Development of an RNA-based theophylline-specific microarray biosensor.

摘要

We are developing an extremely sensitive and compact biosensor that is adaptable to a variety of target analytes. Hammerhead ribozymes have been engineered such that they rearrange from a catalytically inactive to an active conformation upon binding to a target molecule. A donor-acceptor fluorophore pair is coupled to the substrate RNA of such an aptamer, to form a complex referred to as an aptazyme, to monitor real-time cleavage activity in a fluid environment. The fluorophores interact by fluorescence resonance energy transfer (FRET) until binding of the target molecule, when the FRET signal breaks down as the substrate is cleaved and the products dissociate. FRET assays with immobilized aptazymes and using total internal reflection fluorescence (TIRF) microscopy on the single-molecule scale are presented showing an enhancement of substrate cleavage in the presence of theophylline over background. The aptazyme is hybridized onto a DNA microarray and incorporated into a chip specifically designed to allow for measurement in a controlled fluid environment. The use of these microarrays allows for either one spot, or a series of spots, to be addressed independently within the biosensor. This allows for multiple analytes to be tested simultaneously. An enhancement in the substrate cleavage is again observed in the presence of theophylline. Results are presented toward the characterization of a theophylline-specific aptamer-based biosensor using this RNA microarray platform and analogous measurement techniques.