Force spectroscopy of a saxitoxin bindingaptamer
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2017
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Over the last decades optical tweezers have enabled the exploration of pico-Newton forces involved in several single-molecule mechanisms relevant in important biological systems (ie. DNA-folding and molecular motors). This has lead to a new understanding of the mechanical nature of the interactions occuring between biologicaly-relevant molecules. Here, in this thesis we used optical tweezers to study the folding/unfolding dynamics of the Handy-aptamer, a single-stranded-DNA molecule with high affinity for saxitoxin, a potent neurotoxin that is present in red tide algal blooms. This is the cause of substantial economic damage to the shellfish industry. To perform the pulling experiments, we developed a method to chemically attach the Handy-aptamerin between two double-stranded-DNA handles, which themselves were attached tomicron-sized polystyrene beads that were manipulated using the optical tweezers and a micropipette. From the pulling experiments we observed sharp folding/unfolding transitions. None the less, the transition between these states was observed to be erraticand time-dependent, exhibiting changes in sharpness and rate of occurence. Weattempted to stabilize the structure of the aptamer by adding Mg+2 and saxitoxin, both giving similar results; making the rip (sudden transition in extension) force histograms narrower and less abudant at higher forces; implying structure stabilization.These results have been the first steps towards characterizing the folding properties of the Handy-aptamer and identifying the properties that make it bind to saxitoxin.This would prove useful to develop better saxitoxin identifiers and sensors that would have lower toxin surveying costs and prevent deaths.
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Tesis (Magíster en Física)--Pontificia Universidad Católica de Chile, 2017