Beyond the messenger RNA (mRNA) molecules that famously powered the vaccines that helped curb the spread of COVID-19, the human body carries all sorts of RNA that could be used in the molecular therapeutics of the future. For example, group I introns act as molecular scissors that snip and loop themselves into tiny circles, similar to cutting a piece of string and tying its ends together. These circular RNA (circRNA) can serve as templates for protein production.
“CircRNA has higher stability and more durable expression in humans, making them very promising for developing RNA vaccines and therapeutics,” said Yue Wan, a Principal Investigator at the A*STAR Genome Institute of Singapore (A*STAR GIS).
However, looping doesn’t always come easy for these molecules, with different structures and even energy requirements dampening their circularisation efficiency. In addition, the potential to trigger unwanted immune responses poses an important safety consideration for using circRNA.
To tackle these challenges and optimise the formation of circRNA, Wan and first author, Kuo Chieh Liao, teamed up with colleagues from the A*STAR Bioprocessing Technology Institute (A*STAR BTI), A*STAR Institute of Molecular and Cell Biology (A*STAR IMCB), A*STAR Bioinformatics Institute (A*STAR BII), A*STAR Skin Research Labs (A*STAR SRL), National University of Singapore and Duke-NUS Medical School, Singapore.
By adding eight different group I introns to buffers of varying salt levels and temperatures, the researchers determined the best conditions for stimulating the molecules to form tiny circles efficiently. They found that two group I introns traditionally used to make circRNA had a high circularisation efficiency of above 80 percent but also identified four other promising group I introns.
Wan and the team then went on to characterise the immune reactivity of these six group I introns by measuring the expression levels of an immune signalling protein, interferon-beta (IFNβ), and an antiviral protein called IFIT1. With an understanding of the structural interactions that influenced circularisation efficiency, they then performed molecular snipping on these group I introns to create novel hybrid RNAs bearing modified sequences designed to enhance performance.
“Our findings reveal additional possibilities about how RNA sequences can be designed and modified to improve circularisation efficiency and to reduce unwanted immune responses in the human body,” said Liao, a Senior Scientist at A*STAR GIS. For example, the 2-thio-CTP chemical modification led to an increase in protein production.
Upon generating circRNA using two of the low IFNβ-inducing group I introns combined with sequences for the SARS-CoV-2 viral spike protein, the researchers found that these could be effectively used to mount an immune response against COVID-19.
Looking ahead, the team hopes to improve the stability of their circRNA to produce more desired therapeutic proteins. “Cost-effective manufacturing of high-performing circRNA drugs is the key to bringing treatments to patients and making a real-world impact,” said Wan.
The A*STAR-affiliated researchers contributing to this research are from the A*STAR Genome Institute of Singapore (A*STAR GIS), A*STAR Bioprocessing Technology Institute (A*STAR BTI), A*STAR Institute of Molecular and Cell Biology (A*STAR IMCB), A*STAR Bioinformatics Institute (A*STAR BII), and A*STAR Skin Research Labs (A*STAR SRL).
