Unlocking the potential for circular RNA in molecular diagnostics

When circular RNAs were discovered back in the 1970s, they were initially thought to be largely rare, inconsequential curiosities. They seemed to serve no purpose and potentially only existed through aberrant splicing of mRNA. However, two ground-breaking papers published jointly in 2013 revealed that circRNAs could play a functional role in gene regulation.^1,2   

Since then, researchers have found that circRNAs may influence (or be influenced by) disease, creating exciting new opportunities for using these molecules as biomarkers in molecular diagnostic tests. 

Creation and functions of circRNAs

Circular RNAs are typically produced when precursor mRNAs are back-spliced so that a downstream exon is linked to an upstream exon, creating a closed-loop structure. In addition, there are also lariat RNAs formed by the introns as a product of normal splicing. These lariats have a loop structure created by a bond between the 5' end of the intron and a branch point within the intron. 

Circular RNAs have multiple mechanisms for regulating gene expression. They can act as microRNA sponges, binding to these small molecules and preventing them from reaching their target mRNAs.³ Additionally, circRNAs can interact directly with splicing factors to compete with linear transcripts and influence gene expression that way.⁴  

Although they were previously considered to be non-coding, some circRNAs may be translated into functional proteins, despite lacking a 5'-cap.^3,5 This phenomenon is being explored as a way of using synthetic circRNA as a therapeutic agent, potentially offering greater stability than linear mRNA approaches.

circRNA expression is associated with diseases

With the ability of circRNAs to regulate important processes in the cell, it’s unsurprising that dozens have been linked to a variety of diseases.⁶ 

One well-studied circRNA, called cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), contains many binding sites for miR-7.² This relationship appears to play a role in the response to a myocardial infarction (heart attack) and could be used as a diagnostic marker for heart failure.^7,8  

In other aspects of heart health, several circRNAs were found to predict the recurrence of atrial fibrillation after surgical treatment, and circNFAT5 improved predictions of survival after cardiac arrest.³  

Another area of extensive interest is the association between circRNAs and the risk of developing cancer. While some seem to increase the risk through removing miRNAs that are oncogene regulators, other circRNAs may act as tumour suppressors, offering opportunities for new forms of treatment.⁶ 

Research has also found that circRNA can trigger cancer by binding to DNA directly. This can affect transcription and the integrity of the DNA, leading to chromosomal translocations that cause acute leukaemia.⁹ 

Beyond cancer and cardiovascular disease, circRNAs have been proposed as biomarkers for conditions ranging from preeclampsia to pulmonary tuberculosis.^10,11 Researchers have also explored using circRNAs as biomarkers for kidney disease in blood and urine samples.⁶  

CircRNAs are a promising target as diagnostic and prognostic biomarkers. Their stability means they have significantly longer half-lives in blood compared to other RNAs.³ In addition, their tissue-specific expression potentially offers a clearer signal when looking for disease-related changes.³ They can also be extracted from body fluids, so testing could be easily incorporated into liquid biopsies. 

Interested in circular RNA research? Explore enzyme-based strategies to support your work in our in-depth RNA guide.

Techniques for studying circRNA

There is still a lot to learn about circRNA, given how recently it has become an area of interest. Over 100,000 circRNAs have been identified in humans and it’s estimated that circRNA variants have ten times as much diversity as linear RNAs.^9,12 

Research into circRNAs has been boosted significantly by the use of RNase R, a nuclease that breaks down linear RNAs and leaves circRNAs intact.¹³ This essential enzyme can enrich circRNAs in a sample, making them easier to analyse and characterise. This is important to overcome the low amounts of circRNAs present compared to other RNA species. 

RNase R is a magnesium-dependent 3ʹ to 5ʹ exoribonuclease. It requires low magnesium concentrations (0.1-1.0 mM) to be functionally active, which may need to be increased if EDTA is present in substrate RNA solutions.  

Certain linear RNAs can resist RNase R digestion and researchers have found adjustments to the protocol that can help overcome these. Replacing potassium with lithium in the buffer can destabilise G-quadruplex structures making them susceptible to RNase R.¹⁴ RNAs with highly structured 3ʹ ends that withstand RNase R can be polyadenylated using poly(A) polymerase and removed using oligo(dT) beads.¹²  

The RNase R manufactured by LGC Biosearch Technologies™ (formerly Epicentre and Lucigen) has been the go-to enzyme for circular RNA research and development ever since those pioneering studies in 2013 that showed the impact of circRNAs.^1,2,15-18 This high-quality enzyme is manufactured in ISO-13485 facilities and is available in custom formats to meet requirements. 

