Project Summary Development 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. Ozers Normal proteins are often mutated in cancer, generating dominant negative effects as well as gain-of-function activity that contribute to cancer development and to the inhibition of therapeutic responses. Protein detection reagents 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 the mutated forms of the proteins and differentiate these from the normal protein in tumors and cells would have broad-ranging uses in research, therapeutic development, diagnostics, and personalized medicine. Aptamers are an attractive alternative to antibodies as a high affinity reagent for specific binding and detection of proteins due 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 advance rapid identification of high affinity RNA aptamers to important biological targets, such as the three most common missense mutations of p53. The advantages of this platform are a more rapid and cost-effective screening of millions of RNA structures. In addition, our platform will be developed to screen RNA aptamers synthesized with modified ribonucleotides that confer nuclease-resistance, generating tools to probe cells within a much shorter time-frame than the current major technology of Systematic Evolution of Ligands by EXponential enrichment (SELEX). To create and validate the APT-SNAP RNA aptamer array platform, the first goal is to develop a high density RNA microarray and to identify high affinity aptamers with exquisite specificity towards 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 aptamer microarrays from standard DNA microarrays. These RNA microarrays containing rationally designed nucleic acid structures will be used in an iterative process to rapidly identify aptamers of increasing affinity without the use 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 this proposal will be to validate the p53-directed aptamers to identify p53 mutant proteins in their native environment in cancer cell lines and in a cancer tissue microarray. The success of this project will demonstrate the transformative ability of the APT-SNAP technology to rapidly and inexpensively identify aptamers for difficult 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, assessing their presence in heterogeneous tumors, and developing targeted therapeutics.