men In Chinese mythology, the cunning fox with nine tails symbolizes peace and prosperity. Xiao Wang, a chemical biologist at the Massachusetts Institute of Technology, took some scientific inspiration from the multi-tailed fox. Wang and her team were interested in synthesizing different types of RNA, usually in a linear form with a single polyA tail, which is important for protein production. This tail is the limiting step in mRNA degradation. “We wondered why we couldn’t create multiple tails.” said the king. “So if we lose one, we can have a lot of other things.”
mRNA has received much attention since its introduction. modified RNA Vaccines during the COVID-19 pandemic.One However, modified RNA is unstable and inefficiently converted to protein, requiring delivery of high doses that could be toxic to patients when used therapeutically. To address these issues, Wang and her team developed mRNA with multiple polyA tails instead of just one. Recent publications Nature Biotechnology, Scientists discovered that these branched mRNAs last much longer and are translated more efficiently, paving the way for the next generation of mRNA vaccines and treatments.2
Wang and her team first stabilized the polyA tail by adding a few modifications to prevent it from being chewed up by nucleases. To add multiple polyA tails to the mRNA backbone and create a branched structure, the scientists used click chemistry, a series of reactions used to join two molecular entities. Additional modifications prevented the tails from sticking together.
Wang and her team found that the three polyA tails seemed to be the sweet spot. Too many tails made it very difficult to purify branched mRNA using high-performance liquid chromatography. “You can imagine that if there was a constant success rate in ligating one of the polyA tails, the yield would decrease exponentially as the speed increases,” she said.
Next, the scientists attached fluorescent reporters to the mRNA and expressed them in cultured cells to measure how long the branched mRNAs persisted inside the cells and whether ribosomes translated them. By quantifying the number of mRNA transcripts inside the cells and bound to ribosomes, the team found that the branched mRNAs were translated about 1.5 times more efficiently than the linear mRNA control.
These results were replicated inside a mouse model, where scientists injected branched mRNA containing a fluorescent reporter (or a linear mRNA control) into the mice’s eyes. They found that the branched mRNA produced a fluorescent signal three to five times that of the control about two days after injection, and that this strong signal persisted over time, indicating that the branched mRNA was not degraded or translated into protein.
To demonstrate the utility of branched mRNA in a therapeutic sense, Wang and her team decided to improve the CRISPR-Cas9 editing system using branched mRNA. “In the genome editing environment, proteins are more of a limiting factor, so having an extra polyA tail to produce more Cas9 protein would be helpful,” Wang said. When the team tested this in a mouse model, they found that branched mRNA led to a fourfold increase in Cas9 protein expression compared to linear controls, and significantly lower levels of the target protein.
According to , these results are interesting. Alan Jacobsonis a biologist at the Chan School of Medicine at the University of Massachusetts and was not involved in this study. “If you’re vaccinating, it helps because you can do smaller doses,” he said. “And if you’re concerned about side effects, it’s about reducing those side effects.” He is eager to see how scientists overcome the challenges of mass production in the future, given that making branched mRNA requires a lengthy purification process.
In the meantime, Wang and her team are pushing the boundaries of mRNA modification, which they hope could one day help create better vaccines or therapies. “We’re curious about finding different structures and modifications that cells can tolerate,” she said. “It’s fascinating to see how the translation machine responds to mRNA that it’s never seen before.”
references
- Delaunay S, Helm M, Frei M. RNA modifications in physiology and disease: toward clinical applications.. National Pastor Genet. 2024;25(2):104-122.
- Chen H, Liu D, Guo J, et al. Branched chemically modified poly(A) tails enhance the translational capacity of mRNA.. Nat Biotechnology. 2024: 1-10.