After using RNase R to isolate circular RNA molecules, the sample can be analysed. For high-throughput efforts, techniques like RNA-seq or microarrays can review the total circRNA population. To quantify and validate particular circRNA species of interest, RT-qPCR is widely used.^15,19 By comparing RT-qPCR results between samples treated with RNase R and control samples, users can readily confirm if the sequence being amplified is indeed a circRNA. 

In addition, primers can be designed to span the backsplice junction of the circRNA so that the PCR will not amplify any linear counterparts that survived digestion by RNase R. Because of the circular structure, the primers can be divergent, where they face away from each other on the linear equivalent but will successfully amplify the circRNA.¹⁵ 

Reaching clinical applications with circRNA assays

While circRNA shows considerable promise as a new source of valuable disease biomarkers, this field is still in its early stages. The many different circRNAs that are being identified through research will need to be verified in large-scale analyses of clinical samples.  

If successful, these novel biomarkers could offer more accurate diagnoses and prognoses for a wide range of diseases. The relative simplicity of amplifying circRNA from body fluid samples means that these molecular diagnostic assays could see widespread uptake. 

Five decades after its discovery, circRNA could one day soon make its mark in the clinic.

References

1. Memczak S et al. (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338 doi: 10.1038/nature11928  
2. Hansen TB et al. (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388 doi:10.1038/nature11993  
3. Hoque P et al. (2023) Exploring the Multifaceted Biologically Relevant Roles of circRNAs: From Regulation, Translation to Biomarkers. Cells 12(24):2813 doi: 10.3390/cells12242813  
4. Ashwal-Fluss R et al. (2014) circRNA Biogenesis Competes with Pre-mRNA Splicing. Molecular Cell 56(1):55-66 doi: 10.1016/j.molcel.2014.08.019  
5. Yang Y et al. (2017) Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res 27:626–641 doi: 10.1038/cr.2017.31  
6. Geng H-H et al. (2016) The Circular RNA Cdr1as Promotes Myocardial Infarction by Mediating the Regulation of miR-7a on Its Target Genes Expression. PLoS ONE 11:e0151753 doi: 10.1371/journal.pone.0151753
7. Chen C et al. (2020) The Circular RNA CDR1as Regulates the Proliferation and Apoptosis of Human Cardiomyocytes Through the miR-135a/HMOX1 and miR-135b/HMOX1 Axes. Genet. Test. Mol. Biomark. 24:537–548 doi: 10.1089/gtmb.2020.0034 
8. Conn VM et al. Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia. Cancer Cell 41(7):1309 - 1326.e10 doi: 10.1016/j.ccell.2023.05.002  
9. Zhuang ZG et al. (2017) The circular RNA of peripheral blood mononuclear cells: Hsa_circ_0005836 as a new diagnostic biomarker and therapeutic target of active pulmonary tuberculosis. Mol. Immunol. 90:264–272. doi: 10.1016/j.molimm.2017.08.008 
10. Pandey PR et al. (2019) RPAD (RNase R Treatment, Polyadenylation, and Poly(A)+ RNA Depletion) Method to Isolate Highly Pure Circular RNA. Methods 155:41–48. doi: 10.1016/j.ymeth.2018.10.022
11. Suzuki H et al. (2006) Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res 34(8):e63 doi: 10.1093/nar/gkl151 
12. Xiao MS and Wilusz JE (2019) An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends. Nucleic Acids Res 47(16):8755-8769 doi: 10.1093/nar/gkz576
13. Panda AC and Gorospe M. (2018) Detection and Analysis of Circular RNAs by RT-PCR. Bio-Protocol 8:e2775 doi: 10.21769/BioProtoc.2775
14. Ma N et al. (2020) Circular RNAs regulate its parental genes transcription in the AD mouse model using two methods of library construction. FASEB Journal 34(8):10342-10356 doi: 10.1096/fj.201903157R 
15. Jakobi T et al. (2016) Profiling and Validation of the Circular RNA Repertoire in Adult Murine Hearts. Genomics, Proteomics & Bioinformatics 14(4):216–223 doi: 10.1016/j.gpb.2016.02.003
16. Venø MT et al. (2015) Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development. Genome Biol 16, 245 doi: 10.1186/s13059-015-0801-3 
17. Vrommen M et al. (2021) Validation of Circular RNAs Using RT-qPCR After Effective Removal of Linear RNAs by Ribonuclease R. Current Protocols. 1(7):e181. doi: 10.1002/cpz1.1