Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

ABSTRACT

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.