SNAP-X: Development of a Mutagenesis Strategy and High Density Protein Array to Comprehensively Display Protein Variants

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Molecular & Cellular Analysis Technologies
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Project SummarySNAP-X: Development of a Mutagenesis Strategy and High Density Protein Array to Comprehensively Display Protein Variants PIs: Christopher L. Warren and Mary S. OzersPersonalized genomics will be realized when the results of full exome next-generation sequencing (NGS) canbe understood in terms of protein functional effects. Distinguishing causal mutations from passenger mutationsthat have no effect remains the crucial problem to be solved before individual patient exome sequencing canbe applied in the clinic. High-density protein arrays are an emerging solution to assessing functional variants.Preparation of individual mutational clones and spotting of protein variants onto arrays for functional assayusing current methods is costly and time-consuming, not meriting the use of limited research and clinicalresources. A high-throughput methodology for systematic mutational analysis of protein function is needed tospur advancements in clinical application of personalized genomics. We have previously developed a novelhigh density and high throughput peptide microarray platform technology, the SNAP-Tide array (Specificity andAffinity for PepTides), which can display up to one million unique peptides from the human proteome on asingle glass slide, 100 times the peptide density of current commercial products. This increase in peptidedensity is possible because of our innovative synthesis process, in which peptide coding sequences on astandard DNA microarray are converted into RNA-barcoded peptides in vitro and addressed back to the array.In this proposal, we will innovate upon the SNAP-Tide platform to create the first available high-density arraythat displays proteins containing every amino acid substitution. Specifically, we will: 1) Design and synthesizethe SNAP-X system to generate all possible amino acid variants of three cancer-related proteins; 2) Assess thequantity and functionality of the variant proteins synthesized on the SNAP-X array; 3) Using FoxA1 variantsthat have been identified through The Cancer Genome Atlas (TCGA) in breast cancer samples, validate theSNAP-X data by performing secondary experimental assays that segregate these FoxA1 variants bypathogenicity. The array format allows for rapid, simultaneous determination of protein activities such as ligandbinding, protein-protein interactions, and protein-DNA interactions, thus providing information about individualamino acid side chain contributions to these activities. While there are other arrays that display full proteins,they do not display mutated versions of the proteins, nor do they reach the density of the SNAP-X platform.Additionally, our novel cell-free mutagenesis method reduces costs, material, and time to produce in vitro up toa million proteins with single amino acid substitutions. This technology will bridge NGS exome characterization,cancer phenotypes, and clinical outcomes.