| Description |
SELEX technology has been a powerful tool to evolve aptamers that can bind tightly and specifically to a target. Aptamers are nucleic acids with the propensity to assume three-dimensional structures facilitating specific binding interactions with high affinity to the target. Principally, aptamers are selected from a large pool of random sequences by an iterative in vitro method. The main advantages of this technology are that aptamers can be produced in a large scale by a controlled combinatorial chemistry method, easily derivatized regioselectively for detection using dyes, and they are stable for long periods at room temperature when dry. Therefore, single-stranded DNA aptamers are robust binding agents with good shelf-life and easy production and do not require a biological host. One of the most difficult challenges of this technology is that there is no standardized SELEX protocol applicable for all types of target, which in this dissertation includes damaged bases and nucleosides. The first work presented here focuses on identification of products that formed from the reaction of Ni(II)-mediated sulfate radical oxidation of the guanine base. It was observed that a Ni(II)-macrocyclic square-planar complex, NiCR, in the presence of KHSO5 can generate a specific hydantoin lesion called 2-iminohydantoin (2-Ih) in both nucleoside and single-stranded DNA, along with the other well-known guanine oxidation products. The initial goal was to optimize the 2-Ih formation followed by isolation of this iv lesion. Fine tuning the oxidation conditions with respect to the concentration of KHSO5 added in certain time intervals made it possible to generate 2-Ih exclusively. Secondly, SELEX technology was used to select aptamers for the oxidized damaged bases and nucleosides as an approach toward detection of these targets. The structure-switching or capture SELEX technique was used as a successful method to generate DNA aptamers for these targets that are difficult to immobilize or derivatize and purify in the large quantities needed to perform typical SELEX procedures. The targets include the most common oxidation products that are generated as a part of the most important cellular repair pathways, base excision and nuclear excision repair. These products are the damaged nucleobases and 2'-deoxynucleosides of 8-oxo-7,8- dihydroguanine (8-oxo-G) and spiroiminodihydantoin (Sp). This is the first time that aptamers are reported for the damaged diastereomeric lesions, dSp. Lastly, sensors have been developed by truncating these aptamers as a part of post- SELEX modification and the binding affinities for these sensors toward the damaged lesions were determined. The future direction aims toward testing these aptamers by using split aptamer technology and testing these in cellular samples. |
