Browsing by Subject "Receptor"
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Item Aptamers as cross-reactive receptors : using binding patterns to discriminate biomolecules(2013-05) Stewart, Sara, 1980-; Anslyn, Eric V., 1960-; Ellington, Andrew D.Exploration into the use of aptamers as cross-reactive receptors was the focus of this work. Cross-reactivity is of interest for developing assays to identify complex targets and solutions. By exploiting the simple chemistries of aptamers, we hope to introduce a new class of receptors to the science of molecular discrimination. This manuscript first addresses the use designed aptamers for the identification of variants of HIV-1 reverse transcriptase. In this research aptamers were immobilized on a platform and were used to discriminate four variants of HIV-1 reverse transcriptase. It was found that not only could the array discriminate HIV-1 reverse transcriptase variants for which aptamers were designed, it would also discriminate variants for which no aptamers exist. A panel of aptamers was used to discriminate four separate cell lines, which were chosen as examples of complex targets. This aptamer panel was used to further explore the use of aptamers as cross-reactive sensors. Forty-six aptamers were selected from the literature that were designed to be specific to cells or molecules expected to be in the surface of cells. This panel showed differential binding patterns to each of the cell types, displaying cross-reactive behavior. During the course of this research, we also developed a novel ratiometric method of using aptamer count derived from next-generation sequencing as a method for discrimination. This is in lieu of the more commonly used fluorescent signals. Finally the use of multiple signals for pattern recognition routines was further explored by running various models using artificial data. Various situations were applied to replicate different possible situation which might arise when working with macromolecular interactions. The purpose of this was to advance the communities understanding and ability to interpret results from the pattern recognition methods of PCA and LDA.Item Direct measurement of the energy landscape of ligand-receptor interactions(2010-08) Schwemmer, Frank Heinz, 1986-; Florin, Ernst-Ludwig; Shubeita, George T.In this thesis, a novel single molecule technique will be presented that will, for the first time, give direct access to the interaction energy landscapes of small molecules. The technique relies on the interpretation of thermal position fluctuations of a colloidal probe particle tethered to the molecular complex of interest and a geometrical amplification effect that converts Ångstrom scale fluctuations of the ligand in the binding pocket of the receptor to tens of nanometer fluctuation of the bead. The position of the bead is measured with 0.5 MHz bandwidth and 2 nm spatial resolution. The surface characteristic of the substrate was found to be critical for this new technique and various surface effects were observed. Methods were developed to block nonspecific interaction between the surfaces. The mobility of specifically bound particles was found to depend strongly on the density of specific bonds and the length of the molecular complex; low concentration and short linker lead to slow ligand-receptor mediated surface diffusion, high concentration and/or long linkers to an immobilization of the particle. Transient bond formation was observed for the intermediate range. Details of the interaction energy landscape were not resolved. However, a systematic change in the linker length from 22 Å to 29 Å led to a corresponding change in the lateral position fluctuations from 12.9 nm to 13.2 nm in excellent agreement with our theoretical calculations, confirming the geometrical amplification effect. Also, a new phenomenon of nanometer scale friction in the gap between the bead and the surface was discovered. In summary, the results underline that the novel technique might be able to measure details of the interaction energy landscape of a specific ligand-receptor bond and thus test theoretical predictions for its shape.