Nature sub-journal: A new strategy to optimize circular RNA translation levels

Circular RNAs (circRNAs) are a special class of non-coding RNA molecules with a closed circular structure that can express proteins
more efficiently and stably.
Recently, a team of scientists at Stanford University developed a systematic method to rapidly assemble and test the factors that influence circRNA protein production, and increased the yield of circRNA translation and expression proteins by hundreds of times
through optimized design.
The results were published in the journal Nature Biotechnology as "Engineering circular RNA for enhanced protein production.
"

During mRNA translation, the m7G cap recruits eukaryotic initiation factor 4E (eIF4E), which works synergistically with eIF4A and eIF4G to support the recruitment of other initiating factors and ribosomes
.
In contrast, since circular RNAs are end-to-end covalently linked and lack 5' ends, they must rely on cap-independent mechanisms such as internal ribosome entry sites (IRESs) to initiate translation
.
While the ability of circular RNAs containing IRESs to encode proteins has long been known, the principles of circular RNA translation have not been thoroughly dissected
.

In this study, the research team fabricated and tested the synthesized circular RNA
by creating a modular, high-throughput platform.
To improve the protein translation efficiency of circRNA, the research team optimized 5 elements: vector topology, 5' and 3' UTRs, IRESs, and synthetic aptamer initiation translation mechanisms
.
In this study, we succeeded in increasing the protein yield of circRNA translation by a factor of 224 by an optimized combination of these elements
.
It has higher translation efficiency than linear mRNA in vitro, providing longer-lasting translations
in vivo.

The modular circular RNA cloning platform enables fast design-build-test cycling
.

The researchers generated circRNA variants
with different intra-frame interval lengths between translation initiation and splicing.
The peptides encoded by these spacers reflect common viral precursor peptide sequences
from the rhinovirus family.
Testing the expression of these circular RNAs showed that increasing the spacing length was detrimental to translation, and ribosomes were not affected
by td splicing secondary structures.

The researchers also demonstrated that recruiting eIF4G to IRES using aptamers can easily enhance the translation
of circRNA.
Proper localization of aptamers requires placing them near the translational initiation core of the IRES and minimizing disruption of the native RNA structure
.
Future optimizations could allow RNA aptamers to be adapted to other needs, such as enabling small molecules to control the translation of circRNAs or guiding circRNAs to specific intracellular targets
.
In addition, the incorporation of RNA aptamers may provide a pathway
for cell-type-specific expression of circular RNA.

In summary, this study increased the protein output of circRNA translation expression by hundreds of times through various optimizations, which confirmed that the engineered circRNA can have the same strength as linear mRNA in vivo, with more stable expression and longer
translation time.
This study provides new ideas and methods
for future industrial applications.

reference

Chen R, et al.
Engineering circular RNA for enhanced protein production.
 Nature biotechnology, 2022: 1-11.