RNA beacon aptamers are RNA molecules that fluoresce upon binding to a target ligand. In the absence of ligand, a beacon aptamer assumes a hairpin structure in which the 5' and 3' ends are base-paired. In this conformation fluorescence is low because a fluorophore and quencher attached to the ends of the RNA are near each other in space. Fluorescence greatly increases upon ligand binding because an induced conformational change separates the fluorophore from the quencher. In theory, beacon aptamers could provide a fast, simple, sensitive, and specific assay for any molecule of interest. Potential applications include detection and quantification of biochemical reaction products, environmental pollutants, or blood constituents. The long-term objective of this proposal is to develop a reliable method for the rapid production of effective beacon aptamers. Previously, beacon aptamers have been produced in two steps. First, RNA molecules that bind to the target were selected from a randomized RNA pool. Second, a fluorophore and quencher were attached and the resulting molecules were tested for their ability to function as beacon aptamers. The new approach proposed here will select, in a single-step, RNAs that bind to the target and undergo the appropriate conformational change. The selection strategy will be tested by using the antibiotic tobramycin as the target. The results of this """"""""proof-of-principle"""""""" experiment will be used to optimize the selection procedure. Finally, a second selection will use 5'-inosine monophosphate as the target. An inosine beacon aptamer will provide a fast and simple assay for the RNA editing enzymes adenosine deaminases that act on RNA (ADARs). ADARs convert adenosine to inosine in double-stranded RNA and have been shown to be particularly important for the functioning of the nervous system. Current ADAR assays are difficult and time consuming. An inosine beacon aptamer will greatly simplify studies of ADAR mechanisms, substrate specificity and regulation. ? ?
| Vandenengel, Jeffrey E; Morse, Daniel P (2009) Mutational analysis of a signaling aptamer suggests a mechanism for ligand-triggered structure-switching. Biochem Biophys Res Commun 378:51-6 |
| Morse, Daniel P (2007) Direct selection of RNA beacon aptamers. Biochem Biophys Res Commun 359:94-101 |
