Novel Biosensor Uses DNA ‘Barcodes’ to Uncover Disease Signals in Cells

Inside every cell, RNA acts as a vital messenger. Its levels rise when genes are active, while specific fragments can serve as clear warning signs of a viral infection.

Reading these cellular messages gives scientists a direct window into human health, but finding and measuring specific RNA strands inside a cell is often a cumbersome, multi-step process.

To simplify it, researchers at Gladstone Institutes have built a customizable biosensor called the “Detectron.” With mechanics borrowed from retrons, an ancient defense mechanism found in bacteria, the Detectron functions like a smart tripwire: the moment it senses a target RNA strand, it responds by churning out distinct, easy-to-read DNA “barcodes.”

“To build Detectrons, we combined the DNA-production machinery of retrons with a molecular switch that can be programmed to detect pretty much any RNA sequence,” says Gladstone Investigator Seth Shipman, PhD, senior author of the new study published in the journal Nature Biotechnology.  “It gives us a completely new lens for studying biology. And down the line, it could even be used to trigger targeted interventions or treatments in cells.”

Combining the Best of Two Worlds

Detectrons are the latest in a suite of retron-derived tools developed in Shipman’s lab. The tools repurpose retrons’ ability to produce small strands of DNA inside cells—a key step in their traditional role as antiviral agents in bacteria.

“We like retrons because they’re essentially easy-to-use, adaptable DNA barcode generators,” Shipman says. “Our tools work by ensuring the DNA is only made in response to something we choose.”

What sets Detectrons apart from prior retron tools is they have been designed to directly detect RNA itself, something that had not been accomplished until now.

To create Detectrons, Shipman’s group turned to a synthetic biology tool known as a toehold switch—a hairpin-shaped molecule that only opens up if it directly binds to the RNA sequence it is programmed to detect. Typically, opening the hairpin is like flipping on the switch because it unblocks production of a pre-specified color of fluorescent protein, alerting that the RNA was sensed.

“With standard toehold switches, you run out of different possible colors very quickly, so you can only look for a few different RNA sequences at once,” says Jihoon Han, PhD, a postdoctoral researcher in Shipman’s lab and lead author of the study. “In contrast, DNA barcodes can have any possible sequence, so you could theoretically have billions in play simultaneously, each reporting on a unique RNA sequence.”

The researchers set out to combine the best of both worlds: the direct-detection capabilities of a toehold switch with the versatility of retrons’ DNA barcodes—and the Detectron was born.

A Detectron’s molecular switch starts out in the “off” position. Only if an RNA fragment with a particular targeted sequence binds to it, the switch will turn on and prompt the Detectron to begin pumping out small pieces of DNA. The DNA fragments have a known, preprogrammed sequence, which acts as a barcode that can be read back by standard sequencing, revealing the presence of the target RNA.

“We can combine different Detectrons in the same cell at once, each programmed to detect a unique RNA sequence and make a corresponding barcode,” Han says. “That means we can look for multiple RNA targets at the same time, which opens up so many opportunities for discovery.”

Speeding Disease Research with Detectrons

To demonstrate how Detectrons could be applied in disease research, Shipman and Han chose to focus on phage therapy—the use of bacteria-attacking viruses, or phages, to treat bacterial infections.

Phage therapy holds promise as a potential weapon against bacteria that have become resistant to conventional antibiotics, and scientists around the world are studying which types of phages are best suited for it. But typically, narrowing down promising phages for a therapy takes a very long time, as each type must be tested one by one in a dish to see how well it kills a given bacterial strain.

To streamline this process, Gladstone’s team designed several different Detectrons, each programmed to detect RNA from a different phage of interest. They inserted each Detectron into a separate population of E. coli bacteria, and mixed the Detectron-carrying bacteria together in a single tube. The mixture was then exposed to one phage type at a time to determine whether the corresponding DNA barcode was produced.

“We found that, by looking at which DNA barcodes were produced in higher amounts, we could identify which phage had successfully infected the bacteria,” Han says. “This and similar experiments show how Detectrons could be used to help quickly narrow down the right phage for attacking an antibiotic-resistant superbug.”

But potential applications for Detectrons extend far beyond this example.

“When gene editing is used to treat disease, there may be cases where the gene-editing machinery is delivered to unintended cells that should not be edited,” Han says. “With Detectrons, the RNA-triggered switch could give us an extra layer of control, which we could theoretically use to ensure the editing machinery only turns on inside intended cells, while staying off in all other cells.”

Detectrons for All

Shipman’s group is now exploring how to best design Detectrons for use in human cells, which are very different molecular environments from bacterial cells. They are also investigating how to enable Detectrons to reliably sense much longer RNA fragments—up to hundreds of times longer—than the fragments in this study.

All of the lab’s Detectron schematics and programming code are available for use by other laboratories.
“We’re excited to make our own discoveries with Detectrons, but we also really want to make them useful to other researchers so they can design their own versions,” Shipman says. “We want Detectrons to work as well in their hands as they do in ours.”