The human body consists of over 30 trillion cells and over 200 cell types. Within each cell type are different subgroups that reflect the cell's age, activity and other behaviors. Bioengineers have designed synthetic devices that can purify cell types, but the purification of subgroups has proven more challenging. These devices for the most part operate through binary decisions. However, the resolution of subgroups requires numerical operations. A new report in Science Advances describes programmable RNA devices made by CiRA Professor Hirohide Saito and colleagues with this additional capability.

The technology is based on Saito's microRNA (miRNA) switch, or what he calls "miRNA-responsive messenger RNAs (mRNAs)". His research team had previously demonstrated how the switch can be used to purify different cell types made from stem cells. However, Kei Endo, a scientist at the University of Tokyo who coauthored the new study, says the miRNA switch has an important limitation.

"Some cells may show moderate miRNA activity and others very high activity. Even though the activity is different, the cells are considered equal in a logical calculation," he explains.

These different activities could have significant effects for cell therapies.

"We know that transplanting mature cardiomyocytes may have a better therapeutic effect than transplanting immature cardiomyocytes," notes Saito. However, while scientists have designed synthetic devices that can purify cardiomyocytes, none can purify for a specific maturity.

The programmable RNA devices are synthetic RNA molecules that code for a fluorescent protein and include five slots. Each slot codes an RNA sequence that binds to a specific miRNA. Scientists have identified over 2000 types of miRNA, the expression levels of which correlate with the cell type and cell activity.

The miRNA switch also consists of an RNA sequence that codes for a fluorescent protein. When injected into a cell, the switch turns on or off the translation of the fluorescent protein depending on the miRNA expression in the cell. Here, the fluorescence intensity is akin to turning on a light switch; the light is either on or off, with nothing in between.

The programmable RNA devices follow the same strategy but with the added feature of numerical operations. This allows the fluorescence intensity to have a gradation. The binding of a miRNA to one of the slots reduces the fluorescence by a defined factor. The binding of a second miRNA diminishes the fluorescence more by a multiplicative factor. And so on. Further, the value of these factors changes depending on the position of the slot in the RNA.

"The more slots, the more miRNA information," explains Endo. "But the longer the mRNA, the less efficient is the protein synthesis." In the study, Endo tested the device with five slots.

The additivity and tunability allowed the researchers to distinguish not only different cell types from a mixed population, but also stem cells at different days of differentiation. Indeed, inserting 4 RNA devices into a heterogenous cell population resolved five different cell types and differentiating stem cells on five different days.

"We could recapitulate miRNA activity profiles of living cells using numerical operations to distinguish cell states. This will enable the identification and isolation of cells of interest with more complex criteria for medical applications such as cell therapy or drug screenings," says Saito.

• doi: 10.1126/sciadv.aax0835