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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2D layout of bead based thioaptamer/aptamer selection platform for therapeutics and diagnostics. The University of Texas Medical Branch. Available electronically from http : / /hdl .handle .net /2152 .3 /187 .

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