| Description |
Nucleic acid aptamers are short oligonucleotides that can bind specifically to a wide range of targets, making them an analogue to the more widely known amino acidbased antibodies. Aptamers are selected from a random oligonucleotide library by a method called systematic evolution of ligands via exponential enrichment (SELEX), originally described in 1990. The purpose of this dissertation is to highlight new uses for aptamer biosensors, as well as new methods for their selection. We envisioned that a high throughput enantiopurity assay using DNA aptamers (Chapter 2) would be especially powerful in the directed evolution of stereoselective enzymes. As a proof of concept, we used tyrosinamide to demonstrate our enantiopurity analysis method. However, as a compelling target for enzyme evolution, we were drawn to the molecule 2-chloromandelic acid (2-CMA), since it is a key intermediate in the synthesis of Clopidogrel (Plavix) and can be generated asymmetrically using engineered P450 enzymes. In order to select an aptamer for 2-CMA, we used several known beadbased SELEX methods (Chapter 3). While we were able to select for several aptamers using these methods, ultimately we found that conversion of these aptamers into structure switching biosensors was difficult. This inspired us to explore SELEX methods that could directly select for the required biosensor architecture. We initially explored bead-based structure-switching SELEX methodologies. These methods directly link the structure switching architecture to ligand binding. However, we found that we predominately obtained false positives using these methods. Thus, we instead chose to explore capillary electrophoresis (CE) as our separation method due to its high partitioning potential. Using a drag tag and complementary strand, we hypothesize that we will be able to directly select for structure switching aptamers using this method. This method offers the potential to remove the current bottleneck of our enantiopurity analysis method by allowing for rapid development of new structure-switching biosensors for targets of interest. Chapter 4 describes the development of a scalable and cost-effective synthesis for the protected N-[2-(Fmoc)aminoethyl]glycine benzyl ester backbone for peptide nucleic acids (PNA). PNA has emerged as a promising alternative to the native nucleic acids DNA and RNA for a wide variety of applications, including antisense therapy and gene diagnostics. |
