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

Project Summary Development of the High Throughput APT SNAP Platform for Rapid Identificationof Nuclease Resistant RNA Aptamers against pMissense MutationsPIsChristopher LWarren and Mary SOzers Normal proteins are often mutated in cancergenerating dominant negative effects as well as gain of function activity that contribute to cancer development and to the inhibition of therapeutic responsesProtein detection reagents that can selectively recognize single amino acid changes in cancer related proteinssuch as pwithin their cellular context are surprisingly lacking in the fieldThereforetools 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 researchtherapeutic developmentdiagnosticsand personalized medicineAptamers 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 synthesisRNA aptamersin particularcan be made nucleaseresistant by modified nucleotides for use in cellsWe propose a technology platformAPT SNAPto advance rapid identification of high affinity RNA aptamers to important biological targetssuch as the three most common missense mutations of pThe advantages of this platform are a more rapid and cost effective screening of millions of RNA structuresIn additionour platform will be developed to screen RNA aptamers synthesized with modified ribonucleotides that confer nuclease resistancegenerating tools to probe cells within a much shorter time frame than the current major technology of Systematic Evolution of Ligands by EXponential enrichmentSELEXTo create and validate the APT SNAP RNA aptamer array platformthe first goal is to develop a high density RNA microarray and to identify high affinity aptamers with exquisite specificity towards pmutationsusing known aptamer target pairs as positive controlsAn innovative promoterindependent RNA polymerase based method will be employed to generate high density RNA aptamer microarrays from standard DNA microarraysThese 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 amplificationIn the second aimmodified ribonucleotides will be incorporated to create nucleaseresistant versions of these aptamers which will stabilize the aptamers for use in cellsThe third aim of this proposal will be to validate the pdirected aptamers to identify pmutant proteins in their native environment in cancer cell lines and in a cancer tissue microarrayThe success of this project will demonstrate the transformative ability of the APT SNAP technology to rapidly and inexpensively identify aptamers for difficult targetssuch as cancer related protein point mutationsThese aptameric reagents would have broadranging applications towards understanding the function of mutated proteins in a single cell contextassessing their presence in heterogeneous tumorsand developing targeted therapeutics Project Narrative Development of the High Throughput APT SNAP Platform for Rapid Identificationof Nuclease Resistant RNA Aptamers against pMissense MutationsPIsChristopher LWarren and Mary SOzers This proposal will create a high throughput technology platformAPT SNAPto display up to a million uniquenucleotide RNA aptamers on a single glass slide as a rapid and cost effective alternative to current SELEX methodswhich is expected to yield higher success rates for aptamer identification against a proteinThis platform will allow identification of high affinity and high specificity nuclease resistant RNA aptamers to the three mutations of pan important tumor suppressor protein most commonly mutated in cancerfor which selective detection reagents are lackingThe resulting aptamers can be used to selectively recognize mutated cancer proteins in tumors or cellsimpacting cancer research toolstherapeutic developmentand the creation of point of care diagnostics