Nucleic acid based reagentless optical biosensors

Nucleic acid binding species called aptamers are intrinsically well suited for biosensing applications. Not only do they have universal target recognition abilities, but they are also specific, exhibit high binding affinities, and can be easily engineered. Aptamers that contain fluorophores in conformationally labile positions and that signal the presence of their cognate ligands in solution have been termed `signaling aptamers´. An advantage with these optical nucleic acid based sensors is that they are “reagentless biosensors” and can enable real time, continuous, reagentless detection of targets in solution. Although several different strategies have been used to generate signaling aptamers, these methods are not general and have to be adapted to address the individual needs of different aptamers. We have developed a direct selection method that couples ligand binding to signaling, such that all selected aptamers can also of-necessity signal the presence of target analyte. Selection is based on analyte binding mediated conformational changes, vii and consequent release of a complementary quencher oligonucleotide by a fluorescently labeled aptamer beacon, thus generating a fluorescent signal. The selection method was initially proofed using oligonucleotides as target analytes. A novel type of molecular beacons were thus selected that exhibited performance characteristics comparable to those of designed molecular beacons. The selection method could also be successfully adapted to isolate aptamer beacons against small metal ions as analytes. The principal advantage of this method is that it is based on in vitro selection, and can potentially be applied to a wide variety of target molecules, thereby allowing the development of numerous reagentless biosensors. We also employed in vitro selection to isolate RNA aptamers against MEK-1, a protein kinase known to have diagnostic importance for cancer. In addition, signaling aptamers were also designed against various medically relevant proteins which can be used for diagnostic applications.