Method for massively-parallel screening of aptamer switches

Researchers at Stanford have developed a massively parallel screening strategy to screen for target-responsive molecular switches.

Target-responsive RNA- and DNA-based molecular switches have potential utility in a variety of technological applications. Such biosensors are typically based on aptamers that undergo a reversible structure-switching mechanism that is coupled to a detectable readout. Unfortunately, it remains a challenge to generate novel aptamer switches, because most aptamers assume a stably folded structure and do not undergo a binding-induced conformation change. Most approaches to engineer aptamer switches rely on rational design and require prior knowledge of aptamer structure. Such detailed structural characterization has only been achieved for a relatively small number of aptamers. Furthermore, even the most advanced modeling software fails to account for non-canonical base-pairing or can correctly predict three-dimensional folding, which can be critical for target recognition and binding.

Stage of Research
The inventors have developed a system for rapid and massively parallel screening of different switch scaffolds in a single experiment, circumventing the need for rational design. Building upon their recently developed non-natural aptamer array (N2A2) system, the method enables the synthesis and screening of an array of fluorophore-labeled anchored displacement strand (ADS) switch constructs directly onto the flow-cell of a modified Illumina MiSeq instrument. Target-responsive molecular switches are identified by sequential imaging of flow-cell with and without the target molecule, and signal from library members can be linked to their nucleotide sequence by the location of the signal. Imaging data from each ADS construct cluster reveals the presence of switches for which target binding results in increase (signal-on) or decreased (signal-off) fluorescence. This method thereby identifies nucleic acid sequences that are able to act as target-responsive molecular switches. The inventors demonstrate the power of their method in identifying novel molecular switch sequences with a well-characterized ATP aptamer, including several sequences with non-canonical base-pairing. This approach bypasses the time-consuming process of structural analysis, rational design, and optimization by screening nearly every possible 10-nucleotide switch domain sequence in a single assay.

Stage of Development
Research – in vitro