How is selection determined?

Specifically, Batish wanted to know whether there is a special screening process for what RNA cargo can get into the exosomes. Do they have to have a ticket, or can anyone nearby get a free ride? Is it a special pass, like a “golden ticket,” or will any ticket do? 

Some in the field believe the process is passive, whatever is around will get packaged, Batish said.

“But that doesn't fit well when we see very specific cargo being packaged in certain diseases,” she said. “For example, sometimes there are some RNAs that are not highly expressed in the cell, but they're highly packaged into the EVs. The cell is making something to send out, not to use.”

Experimental and computational studies agree

In the study, the research team isolated and characterized EVs from cells, then sequenced the RNA found inside to determine what was highly enriched into the exosomes as compared to what was found inside the cell itself. The researchers found the most abundant type of RNA in the exosomes is circular RNA.

At first, the research team thought maybe the circular RNA had a special sequence that is recognized by proteins and provides a zip code, of sorts, for where it gets delivered, but they didn't find anything out of the ordinary to explain the enrichment of these selected RNAs in EVs. Next, they considered size, but found that alone cannot explain the enrichment, so the team turned to RNA structure — and started to piece the puzzle together.

“With advances in structural biology of RNA, we now know that RNA has a complex shape. So, we hypothesized that it is because the circularity gives a unique structure to the RNA that it can be packaged into exosomes,” Batish said.

The researchers used bioinformatics techniques to calculate the “structuredness” of linear RNA compared to circular RNA and they found that circular RNA are far less structured than their linear RNA counterparts. They confirmed this experimental finding computationally, by switching the linear and circular RNA characteristics on a computer model and running the structure test again. The computer recognized the linear RNA, which Batish had coded as circular, as less structured, simply because of the way she identified it. 

Additional proof came from test results showing that the RNA appeared in the exosomes only when expressed in a circular form, not when expressed in a linear form. According to Batish, this verified that just making an RNA sequence circular increased its likelihood to be packaged into the exosome, even when all other factors are the same. 

“So, if you have a piece of RNA that you want to deliver as a therapeutic, make it circular to reduce its structure and increase the likelihood of it being packaged into the exosome. Then it will be stable, more likely to stay, and more efficient to package. That's one translational application of our findings,” Batish said.

Further work is needed, but Batish said the finding has the potential to help researchers engineer effective RNA-based therapeutics outside the cell and deliver them at will. 

“If we can deliver RNA therapeutics using exosomes, we will have better outcomes in terms of efficacy of the treatment,” Batish said. “Now that we know the RNA parameters, it may give us more levers to turn. Next steps include teasing out what proteins help carry RNA cargo into these exosomes and exploring whether we can manipulate and change those keys and locks to improve therapeutic efficiency.”