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Browsing by Subject "HIVRT"

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    Aptamer selection using capillary electrophoresis-SELEX
    (2008-10) Mosing, Renee Karen
    SELEX is a method used for the combinatorial selection of aptamers, or single stranded nucleic acid sequences that bind with high affinity and specificity to target molecules. Although successful, SELEX is a very time consuming, laborious process. We introduced a modification to this process called capillary electrophoresis-SELEX. This protocol proved to be significantly more efficient, which greatly decreased the time requirement of the process from weeks to days. This improvement was observed despite the lower loading capacity and resolution limited injections on CE which introduced approximately 1013 sequences to the separation instead of the 1015 sequences introduced in more traditional selections protocols. In fact, ssDNA aptamers with picomolar affinity for HIV-1 RT were identified in 4 rounds. Further sequence/structure characterization of these sequences demonstrated no homology, indicating that several sequences can bind with high affinity to the target. Interestingly, the aptamers were 10 fold more selective for the original target (HIV-1 RT) than other reverse transcriptases. Despite this result, the aptamers did not demonstrate inhibition of reverse transcriptase activity. The success of these collections prompted investigation of more challenging targets such as mitochondria and bacteria. These targets are difficult to purify and have surface chemistries that are constantly changing. Aptamers for these targets must identify a feature on the surface that is consistent in order to be conserved throughout the process. Our experiments indicated that the aptamers may have bound to features on the surface that are not very abundant, making affinity characterization cumbersome. Future experiments aim to determine if the aptamers are becoming more refined for specific features rather than just focusing on increase in affinity. Finally, initial experiments were performed for a model that will drive selections toward a specific binding site on the surface of the HIV assembly protein, capsid. Further experiments are proposed to allow aptamer binding to specific sites, and for aptamer characterization.