We propose to develop high-throughput methods for the generation of nucleic acid biosensors akin to molecular beacons that can reversibly detect and quantitate a much wider variety of analytes, including ions, small organic compounds, and proteins. It has previously proven possible to select nucleic acid binding species (aptamers) from random sequence libraries that can bind to a diverse range of ligands, and we have previously adapted aptamers to function as optical biosensors. However, these methods are not generalizable, and thus cannot be applied to the high-throughput generation of multiple biosensors in parallel. Methods for the high-throughput generation of nucleic acid biosensors will ultimately enable a variety of applications, including making diagnostic reagents, proteome arrays, and eventually nanotechnology components such as nucleic acid switches and read-outs. We propose two novel methods to generate aptamer beacons, and then propose to ultimately proof these methods by generating arrays that can detect proteins and metal ions. First, like the molecular beacons for which they are named, aptamer beacons can be designed to assume two states: a hairpin structure that does not bind to an analyte and that poises a fluorescent reporter adjacent to a quencher, and an analyte-binding conformation that splits the fluorescent reporter away from the quencher and thus yields a fluorescent signal. We have previously used computational methods to design ligand-activated ribozymes, and now propose to adapt these methods to the computational design of aptamer beacons. We will probe the conformations and thermodynamics of designed beacons, in order to optimize the computational models and to design a variety of protein-responsive beacons. Second, we propose novel methods that directly couple selection for analyte-binding to the generation of fluorescent signals. These methods will initially be developed with both metal ion and protein targets. Third, to demonstrate that the high-throughput generation of aptamer beacons by either design or selection will yield biosensors that can function in arrays, the various aptamer beacons that emerge from the first two specific aims will be immobilized, and their sensitivities and specificities characterized.
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