Circular RNA (circRNA), a covalently closed single-stranded RNA molecule, has rapidly gained traction in the research and discovery setting. Initially considered a result of erroneous RNA splicing since the mid-1970’s, circRNAs have subsequently demonstrated a role in a wide array of biological functions. These functions include transcriptional/translational regulators, microRNA sponges, protein scaffolds, and encoding small proteins. More recently, circRNA emerged as a potential candidate for several therapeutic applications, including vaccine development and diagnostic tools.
Key Benefits and Functions of circRNA
In eukaryotic systems, linear mRNA transcripts are typically generated through several processing and maturation steps, including 5’ capping, splicing to remove non-coding regions, and 3’ polyadenylation. These steps are essential for creating a stable transcript that is correctly transported out of the cell nucleus and translated into a functional protein.
However, the maturation process of circRNAs, is vastly different. Endogenously, circRNA is generated via back-splicing, where the 3’end of an exon ligates to the 5’end of its own or an upstream exon through a 3’, 5’-phosphodiester bond (Figure 1)5.
Through back-splicing, the steps of capping and polyadenylation are eliminated. Despite lacking these features, circRNA boasts a longer half-life than its linear counterpart, owing to the absence of free ends available for exoribonuclease degradation.
The longer half-life is also often attributed to the reduced immunogenic response that circRNAs stimulate within the host. Linear mRNAs are naturally found to possess several modified nucleosides, with one of the most abundant being pseudouridine. Nucleoside modification is commonly described as a mechanism to improve stability and reduce immunogenicity of the mRNA transcript. In contrast, the same nucleoside modifications are less important for circRNA and may, in fact, be detrimental in circularization efficiency1.
In many cases, endogenous circRNAs display cell- and tissue-specific expression. With over 10% of human circRNAs showing tissue specificity, it has been suggested there may be a key function of circRNAs in the development and differentiation of tissues/organs2, although the mechanisms behind this are still largely unexplored.
CircRNAs for Biomedical Applications
Aside from the documented endogenous functions, circRNAs have been implicated in a growing number of pathophysiologies, including cardiovascular and neurological disorders, and cancers. One example is circ-MYBL2, a variant of the MYBL2 transcription factor, evident to be upregulated in acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase-3-Internal tandem duplication (FLT3-ITD) mutations, with selective knockdown of circ-MYBL2 impairing the proliferation and progression of carcinogenic cells4. Similarly, circ-MYBL2 has been observed to be upregulated and promote disease progression in cervical cancer patients3. Together, these and other studies demonstrate the benefit of circRNAs as both a novel therapeutic target and as a valuable tool for diagnostic purposes.
Alongside their implication in pathophysiologies, mRNAs have also gained significant traction in the field of immunology. With the successful use of mRNA in the COVID-19 vaccine, multiple mRNA vaccines are currently in development targeting influenza, respiratory syncytial virus (RSV) and cytomegalovirus (CMV). It is important to note that these are all based on the canonical linear mRNA design. While circRNA vaccines are still in their infancy, they offer a promising tool in adaptive immunity. CircRNA, with their higher stability and longer half-life, has the potential to express antigens over a more extended period, resulting in an improved antibody response.
The mechanisms involved in both native circRNA function and metabolism are still largely unclear, consequently limiting current applications in both diagnostic and therapeutic settings. Our scientists are dedicated to advancing circRNA research and helping the scientific field address these knowledge gaps. We recognize that the production of high-quality research grade circRNA starts with an optimized and robust IVT workflow.
circRNA Synthesis Best Practices
Best practices for circRNA synthesis have evolved over the past several years. Scientists have exploited the abovementioned features of circRNA in vitro for several years; initially synthesizing circRNA using chemical ligation and more commonly now using enzymatic ligation and permuted intron-exon (PIE) strategies. Additionally, the inclusion of internal ribosomal entry site (IRES) to facilitate ribosomal binding and protein translation in a cap-independent manner is a feature frequently included in circRNA design to promote expression in the host system.
Aside from the design features and synthesis strategy, a key consideration when generating circRNA is to ensure the sample is a homogenous mix of circRNA products, without any linear RNA populations (Figure 2). It is critical to eradicate linear RNA in the sample if the primary focus is the functionality of the circRNA (Figure 3). Our team of experts verifies the RNA sample across multiple steps: first, treating it with RNAseR to remove any linear populations, secondly, performing an RNAseR tolerance test to verify only a circular population, and lastly, using RT-PCR and sequencing across the circularized junction to confirm homogeneity of circular products.
We also incorporate other important practices to ensure alignment with your downstream needs:
- Delivery in lyophilized format ensures stability during transit and provides the flexibility to reconstitute in a buffer suitable for your downstream applications.
- Multiple yield and aliquoting options providing flexibility in your downstream project design and enabling you to avoid freeze-thaw cycles of circRNA, which can dramatically impact the sample integrity.
- Retention of a small aliquot of the starting template plasmid free of charge, allowing for convenient repeat IVT to generate more circRNA in future projects if needed.
- Flexible design options to accommodate various needs. Have a GOI but not sure where to start? Need circRNA without IRES? We can adjust designs based on your experimental requirements.
- Rigorous quality control (QC) is paramount. All circRNA samples are delivered with a comprehensive data package including all QC parameters tested by our expert production team.
Conclusion
While circRNA research has increased significantly over the past decade, there remains a significant knowledge gap in its endogenous function and use in a therapeutic setting. To help you explore the function of circRNA in your experimental applications, we have developed a first-to-market IVT circRNA service. Connect with us today to discuss how we can help you achieve the goals of your circRNA project!
References
1. Wesselhoeft et al., 2019. Mol Cell. 74(3): 508-520. doi:10.1016/j.molcel.2019.02.015
2. Xia et al., 2017. Briefings in Bioinformatics. 18(6):984-992. doi: 10.1093/bib/bbw081
3. Wang et al., 2019 OncoTargets and Therapy. 12: 9957-9964. doi: 10.2147/OTT.S218976
4. Sun et al., 2019. Blood. 134(18):1533-1546. doi: 10.1182/blood.2019000802
5. Yu, CY., Kuo, HC. The emerging roles and functions of circular RNAs and their generation. J Biomed Sci 26, 29 (2019). https://doi.org/10.1186/s12929-019-0523-z