Running in Circles? A Faster Way to Analyze Circular RNAs

Circular RNA (circRNA) is a covalently closed loop of single stranded RNA. Recently, the use of circRNA molecules in clinical settings has gained significant traction. This has driven the need to produce synthetic circRNA and identify straightforward and scalable analytical techniques to assess the efficiencies of circularization reactions. While standard agarose gel electrophoresis is a staple molecular biology method granting the ability to quickly visualize results, it fails to differentiate circRNAs from linear forms of RNA without additional sample preparation. Intriguingly, the Invitrogen E-Gel EX Agarose Gels were found to be uniquely capable of differentiating circRNA from linear RNA with minimal sample prep, helping save time and cost while simplifying workflows for circRNA analysis.

Circular RNAs (circRNAs) have become a topic of intense research to better understand their function, role in diseases, and potential therapeutic value. These molecules consist of single stranded RNA in a covalently closed loop structure.[1] The first circRNAs were isolated from viruses in 1976.[2] Later, in 1991, Nigro et al. were the first ones to discover circRNA production via non-canonical splicing of a deleted gene associated with colon cancer.[3] However, further research was not pursued as circRNAs were presumed to be rare and functionally inert byproducts of atypical splicing (Figure 1).[3] It was only with the advent of RNAseq that the true scope and importance of circRNAs started to emerge. 

Further studies provided a clearer understanding of both the prevalence and functional role of circRNAs in molecular and cellular biology. They are created through alternative splicing of pre-mRNA molecules and exhibit a significantly longer half-life, exceeding 48 hours.[4] This enhanced durability of circRNAs arises from the absence of free 5’ or 3’ ends, present in linear forms, which are subject to exonuclease-mediated degradation.[5] Additionally, the sequence of origin determines the location of circRNA with intron-derived being found in the nucleus and exon-derived being found in the cytoplasm (Figure 1).[3] Their potential role in important cellular processes is emphasized by demonstrated conservation in mammalian genomes (Figure 1). 

In humans, circRNAs were found to be expressed in thousands of genes and sometimes exhibit even higher expression levels than their linear mRNA counterparts.[4] This finding indicates that circRNAs are not a rare occurrence but are highly abundant molecules playing an important role in cell homeostasis.[4] Possible functions of circRNAs are being explored with existing studies suggesting their importance in regulation of protein expression, regulation of mRNA transcription, serving as scaffolds in protein biogenesis, and serving as sponges for proteins and microRNA (miRNA).[6–8] Given their suggested involvement in diverse cellular processes (Figure 1), it is no surprise that circRNAs were shown to contribute to cancer, neurological disorders, and diabetes.[8]

The long half-life of circRNAs in vivo is fueling interest as an avenue to enhance the potency of RNA-based therapeutics. Synthetic circRNAs, produced in vitro, were demonstrated to be translatable when an internal ribosomal entry site (IRES) was engineered into the sequence and used in vaccine development.[4] Also, they have shown potential as miRNA sponges as well as in other therapeutic applications.[9] While there is immense potential in circRNAs, our understanding of them is in its infancy with many challenges still present.[7]

While circRNA have a potential role in disease pathogenesis, they are also promising candidates in novel therapeutics.

Given the potential role that circRNAs play in disease, and their inherent advantages from a therapeutics perspective, there is a growing need for methods of generating circRNA and for conducting subsequent quality control. Currently, synthetic circRNAs are manufactured from a template plasmid DNA via an in vitro transcription (IVT) and subsequently circularized by enzymatic or self-catalytic reactions. Method success is determined by assessing the resulting RNA sequence length and characteristics, including what fraction of the product is circular versus linear.[10]

Determining the degree of circularization is an important quality control step in this process. This can be achieved through size exclusion high-performance liquid chromatography (SEC-HPLC) or agarose gel electrophoresis. Each method has advantages and disadvantages, with SEC-HPLC being highly accurate yet time consuming and costly. On the other hand, electrophoresis is a familiar and simple technique, but requires and additional step of “nicking” the circularized molecule which then produces one (if circularized) or two (if linear) products.Agarose gel electrophoresis represents an attractive method for circRNA analysis given its workflow simplicity, lower costs, and times savings advantages over SEC-HPLC. However, a standard agarose gel cannot differentiate between circular and linear forms of the same RNA molecule without the additional enzymatic step. [10]

The Invitrogen E-Gel EX Agarose Gels, run on the Invitrogen Power Snap Plus Electrophoresis System, are the only gel electrophoresis system demonstrated to be able to differentiate between linear and circular forms of RNA with minimal sample prep and no need for enzymatic “nicking.”[5,10] In E-Gel EX agarose gels circRNA was demonstrated to migrate more slowly than linear forms. Furthermore, the usefulness of a E-Gels to differentiate circRNA from linear RNA has been called out in other published reports of circRNA electrophoresis patterns.[11,12] 

When paired with E-Gel Power Snap Plus Electrophoresis Systems, E-Gel EX gels offer facilitated workflows and convenient flexibility for circRNA analysis, including: 

• Fast resolving time—Complete separation, with minimal prep, possible in just 10 minutes. 
• Convenient options—Precast agarose gels  available in 1%, 2%, and 4%  agarose concentrations to accommodate variety of small and large fragment analysis and throughput capacities.
• Ease of analysis—Enhanced band intensity analysis capabilities through use of iBright Analysis Software (available to run on local PC or cloud).

Circular RNAs, once thought to be inert byproducts of RNA splicing, have emerged as having roles in diverse cellular functions as well as an attractive tool for both RNA-based vaccines and therapeutic applications.As a result, there are now multiple development efforts to help improve the workflows for manufacturing and analyzing circRNAs.

The E-Gel EX Agarose Gels, combined with the E-Gel Power Snap Plus Electrophoresis System, are the only electrophoresis system currently capable of differentiating between circular and linear RNAs in their un-processed forms.This system can facilitate your circRNA workflows with minimal sample preparation and rapid run times, all while offering flexibility and convenience of precast gels with multiple agarose concentrations and formats. You can stop running in circles with Invitrogen solutions that can help provide rapid and straightforward circRNA analysis options. 

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