Graduate Thesis Or Dissertation

 

RNA and DNA aptamers as affinity stationary phases for liquid chromatography and capillary electrochromatography Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/6q182p680

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  • Due to a number of disadvantages associated with the use of antibodies as affinity stationary phases, researchers have recently began to explore the use of RNA and DNA aptamers for use as affinity stationary phases. These molecules have been shown to be a viable choice for applications in many methods relying on molecular recognition. In this work, the use of aptamers as affinity stationary phases in several chromatographic formats and the effect of different mobile phase compositions on aptamer-target interactions were explored. Our first work with aptamers involved the use of them in open-tubular liquid chromatography (OTLC) and open-tubular capillary electrochromatography (OTCEC). Different immobilization schemes were employed in order to use aptamers in both of these formats. The effect that mobile phase compositions exerted on the chromatographic retention factor (k') of an RNA aptamer for its target molecules was explored. We next moved onto applying an aptamer stationary phase in a packed bed format. We found that immobilizing an amine-modified DNA aptamer onto aziactone/bis-acrylamide copolymer particles was an easy one-step method. The resulting stationary phase was capable of selectively retaining the protein target of the DNA aptamer, a-thrombin. We found the interaction between the aptamer and its target to be largely dependent upon ionic strength. Our final study of this research project involved creating a monolithic porous polymer aptamer-derivatized stationary phase. Monolithic stationary phases have been found to offer enhanced efficiency when compared to more traditional packed bed stationary phases. Within this research group it was desirable to create a monolithic stationary phases for their compatibility with a microchip format. We created a porous polymer containing a functional group on the surface of the pores which was capable of reacting with the amine-modified DNA aptamer. The result was the DNA aptamer immobilized onto the surface of the monolith. This stationary phase was found to be capable of retaining ct-thrombin (the aptamer target). The monolith was also characterized as to the dissociation constant (KD) of the aptamer-thrombin complex, and the total number of active binding sites (Bt) immobilized on the monolith surface.
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