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    Anti-cancer and anti-viral aptamers

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    chut65850.pdf (2.383Mb)
    Date
    2006
    Author
    Chu, Ted Chitai
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    Abstract
    Aptamers generated by two approaches, from purified protein and whole cell surface selections, have the potential to serve as detection and therapeutic agents against prostate cancer. In addition, aptamers generated from the purified viral protein NS1 have been shown to inhibit influenza virus replication in cell culture. Anti-prostate specific membrane antigen aptamer A9 was used with high specificity for common detection methods such as flow cytometry and confocal microscopy and for novel detection methods such as proximity dependent ligation assay. Moreover, the PSMA aptamer was shown to function as a delivery vehicle for siRNA and toxin. Cell-type specific GAPDH and lamin A/C gene knockdown were demonstrated using streptavidin as a bridge to connect biotin-conjugated aptamers to biotin-conjugated siRNA. Cytotoxicity assays also revealed that aptamers were able to specifically deliver the recombinant plant toxin rGelonin specifically to LNCaP cells when conjugated with the toxin. Furthermore, aptamers were generated from whole cell surface selections of the prostate cancer cell lines LNCaP and PC3. This suggested that anti-PC3 aptamers could target cell surfaces through indirect immunostaining and images taken by confocal microscopy. Membrane binding assays, FACS, and proximate dependent ligation assays also revealed that the aptamers were able to distinguish with great sensitivity and specificity the two non-prostate cancer cell lines, A549 and H526 as well as LNCaP and PC3 cells from DU145, another common prostate cancer line. More importantly, these experiments demonstrated that aptamers could be designed to have specificities to a certain subtype of cancer cells or to the general population. In other studies, aptamers vi were also generated from purified NS1 protein encoded by the influenza virus. The calculated Kd for these aptamers was in the range of 10 nM for the full length NS1 protein. Competition and binding assays suggested that the aptamers also targeted the dsRNA binding domain of the NS1 protein. Transfecting aptamers to human lung cancer cells, A549, infected with influenza virus revealed that the aptamers were able to inhibit the replication of both the human and avian strains of influenza virus in cell cultures. These findings strongly demonstrated the broad range and effectiveness of aptamers as tools for detection, diagnostics, and therapeutics.
    Department
    Institute for Cellular and Molecular Biology
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    text
    URI
    http://hdl.handle.net/2152/2851
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    © The University of Texas at Austin