By Léo Soares
From wheels to molecules
Looking back at mankind’s history, we can see how the concept of circularization influenced the development of our society. Wheels, for example, are one of the most important first human inventions. Across the centuries, wheels (Fig.1) have been used in carriages, trains, cars, bikes, and buses to facilitate people’s lives. Yet the concept of circularity is also prevalent naturally in biology. Molecules such as RNA, proteins, and nucleotides are apparently more stable when circularized1–3. But what exactly does the circularization of molecules mean?
To answer those questions, circular RNA (circRNA) – a type of endogenous RNA- could serve as a model for the artificial circularization of messenger RNA (mRNA). This article will dive into the universe of these molecules and unravel nature’s tricks used for the artificial circularization of messenger RNA (mRNA) and its application in biotechnology and drug discovery.
Figure 1 (a)The wheel. (b) mRNA structure.
Image source: L. Soares
Why circularize molecules?
The circularization of molecules such as mRNA, proteins, or nucleotides has become an important focus in drug development and biotechnology due to its potential to increase stability1–3. Pharmaceutical companies invest millions of dollars searching for stable molecules with improved pharmacokinetics. A more stable protein, for example, could be used in a reaction at a higher temperature2. This process is beneficial for higher yield of biochemical reactions, highlighting its potential application in biotechnology.
mRNA is a molecule that serves as a template for protein synthesis and is made up of nucleotide blocks that hold two distinctive ends: known as the the 5’ and 3’ ends. From its synthesis to degradation, mRNA is always surrounded by proteins, especially nucleases, special enzymes4. One of these nucleases can degrade the mRNA, which limits its availability. However, there is a way to make the mRNA resistant to degradation by nucleases. By bringing the 5’ and 3’ ends together and forming a loop, the mRNA becomes more resistant to enzymatic degradation and increases its overall stability1. Likely, mRNA circularization impacts the interaction between nucleases and mRNA. There is evidence that naturally occurring circular molecules, such as circular RNA (Fig. 2)5, provide us with valuable insight into the benefits of artificial circularization of mRNA.
Circular RNA and its action
mRNA is a well-known type of RNA, but there is another type of RNA which is not so famous to researchers: the circular RNA. This molecule plays a crucial role in the regulation of gene expression in eukaryotes. Unlike mRNA, which is ‘’linear”, circular RNA has no typical 5’ and 3’ ends (Fig.5). This circular structure makes it incredibly stable and resistant to nucleases. Circular RNA also works as a sponge for micro-RNAs, which are small non-coding RNAs that can regulate up to 30% of genes that encodes proteins6. Circular RNA binds to micro-RNAs (Fig.2) preventing them from pairing with mRNA, by doing so, mRNA can be fully translated into a protein 6. This is how circular RNA can act as a powerful regulator of gene expression6. Scientists have taken inspiration from circular RNA and are now exploring ways to artificially circularize mRNA to enhance its stability, which could have exciting implications for drug development.
Figure 2. circular RNA, surrounuded by micro-RNAs. Image source: L. Soares.
Engineering artificially circularized mRNA
Artificial circularization of molecules such as nucleotides has been around for a long time. Several methods to achieve this, including enzymatic and chemical approaches,1,7 have been used to circularize molecules. However, when it comes to mRNA, which is a large molecule, bringing the 5’ and 3’ ends together is challenging1. In 2018, Wasselhoet et al. proposed a new method to circularize mRNA into a circular form with a significant protein expression: a “ribozimatic method’’1. This method uses RNA as an enzyme combined with genetic engineering to create what’s known as a phosphodiester bond between the two chemical (hydroxyl) groups at each of the 5’ and 3” ends (Fig. 4b bond in red)1. The protocols and technique details are rather complex to explain, but this was the first time that a mRNA had been artificially circularized.
Because the artificial circular mRNA is more stable than its linear counterpart, this new molecule displays superior pharmacokinetics than the original, that is, the new molecule would last longer to perform its functions in our bodies, because nucleases would not be able to degrade it.
Figure 3. (a) mRNA linear with the two ends depicted (5′ and 3 ends). (b) mRNA circularized, highlighting the phosphodiester bond (bond between two hydroxyl groups) in dark red.
