Enhancing selection and biological applications of oligonucleotide affinity reagents

Abstract

Aptamers are unique sequences of single-stranded DNA or RNA that exhibitsignificant affinity toward a particular target of interest. They have a number of advantages over antibodies – ease of modification, cell-free synthesis, and spontaneous renaturation – yet the preference of most researchers for antibodies continues due to prevalence of commercial antibody sources, hesitance adjusting existing research protocols, and a subconscious association of binding behavior with proteins rather than nucleic acids. While aptamers have already shown their technical advantages in the literature, they will also need to demonstrate their practicality via a broader range of targets and useful applications in order for aptamers to become more widespread in bioanalytical research. One challenge aptamers have faced is the inconsistency of SELEX, the combinatorial method by which new aptamers are identified, as well as a small pool of potential aptamer targets. This dissertation demonstrates the feasibility of aptamer selection against intact membrane proteins using a capillary electrophoresis-based SELEX technique, an achievement not yet demonstrated in the literature that opens SELEX to a much wider range of biologically significant potential targets. In addition to high affinity binding (Kd < 10 nM), these aptamers should undergo cellular internalization with their membrane receptor target, allowing hybrid aptamers to reach previously inaccessible intracellular targets as well. Additionally, this dissertation serves to increase the body of work demonstrating the bioanalytical capabilities of aptamers and other oligonucleotide affinity reagents. First, progress is shown toward aptamers targeting mouse leptin for use in a real-time aptamer- based microfluidic assay to quantify leptin secretion in cultured adipocytes. This proposed device would allow measurement of leptin, an important metabolic protein related to obesity, on a sub-minute time scale in response to metabolic stimuli. Quantification involves on-line separation of aptamers and aptamer-leptin complexes by electrophoresis, a technique that lends itself well to oligonucleotide affinity reagents due to their highly negative charge. Finally, a heart model based on cardiomyocyte differentiation from induced stem cells is demonstrated in preparation for measurement of physiological effects of aptamer-like SPIDRs (small protein-interacting DNAs and RNAs) on cardiomyocyte beating. SPIDRs have been shown to bind to the calcium pump SERCA and its control protein phospholamban, increasing the calcium affinity of SERCA. The cardiomyocyte model will be used to evaluate the hypothesis that this increase in calcium affinity leads to measurable physiological changes in beating, namely the beat rate and fall time, demonstrating the therapeutic potential of SPIDRs. Herein, optimization of stem cell growth and differentiation to cardiomyocytes are shown, as well as preliminary quantitative data regarding the reproducibility of cell impedance measurements.