Selection and Characterization of DNA Aptamers for the Detection of Antibiotics

Abstract

DNA is a naturally occurring biomacromolecule that plays many roles in living organisms. While the majority of natural DNA is double stranded, chemical synthesis allows the production of single-stranded DNA oligonucleotides that can carry chemical functions for molecular recognition, and these are known as DNA aptamers. There is wide range of targets that DNA can bind, from metabolites, drugs, toxins, and other small molecules to proteins and even cells and tissues. Aptamers are generally selected through an iterative process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). After this process is completed, the newly selected aptamers can then be subject to binding assays for characterization before finally becoming useful for sensing. Compared to the traditional methods of sensing such as HPLC, mass spectroscopy, or antibodies, aptamers are cheaper, easier to transport and use, have longer shelf lives, and can have a wider range of targets. This focus of this thesis is to use a method called capture-SELEX to isolate new aptamers for a few important antibiotics. While aptamers have been reported for them, they were mostly obtained by the immobilization of target molecules. In capture-SELEX, the DNA library is immobilization allowing the use of free target molecules. In Chapter 1, background information is given about nucleic acid structure, DNA, and the current state of aptamers. The SELEX process is also discussed in detail as well as some characterization methods and sensor applications. In Chapter 2, a new DNA aptamer for the family of tetracycline antibiotics was selected using capture-SELEX and oxytetracycline (OTC) as the target. This new aptamer was called OTC5 and had a dissociation constant (Kd) of 150 nM OTC measured using ITC. This aptamer could also enhance the intrinsic fluorescence of the tetracycline antibiotics and this property could be exploited for label free and dye free sensing. Follow-up studies were done on the OTC5 aptamer. vii It was found that metal ions (specifically Mg2+) had an effect on the binding of OTC5 to the tetracyclines. pH also affected binding with pH 6 promoting binding more than higher pH values. Studies were also done on splitting the OTC5 aptamer. The split aptamer retained its binding with doxycycline. In Chapter 3, from the remaining SELEX pool of the selection in Chapter 2, 10 other aptamers were identified to have binding with the tetracyclines. Some of these could distinguish between tetracycline, doxycycline, and oxytetracycline which led to the development of an aptamer sensor array that could differentiate these antibiotics with statistical significance. In Chapter 4, a new aptamer called CAP1 was selected for chloramphenicol (CAP) using capture- SELEX. Previous aptamers for CAP were selected by target immobilization which omitted a portion of the CAP molecule. When subjected to ITC, this previous aptamer did not show any indications of binding to the full CAP molecule. The newly selected CAP1 showed a fitted Kd of 9.8 μM using ITC. A sensor was also developed using Thioflavin-T fluorescence and had a limit of detection of 1.8 μM in lake water and 3.8 μM in wastewater. In Chapter 5, the effect of pH on aptamer selection was studied by a parallel selection at pH 6 and pH 8 for the aminoglycoside antibiotic kanamycin A. The selection at pH 6 showed better convergence than pH 8 and yielded an aptamer (KAN6-1). This aptamer had a Kd of around 300 nM from ITC. pH and salt studies were done using Thioflavin-T fluorescence assays and the optimal condition for binding was at pH 6 with no added salts. When KAN6-1 was tried with thioflavin-T under pH 8 selection conditions, there was no evidence of any binding. A sensor was then designed using KAN6-1 and had a limit of detection of 0.1 μM in lake water.