Scientists simulate evolution in the lab by introducing mutations iteratively into biomolecules such as nucleic acids and selecting for desired properties. When carrying this process out specifically on RNA molecules, they can evolve the RNAs to bind specific small molecules. But many of these so-called aptamers don’t bind well to their targets when put inside cells because they don’t fold into stable structures.
“As we solved the structures of naturally occurring aptamers, we noticed they had much more complex secondary and tertiary structures” than versions made in the lab, says Robert T. Batey of the University of Colorado, Boulder. “So we decided to use these naturally occurring RNA folds as starting points” for producing more stable artificial aptamers.
To prove their concept, Batey and coworkers used RNA sequences from naturally occurring ribozymes and riboswitches as scaffolds to evolve aptamers that bind amino acids and other small molecules used to make neurotransmitters (Nat. Chem. Biol. 2017, DOI: 10.1038/nchembio.2278). The resulting aptamers are selective for these precursor molecules over structurally similar amino acids and the neurotransmitters themselves.
One challenge, Batey says, was finding an enzyme that would not introduce mutations that disrupt the scaffolds. For instance, he says, the original reverse transcriptase the team tried quickly destroyed the scaffold by introducing mutations that caused the RNAs to misfold. The team had better luck with a recently discovered reverse transcriptase.
To convert their aptamers into sensors, the researchers connected an evolved aptamer to a second fluorophore-binding aptamer. When the first aptamer binds its target, the second aptamer is able to bind the fluorophore, which emits light in response. A third piece—an attached transfer RNA scaffold—stabilizes the structure in cells. The researchers characterized three sensors that worked inside and outside cells.
“Till now there hasn’t been a robust set of design principles that one could use to quickly generate a diverse set of aptamer-based biosensors that also fold properly in cellular contexts,” says Herman O. Sintim, who develops aptamer-based sensors at Purdue University. “Using aptamer folds that have undergone extensive biological evolution as scaffolds and developing methodologies to limit mutations of such scaffolds during the aptamer evolution and selection process is a surprisingly simple approach, yet it hadn’t been demonstrated until now.”
“Given the great diversity of natural riboswitch and ribozyme systems that have been characterized, this idea really has the potential to be transformative and widely applicable to a whole host of small molecule sensors and devices in the cell,” says Maria DeRosa, who studies aptamer-based sensors and catalysts at Carleton University.
Ming Chen Hammond, who develops RNA-based biosensors at the University of California, Berkeley, says the study provides a “master class” on how to make such novel aptamer sensors. “Many of us dream of having our own riboswitch or biosensor for measuring any given metabolite in cells,” she says. “This takes us a step closer.”