Circular RNA

This week I came across a new biotech company (Orna Therapeutics) that is focusing on circular RNA (circRNA) for mRNA therapies and read their 2018 research paper. Circular RNA connects the 3’ and 5’ ends. Circular RNA came up when we were discussing the immune response to mRNA and has been shown as a promising method for evading immune response.

I began thinking about circular RNA when we were working on the Spike protein puzzle, and I commented in a town hall that SARS-CoV-2 is linear. I was wondering if our designs needed to be linear in order to function well or if they could be circular, especially since most of the designs submitted were circular. The OpenVaccine lab instructions recommended that the UTRs not bond with any other portion of the sequence and keeping the first 14 nucleotides unpaired, which suggests to me that the researchers were envisioning linear designs as desirable. (A side note, why is it so difficult to create large linear designs in our folding engines and does that point to a potential flaw that can be addressed?) Subsequent literature review discovered that the ends and first 14 do not need to be unbound to translate efficiently. And in fact, this paper provides evidence that circular mRNA can be more stable and thus produce protein at higher rates than linear mRNA. The circular RNA in this study had twice the half-life as linear RNA and produced 4x protein, but it also involved lab techniques that boosted results, so not exactly an apples to apples comparison. (BTW, the researchers used Gaussia luciferase (Glac) not Nanoluciferase.)

There are several additional findings in the paper for improving translation that might be relevant for future mRNA labs.

What about mRNA designs that take a more complex path, as Amy Barish put it? Is there a more specific term for this beyond circRNA? I’ve not seen such designs addressed in research and would like to read such research, if anyone has come across it.

A separate issue mentioned in this paper that I’ve been wondering about is the role played by secondary structure. The researchers note, “Permissive spacers were designed to conserve secondary structures present within intron sequences that may be important for ribozyme activity…”, and point to the structures in Figure 2. Secondary structure was not mentioned as playing a role in our Nluc experiments. Maybe secondary structure does play a role in Nluc function? Could that explain the sub-par translation of my Round 6 design?


The circular RNA they’ve designed have a few important features necessary for circularization. The first is that the 5’ and 3’ ends are two halves of an intron that are needed to chemically circularize the RNA using its ribozyme activity. The second is that IRES sequences/structures are embedded in the RNA to allow ribosomes to bind to the circular RNA and produce protein. So secondary structure is an important feature in the constructs they’ve designed,

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