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Development of the High Throughput APT-SNAP Platform for Rapid Identification ofNuclease-Resistant RNA Aptamers against p53 Missense Mutations

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Molecular & Cellular Analysis Technologies
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Project SummaryDevelopment of the High Throughput APT-SNAP Platform for Rapid Identification of Nuclease-Resistant RNA Aptamers against p53 Missense Mutations PIs: Christopher L. Warren and Mary S. OzersNormal proteins are often mutated in cancer, generating dominant negative effects as well as gain-of-functionactivity that contribute to cancer development and to the inhibition of therapeutic responses. Protein detectionreagents that can selectively recognize single amino acid changes in cancer-related proteins, such as p53,within their cellular context are surprisingly lacking in the field. Therefore, tools that can selectively identify themutated forms of the proteins and differentiate these from the normal protein in tumors and cells would havebroad-ranging uses in research, therapeutic development, diagnostics, and personalized medicine. Aptamersare an attractive alternative to antibodies as a high affinity reagent for specific binding and detection of proteinsdue to their structural diversity and ease of synthesis. RNA aptamers, in particular, can be made nuclease-resistant by modified nucleotides for use in cells. We propose a technology platform, APT-SNAP, to advancerapid identification of high affinity RNA aptamers to important biological targets, such as the three mostcommon missense mutations of p53. The advantages of this platform are a more rapid and cost-effectivescreening of millions of RNA structures. In addition, our platform will be developed to screen RNA aptamerssynthesized with modified ribonucleotides that confer nuclease-resistance, generating tools to probe cellswithin a much shorter time-frame than the current major technology of Systematic Evolution of Ligands byEXponential enrichment (SELEX). To create and validate the APT-SNAP RNA aptamer array platform, the firstgoal is to develop a high density RNA microarray and to identify high affinity aptamers with exquisite specificitytowards p53 mutations, using known aptamer-target pairs as positive controls. An innovative promoter-independent RNA polymerase based method will be employed to generate high density RNA aptamermicroarrays from standard DNA microarrays. These RNA microarrays containing rationally designed nucleicacid structures will be used in an iterative process to rapidly identify aptamers of increasing affinity without theuse of PCR amplification. In the second aim, modified ribonucleotides will be incorporated to create nuclease-resistant versions of these aptamers which will stabilize the aptamers for use in cells. The third aim of thisproposal will be to validate the p53-directed aptamers to identify p53 mutant proteins in their nativeenvironment in cancer cell lines and in a cancer tissue microarray. The success of this project will demonstratethe transformative ability of the APT-SNAP technology to rapidly and inexpensively identify aptamers fordifficult targets, such as cancer-related protein point mutations. These aptameric reagents would have broad-ranging applications towards understanding the function of mutated proteins in a single cell context, assessingtheir presence in heterogeneous tumors, and developing targeted therapeutics.