Source: L. Soares, ChemDraw
Circularization’s potential for existing mRNA therapies
When we think about mRNA therapy, the first thing to come to mind is the recent creation of mRNA vaccines. The COVID-19 pandemic popularized mRNA therapy use in many of the vaccines, but its history already dates back to 1990 when Wollff et al.8 injected mRNA into mice muscle, producing the corresponding protein. There are several mRNA therapies currently in clinical trials where circularization could improve efficiency, one of which is used to treat cystic fibrosis (CF)9–12.
CF is a lifelong disease caused by a recessive gene mutation resulting in a cystic fibrosis transmembrane conductance regulator protein (CFTR) disfunction 13 causing among others, respiratory symptoms. The current mRNA therapy strategy is based on the substitution or replacement of the ineffective membrane protein CTFR12. The current mRNA therapy uses a linear molecule; however, developing and employing an artificially circular mRNA could benefit or enhance existing mRNA therapy strategies. Due to its stability, there is potential for the circularized mRNA to outlast its linear counterpart in the body. In treatment of chronic diseases such as CF, longer lasting substitution of CTFR protein could be very effective. In theory, fewer artificial circular mRNA would be needed to produce the same effect as the linear mRNA1
Figure 4. (a) Defective CTFR protein present in fibrosis cystic. (b) CTFR protein replaced after regular mRNA therapy. (c) CTFR protein replaced after circularized mRNA therapy.
Source: L. Soares, ChemDraw
Circularization brings the idea of unity and cohesion. It’s as if all the nucleotide blocks of mRNA have joined together, ready to fight the same fight. The simple step of bringing the two ends together impacts how certain enzymes recognize and interact with these molecules. Most important is to use a successful concept or natural phenomenon and apply it to another molecule to make it more stable. It’s a reminder that in science, we should always first observe stable structures and processes in nature for inspiration to our own medical research and applications. By doing so, we could apply this to other processes as our search for more stable biomolecules continues.
About the author:
Léo Soares is finalizing his master’s at VU Amsterdam. His main interests include synthetic organic chemistry, chemical biology, and immunology. He is also really captivated by RNA biology, peptide synthesis, protein 3D structure stabilization, and its application in medicine and industry
Further reading:
1. Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018).
2. Pelay-Gimeno, M., Bange, T., Hennig, S. & Grossmann, T. N. In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme. Angew. Chem. Int. Ed. 57, 11164–11170 (2018).
3. Liu, M. et al. An Efficient, Site‐Selective and Spontaneous Peptide Macrocyclisation During in vitro Translation. Chem. – Eur. J. 29, e202203923 (2023).
4. Gupta, S. K., Haigh, B. J., Griffin, F. J. & Wheeler, T. T. The mammalian secreted RNases: Mechanisms of action in host defence. Innate Immun. 19, 86–97 (2013).
5. Bolha, L., Ravnik-Glavač, M. & Glavač, D. Circular RNAs: Biogenesis, Function, and a Role as Possible Cancer Biomarkers. Int. J. Genomics 2017, 1–19 (2017).
6. Circular RNAs: Biogenesis and Functions. vol. 1087 (Springer Singapore, 2018).
7. Petkovic, S. & Müller, S. RNA circularization strategies in vivo and in vitro. Nucleic Acids Res. 43, 2454–2465 (2015).
8. Zeng, C., Zhang, C., Walker, P. G. & Dong, Y. Formulation and Delivery Technologies for mRNA Vaccines. in mRNA Vaccines (eds. Yu, D. & Petsch, B.) vol. 440 71–110 (Springer International Publishing, 2020).
9. Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014).
10. Liang, Y., Huang, L. & Liu, T. Development and Delivery Systems of mRNA Vaccines. Front. Bioeng. Biotechnol. 9, 718753 (2021).
11. Sahu, I., Haque, A. K. M. A., Weidensee, B., Weinmann, P. & Kormann, M. S. D. Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases. Mol. Ther. 27, 803–823 (2019).
12. Qin, S. et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct. Target. Ther. 7, 166 (2022).
13. Hanssens, L. S., Duchateau, J. & Casimir, G. J. CFTR Protein: Not Just a Chloride Channel? Cells 10, 2844 (2021).
- Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). ↩︎