Fluorescent Energy Transfer Readout of an Aptazyme-based Biosensor

Biosensors are devices that amplify signals generated from the specific interaction between a receptor and an analyte of interest. RNA structural motifs called aptamers have recently been discovered as receptor components for biosensors owing to the ease with which they can be evolved in vitro to bind a variety of ligands with high specificity and affinity. By coupling an aptamer as allosteric control element to a catalytic RNA such as the hammerhead ribozyme, ligand binding is transduced into a catalytic event. We have made use of fluorescence resonance energy transfer (FRET) to further amplify ligand induced catalysis into an easily detectable fluorescence signal. This chapter reviews in detail the methods and protocols to prepare a theophylline specific aptazyme and to label its substrate with fluorophores. We also include detailed protocols to characterize by FRET the binding affinity of the target, theophylline, as well as the external substrate to the aptazyme. The chapter should therefore facilitate the implementation of RNA-based biosensor components for other analytes of interest.