to enhance potency and improve therapeutic outcomes remain an area of active research.
Most current approaches rely on stabilizing the polyadenylated (polyA) tail, which is a simple string of adenosines. Its length, structure and stability have a profound impact on mRNA quality, translational efficiency, and therapeutic performance (2). Although a longer polyA tail enhances stability and expression, encoding a long homopolymeric A sequence directly into the DNA template remains technically challenging, especially when using traditional plasmid DNA workflows.
The polyA tail: A minor sequence with major influence
PolyA tails are typically between 70 -200 nucleotides in length and are found on the 3’ end of nearly all eucaryotic mRNAs. The polyA tail of the mRNA binds to polyA-binding proteins (PABP) involved in translation and enhances stability of the mRNA (1). In many mRNA applications, longer and more stable polyA tails not only enhance transcript stability but also improve consistency, durability, and therapeutic performance.
During bacterial fermentation, these long homopolymeric regions are particularly prone to recombination and deletion. Recombination events can shorten the polyA tail, which can compromise the integrity of the template (3). This is often a concern in a GMP setting, where lack of consistency can ultimately lead to batch failures and repeats. By ensuring a stable polyA sequence with a pre-defined length, researchers can significantly improve the overall quality and predictability of mRNA-based therapeutics.
Challenges of encoding long polyA tails in plasmid DNA manufacturing
Selection of a strategy to incorporate the polyA tail is a critical design decision in mRNA manufacturing. Often it directly influences process robustness, scalability, and final product quality. Although encoding the polyA tail within the plasmid DNA template can simplify the overall workflow, this approach introduces distinct manufacturing challenges when long polyA sequences are required, as it is typically limited to ~120bp.
PolyA sequences are inherently unstable in plasmid DNA and are prone to homologous recombination during bacterial propagation. To mitigate this risk, researchers rely on enzymatic polyadenylation directly to the mRNA after transcription, antranscription, an approach primarily adopted to bypass plasmid instability rather than for process simplicity. A third, commonly used strategy is to encode the polyA tail within the DNA template but segment the polyA sequence to reduce recombination and preserve tail length; this approach has been successfully implemented in licensed mRNA.
While these strategies can enable production of the desired polyA tail length, the primary concern remains batch-to-batch consistency. Variability in polyA tail length can complicate GMP manufacturing, increasing the risk of batch failures and production delays. Inadequate control of polyA heterogeneity may adversely affect critical quality attributes (CQAs), and in turn, compromise quality target product profiles (QTPPs), raising regulatory concern. Poor robustness also carries significant cost and timeline implications for drug manufacturers. Batch failures or rejections resulting from unmet CQAs can lead to material waste, increased manufacturing costs, and delays in clinical or commercial supply.
Cell-free DNA manufacturing: A modern solution
Due to the importance of the polyA tail in mRNA stability, there are new methods emerging to overcome these challenges. Technologies such as cell-free DNA manufacturing address these concerns and improve process efficiency. As an alternative to pDNA, synthetic cell-free DNA is manufactured through a fully enzymatic process, meaning it doesn’t involve bacterial fermentation processes. 4basebio’s cell-free opDNA® template is ideally suited for mRNA applications due to its 3’ open-end which can feed directly into IVT processes without the need for linearization or de-ending. Importantly, this approach is already enabling clinical trials, demonstrating its practical applicability and readiness for therapeutic development.
What are the advantages of using synthetic DNA for your mRNA therapeutic programs:
No bacterial fermentation processes
4basebio’s enzymatic manufacturing process doesn’t require bacterial fermentation steps which are needed for pDNA manufacturing. This ensures that long homopolymeric regions remain intact and don’t undergo homologous recombination, as often seen with pDNA.
PolyA tail encoded in DNA template
Thanks to the unique enzymatic process, long continuous polyA sequences up to 180bp, can be directly encoded into the template. This ensures a homologous polyA tail and eliminates risks of batch failure and the need for enzymatic tailing step or breaking up the polyA tract.
Faster manufacturing process
Cell-free manufacturing processes allow for much faster DNA amplification compared to plasmid-based processes. With platforms like 4basebio, GMP-grade DNA templates can be delivered up to four times faster than traditional pDNA workflows.
No need for linearization
mRNA workflows that use pDNA require an additional step where the circular DNA needs to be linearized to input into an IVT reaction. When using opDNA® constructs this step is eliminated as the template is linear with a 3’ open end.
Why the field should move beyond plasmid DNA for mRNA manufacturing
As mRNA technologies advance toward larger clinical programs and commercial-scale production, the limitations of plasmid DNA are becoming more apparent. The need for stable polyA tails, rapid GMP timelines, and consistent control of critical quality attributes has become standard in mRNA therapeutic development.
In a GMP environment, fermentation-based DNA manufacturing is increasingly showing its limitations. Dependence on bacterial propagation, extended production timelines, and complex process optimization make it difficult to meet the demands of modern mRNA development. As programs accelerate toward the clinic, these challenges can slow progress and introduce unnecessary risk.
Cell-free DNA manufacturing addresses these challenges in a more fundamental way. Rather than offering a single process improvement, it realigns DNA template production with how mRNA therapies are now developed. Faster turnaround times, simplified processes, and improved sequence integrity transform DNA manufacturing from a bottleneck into a strategic advantage.
Moving beyond plasmid DNA is therefore not just an optimization of existing workflows, but a shift in manufacturing strategy. Cell-free approaches provide a stronger foundation for the precision, scalability, and regulatory rigor required for next-generation mRNA therapeutics.
Interested in finding out more about the use of synthetic DNA for mRNA production? Download our technical note and see how synthetic DNA performs against plasmid DNA.
References:
1. World’s first patient treated with personalized CRISPR gene editing therapy at Children’s Hospital of Philadelphia | Children’s Hospital of Philadelphia. Children’s Hospital of Philadelphia. (2025, May 15). https://www.chop.edu/news/worlds-first-patient-treated-personalized-crispr-gene-editing-therapy-childrens-hospital
2. Nikita Biziaev, Alexey Shuvalov, Ali Salman, Tatiana Egorova, Ekaterina Shuvalova, Elena Alkalaeva, The impact of mRNA poly(A) tail length on eukaryotic translation stages, Nucleic Acids Research, Volume 52, Issue 13, 22 July 2024, Pages 7792–7808, https://doi.org/10.1093/nar/gkae510
3. Trepotec Z, Geiger J, Plank C, Aneja MK, Rudolph C. Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA. 2019 Apr;25(4):507-518. doi: 10.1261/rna.069286.118. Epub 2019 Jan 15. PMID: 30647100; PMCID: PMC6426288