RNA aptamers light up after ligand reacts

Researchers have figured out a way to make an RNA biosensor that fluoresces when it encounters small inorganic metabolites—not by engineering the RNA but by adjusting its fluorogenic ligand molecule.

Back in 2011, scientists engineered fluorescent RNA molecules made up of an RNA aptamer that binds to a hydroxybenzylidene imidazolinone (HBI) ligand (Science 2011, DOI: 10.1126/science.1207339). Neither molecule fluoresces on its own—but when they bind, the complex lights up, enabling researchers to image RNA molecules in living cells.

Researchers have tested strategies to combine riboswitches—RNA motifs that change conformation in response to binding another molecule—with these aptamers to create biosensors that fluoresce only when they bind both an HBI and a biomolecule such as S-adenosylmethionine.

But according to Rutgers University, New Brunswick, chemist Enver Cagri Izgu, such sensors have been tough to design, especially for inorganic molecules that are not prone to interact with RNA. “RNA has a folding problem, especially in cells,” he says. “It’s really difficult to control these things and really make them useful biomedically.”

Therefore, he says, he reasoned, “Why don’t we just make an organic molecule to interact with the metabolite of interest?” In a recent paper in Angewandte Chemie, Izgu and colleagues describe several preligands that react to form a fluorogenic HBI in the presence of inorganic redox-signaling molecules and that can be used as biosensing aptamers (2024, DOI: 10.1002/anie.202421936).

The researchers added a caging group to an HBI structure that is labile to hydrogen peroxide but does not cross-react with other reactive oxygen species. The preligand molecule does not fluoresce, even when bound to the aptamer; it has to be cleaved first. The team repeated the process with a functional group that could be cleaved by reacting with hydrogen sulfide but not other reactive thiols; it, too, converts to an HBI after reacting with its analyte.

When the first preligand is added to bacteria that express the aptamer, introducing hydrogen peroxide makes the cells light up, the researchers found.

Izgu says that his team is working on more HBI-derived compounds to sense an array of analytes.