FRET Aptamer-Based Biosensor Array for Homeland Defense

Fluorescence Resonance Energy Transfer (FRET)-DNA aptamers are oligonucleotides that have been modified with fluorophores and quenchers in their structures so that upon binding to their target analytes, the fluorophore and quencher become separated and capable of emitting light to indicate that binding has occurred. In Phase I, Operational Technologies Corporation (OpTech) will compare a ¿rational design¿ method for the engineering of FRET aptamers in which known aptamer sequences will be selectively modified with fluorophores and quenchers with the intent of achieving optimal FRET. Unfortunately, selective modification may also effect binding affinity or specificity of the aptamers. Therefore, OpTech will compare the ¿rational design¿ method with its own proprietary ¿random selection¿ method of generating FRET aptamers. Under the random selection method, the starting library of random DNA sequences will be doped with fluorophore- and quencher-labeled nucleotides that become incorporated into the final aptamer structures. One major advantage of this approach is that potential effects of the fluorophores and quenchers on aptamer binding affinity and specificity are nullified (i.e., not chosen) in the selection process. By the end of Phase I, OpTech will choose one method (rational or random selection) to proceed with into Phase II. In Phase II, OpTech will use the chosen method to generate FRET aptamers to several pesticides (chemical warfare agent simulants) and will clone and sequence the anti-pesticide aptamers. OpTech will also construct and optimize an aptamer array chip and build a compact FRET array scanner prototype for field use by first responders. The compact array scanner will be constructed in conjunction with Taboada Research Instruments, Inc. (San Antonio, TX). FRET aptamers can release their bound analytes when heated and flushed and resume their original conformations at lower temperatures. Therefore, the multianalyte array should be reusable for many cycles of detection. In Phase III, OpTech will pursue other commercial applications of the technology such as medical and veterinary diagnostics, environmental monitoring, food safety, and other applications.