<p> This thesis focuses on fundamental and applied aspects of sol-gel based affinity assays for screening membrane receptor targets. Fundamental studies investigate the role of non-specific interactions between polycationic polymers and sol-gel derived monoliths prepared from sodium silicate precursors. Previous studies from our group using time-resolved
fluorescence anisotropy (TRFA) have shown that both the sidechain motion (Φ1) and backbone motion (Φ2) of polycationic polymers, such as poly-D-lysine, can be measured by fitting anisotropy decays of fluorescein labeled polymers to a two component hindered rotor model. These studies demonstrated that polycationic polymers remain fairly mobile in sol-gel derived materials made from sodium silicate precursors, despite the strong electrostatic interactions between the cationic polymers and the anionic silica materials. The first objective of my work was to assess the nature of the electrostatic interactions between several polycationic polymers and a silica material made from sodium silicate using a novel two-point labeling technique with a pyranine dye to determine if previous studies were indeed correct. Our results show that the two-point labeling technique with pyranine provides a more rigid interaction between the polymer and the dye compared to the previous labeling method using fluorescein, allowing for more accurate monitoring of dynamic motions of cationic polymers in sol-gel derived materials. The dynamics of poly-D-lysine entrapped in sol-gel derived materials was indeed seen to be highly restricted, contrary to results obtained in previous studies.</p> <p> While the first project provided a framework for understanding the effect of electrostatic interactions on the dynamics of biomolecules, it also provide valuable insight into the effect of non-specific interactions between cationic species and sol-gel derived materials in general. These considerations are important when using sol-gel based affinity columns for small molecule screening. The second study outlines a competitive affinity-based screening method using mass spectrometry (MS) detection that can help reduce the effect of non-specific interactions between test compounds and the column matrix in small molecule screening applications. This technique relies on the use of a high-affinity indicator compound pre-equilibrated on column to identify weak affinity ligands in mixtures through transient spikes in the indicator signal that result from the competition between the indicator and test compounds. The results of this study demonstrate the ability to identify weak affinity ligands for the nicotinic acetylcholine receptor (nAChR) using low to sub-picomole amount of active receptor on column. The technique results in a reproducible signal output that can potentially be used to obtain quantitative data on the binding affinities of target-ligand interactions. The assay is amenable to automation and can be performed at high speeds, thereby demonstrating the potential of this technique as a high-throughput screening tool for screening membrane receptors.</p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21610 |
| Date | January 2007 |
| Creators | Sharma, Jai |
| Contributors | Brennan, John, Biochemistry |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
Page generated in 2.0195 seconds