| Nucleic acids hold unique molecular recognition properties that recommend them as ideal candidates in the construction of biosensing devices. Nucleic acids can inherently recognize antisense sequences of DNA or RNA and engage in double helical structures through Watson-Crick interactions. Moreover, DNA aptamers are in vitro selected single-stranded nucleic acid molecules that have specific ligand-binding capabilities for biological entities such as metabolites, hormones, proteins or even whole cells.; This thesis describes our efforts in combining molecular recognition properties of nucleic acids with the flexibility of fluorescence spectroscopy as detection method for the creation of two DNA-based biosensing devices, called "tripartite molecular beacons" and "structure-switching signaling aptamers".; Molecular beacons are hairpin-shaped fluorescent DNA probes designed to detect specific sequences of nucleic acids through a fluorescence quenching/dequenching mechanism. We demonstrate in this thesis that "tripartite molecular beacons", a novel format of fluorescent DNA probes for nucleic acid detection, can have detection performances similar to molecular beacons, but a significantly reduced production and purification costs.; Furthermore, this thesis describes a strategy of creating fluorescence signaling aptamers for the detection of non-nucleic acid molecules. The 'structure-switching' method is based on target-induced conformational reorganization between two possible states of a DNA aptamer: a low-fluorescence duplex state between a fluorescent aptamer and a complementary quenching DNA, and a high-fluorescence ligand-aptamer complex state between the fluorescent aptamer and the target of interest. In this thesis we explain in detail how structure-switching signaling aptamers can be created from existent aptamers through rational design or isolated from vast libraries of random oligonucleotides through in vitro selection. Moreover, we demonstrate that structure-switching signaling aptamers can be used not only for simple ligand-binding detection assays, but also for controlling the progress of reactions, tools for enzyme characterization, sensitive fluorescent probes for drug discovery, or flexible molecular switches for nanotechnology. |
