2D layout of bead based thioaptamer/aptamer selection platform for therapeutics and diagnostics

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2D layout of bead based thioaptamer/aptamer selection platform for therapeutics and diagnostics

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Title: 2D layout of bead based thioaptamer/aptamer selection platform for therapeutics and diagnostics
Author: Xu Zhao
Abstract: Nucleic acid research has expanded the way we can intervene with biological systems. Especially, oligonucleotide agents (ODN or aptamers) are believed to affect cell function via complementary recognition or binding to specific proteins by forming tertiary structure. This opens new ways in therapeutics and diagnostics. The phosphoro- mono-/di- thioate substitutions in the backbone (termed “thioaptamer”) grants ODN nuclease resistance and higher binding affinity. \r\nA bead-based combinatorial library, in which every bead contains a unique species of aptamers, provides a promising platform for selection of aptamers and thioaptamers. To successfully screen the bead-based library, 2D layout of beads in gel and on bead screening model is proposed. To develop the 2D layout of beads and its corresponding functional assays, a model system is first established: NF-kappa B proteins were expressed, purified and characterized. Thioaptamer XBY6, which specifically targets NF-kappa B protein, and its natural origin, I-kappa B were synthesized and verified. Thioaptamer purification using FPLC and HPLC was also investigated, and several 5’-funtionalized thioaptamers were successfully purified. Electrophoretic mobility shifting assay (EMSA) has been used to verify XBY6 binding, and ELISA assay has been used to verify I-kappa B binding towards human recombinant NF-kappa B protein. Preliminary study of bead in 2D gel showed applicability of bead-based selection and thus on bead functional assays were developed. Both double strand one species library with I-kappa B sequence and a 212=4096 different species beads library were constructed and verified. The library was then tested using on-bead EMSA like assay and ELISA assay. Both assays showed encouraging results for 2D layout selection and further enhancement of visualization (signal/noise improvement) is discussed. \r\nThe project suggested that 2D layout of beads in gel (PAGE) is well suited for parallel high-throughput selection of thioaptamers and aptamers, thus paving a new way for drug discovery and future therapeutics and diagnostics.\r\n
URI: http://hdl.handle.net/2152.3/187
Date: 2007-07-20

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