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