Successful mRNA therapeutic design: optimizing for stability and translation | eBlog

mRNA therapeutics have unlocked the potential for rapid responses to infectious disease, personalized treatments for cancer, and protein replacement for different diseases. To ensure efficacy, specific features need to be included in the therapeutic to promote stability, efficient translation, and to minimize endogenous regulation. In a traditional mRNA therapeutic, such as the SARS-CoV2 vaccine, the structure mirrors mRNAs found in the body. There is a cap on the 5’ end, followed by an untranslated region (UTR), the sequence encoding the therapeutic protein, a 3’ UTR, and ending with a polyA tail (figure 1A)1. Newer approaches are changing this paradigm however, with growing interest in self-amplifying (figure 1B) and circularized (figure 1C) RNAs.

In this blog we review some of the key features that go into designing and optimizing a successful mRNA therapeutic that is stable and produces appropriate levels of protein product for vaccination and disease treatments.

Schematic showing the typical structures of mRNA therapeutics

Figure 1: Three design strategies for mRNA therapeutics. A. A traditional linear construct, such as the SARS-CoV2 vaccine, designed to mimic a typical mRNA. It has a 5′ cap, a polyA tail, UTRs, and a coding sequence for the therapeutic protein. B. A self-amplifying RNA which includes nsP1-4, proteins from alphavirus, for increased translation of the therapeutic protein. C. A circular construct that includes a site for ribosome binding (IRES) and the coding sequence.

5′ caps and 3′ tails are essential for stability and translation

Linear mRNA therapeutic with cap and tail highlighted

Both the 5’ cap and 3’ tail are involved in promoting translation and maintaining stability to maximize protein production. The 5’ cap is designed to mimic endogenous 5’ mRNA capping to avoid recognition by the innate immune system and limit degradation, while the tail is typically a stretch of over 100 adenines to limit degradation by exonucleases1.2. The combination of both features allows for the recruitment of the ribosome complex for translation and the generation of the required protein for therapeutic efficacy2.

To improve stability, several artificial cap analogs have been developed that can limit degradation while mimicking natural 5’ cap behaviors2. Tails are often added enzymatically which can produce variable lengths or are directly included in the DNA template to achieve a fixed length3. With 5’ caps it is critical to measure the capping efficiency to ensure that the therapeutic will remain stable and can be translated, although there are limitations with current analytical methods4. Tail addition also needs to be evaluated to ensure therapeutic efficacy, and current methods typically require specialized equipment and expertise5.

Proper UTR selection is critical for therapeutic success

Linear mRNA with UTRs highlighted

UTRs are untranslated regions of an mRNA that play key roles in protein translation, stability, and localization within the cell1. The 5’ UTR (upstream of the coding sequence) is involved in mRNA stabilization and translation initiation at the start codon3,6. The 3’ UTR is associated with mRNA stability and endogenous regulation, such as by miRNAs3.

One approach for UTR selection is to identify UTRs from a highly expressed gene that has limited miRNA regulation. This approach was used with the Pfizer/BioNTech SARS-Cov-2 vaccine, the 5’ UTR is a modified version of the human α-globin gene and the 3’ UTR is a fusion between a mitochondrial UTR (mtRNR1) and the AES/TL5 gene that is predicted to have low miRNA binding6. However, the activity of a given UTR will depend on the cell type it is acting in and there is growing interest in designing UTRs to be more efficient and targeted3,7.

RNA structure is a critical determinant of translation initiation in the 5’ UTR, and recent work has shown that modifying the UTR sequence to adjust RNA structure can have significant impacts in the rate of protein production1,8. For example, deletion of a stem loop in the SARS-CoV-2 UTR can increase the ribosome load8. This result shows how measurements of RNA structure in the UTR are critical for therapeutic success.

Coding sequence optimization can improve therapeutic efficacy

Linear mRNA therapeutic with the coding sequence highlighted

Although a specific protein product is required, there are several optimizations that can be done to improve the efficacy of the therapeutic without altering the final protein product including adding modified bases, optimizing codons, and altering structure.

Most mRNA therapeutics incorporate modified bases, such as N1-methylpseudoridine, to limit innate immune responses1. These modifications can induce changes in the RNA structure which can affect stability and translation9, making it important to evaluate structure with the bases included.

Codon optimization is where a specific codon for a given amino acid is selected over other codons that encode the same amino acid to ensure that translation proceeds at an appropriate pace and to avoid frameshifting9. This optimization is performed by looking at the relative usage of the codon in the human genome, and then selecting codons that are used more often with an assumption that their higher usage indicates higher abundance of the paired tRNA9. A major limitation of this approach is that tRNA abundance can vary across cell types, which could lead to the selection of an inefficient codon where the therapeutic is acting9.

In addition to changing codons to optimize translation, codons can also be selected to guide specific RNA secondary structures9. Different structures are associated with different degrees of stability, and typically the more base pairing there is the more stable the RNA will be9. Research into how to best design a therapeutic’s structure is occurring at a rapid pace8,10, although there is still work needed to determine the effect of the cellular environment and RNA-binding proteins on the final structure that is formed after delivery.

Other design strategies can improve stability and translation

The above features are for a traditional, linear mRNA therapeutic as used with the approved vaccines. However, interest is growing in alternative approaches that can increase stability and/or improve translation.

Self-amplifying RNAs are RNAs that have a replication-competent vector (typically using nsP1-4 from alphavirus) that enables amplification of the therapeutic, making them useful for gene replacement therapies7,11. This type of approach is being used by Replicate Bioscience in their Rabies vaccine which is currently in Phase 1 trials12 and it was used in a clinically approved vaccine for SARS-CoV-2 in Japan in 202313.

Circular RNAs are a closed loop without a 5’ cap or a polyA tail, however by being a single loop they induce limited immunogenicity and are protected from degradation leading to an extended half-life compared to linear therapeutics7,14. To allow for translation, an internal ribosome entry site (IRES) needs to be included next to the coding sequence14. Although circularization has the potential to produce stable, long-lasting vaccines they are still in preclinical development15.


mRNA therapeutics have already led to rapid, global immunization against SARS-CoV-2 and are expected to continue to grow as a robust and effective vaccination platform in addition to treating cancers and other diseases. To have an effective therapeutic, it is critical to have a design that enables sustained expression and high protein production. At Eclipsebio, we have a portfolio of solutions that can be used to optimize and characterize mRNA therapeutics including eSHAPE for RNA structure determination and eRibo Pro for measurements of expression and translation. Contact us today to learn how we can help you reach the clinic faster and with more confidence.

  1. Chaudhary et al.
  2. Qin et al.
  3. Fang et al.
  4. Tu et al.
  5. Gilar et al.
  6. Xia
  7. Rohner et al.
  8. Leppek et al.
  9. Metkar et al.
  10. Zhang et al.
  11. Bloom et al.
  12. PR Newswire
  13. Dolgin
  14. Niu et al.
  15. Qi et al.

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