The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants

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Title: The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants
Author: Syrett, Heather Angel
Abstract: The work described here details the experimental progress toward an improved means of HIV -1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV -1 reverse transcriptase detection assay using RNA aptamers for protein capture . After characterization of the identity and function of the aptamer samples to be used , we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA ) such that through the course of this work we might bring the assay to a point of high reproducibility . In doing so , we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement . Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV -1 reverse transcriptase protein and its mutant variants . Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct . The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding , essentially serving as a simple internal control for aptamer -protein binding . After optimizing the AASA aptamer construct and experimental procedure , the AASA was extended to a multiplexed array format . Using four groups of aptamers selected against two HIV -1 RT variants (wild -type and mutant 3 ) we tested the hypothesis that immobilized anti -HIV -1 aptamers might be capable of binding HIV -1 RT variants and regardless of their selective target . The experiments described here are the first example of these aptamers being used in a multiplexed array format , and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel , immobilized assay format , but also an indicator of the utility and flexibility of RNA aptamer functionality . The promising results described in these preliminary studies are the starting block from which several interesting aptamer -protein interaction and drug -competition studies have begun .
URI: http : / /hdl .handle .net /2152 /ETD -UT -2010 -08 -1863
Date: 2010-11-09

Citation

The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants. Doctoral dissertation, University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /ETD -UT -2010 -08 -1863 .

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