Aryl-Deoxyguanosine Probes: Application in G-Quadruplex Forming Aptamers
Aptamers are laboratory created single-strands of DNA or RNA that have been designed to bind to a specific target with high affinity and specificity, and are gaining popularity over antibody detection due to their economical and ethical advantages. With recent developments in the field of aptamer technology, the desire for fluorescent probes that can be incorporated within an oligonucleotide strand, without disrupting it's activity, has increased. Addition of an aryl group at the C8 site of deoxyguanosine (dG) affords addition products (adducts) which can have a variety of fluorescence capabilities. These fluorescent nucleosides have been known to be sensitive to their solvent environment, base stacking, and H-bonding interactions, making them ideal candidates for development as fluorescent probes within aptamers. Aside from possessing fluorescence capabilities, the formation of a C8-aryl-dG adduct alters the conformational preference of the nucleoside. The probes are known to prefer the -syn¬-conformation, where rotation about its glycosidic bond minimizes steric interactions with the bulky aromatic group. This conformational preference can be exploited to influence the stability of a folded aptamer, and probe the structural features of the aptamer-target complex. A common folded motif within aptamers is the G-quadruplex (GQ). Insertion of probes within the core structure of the GQ has been fairly limited to date because current probes available are not able to be inserted in the place of a dG base, without disruption of the folded structure. The C8-aryl-dG probes presented in this thesis stabilize the folded GQ when placed within a syn-G, and have been optimized within a duplex to GQ exchange detection platform with thrombin binding aptamer (TBA). The utility of these probes were highlighted with insertion of 8-(2"-thienyl)-dG within the ochratoxin A aptamer to determine its structural properties, as well as probing the structural polymorphism of the human telomeric repeat sequence (HTelo). In order to create a series of probes that could be excited by visible light, further probe development was applied towards the creation of a series of push-pull adducts that contained molecular-rotor light switching abilities.