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

项目摘要 Snap-X：突变策略的发展和高密度蛋白质阵列的全面展示 蛋白质变异体 PIS：克里斯托弗·L·沃伦和玛丽·S·奥泽斯 当完整外显子组下一代测序(NGS)的结果能够 从蛋白质功能效应的角度来理解。区分因果突变和乘客突变 在单个患者外显子组测序之前，没有影响的仍然是需要解决的关键问题 可应用于临床。高密度蛋白质阵列是评估功能变异的一种新兴解决方案。 用于功能分析的单个突变克隆的制备和蛋白质变异体在阵列上的定位 使用目前的方法既昂贵又耗时，不值得使用有限的研究和临床 资源。需要一种高通量的方法来系统分析蛋白质功能的突变 促进个性化基因组学在临床应用中的进步。我们之前开发了一部小说 高密度和高通量的多肽微阵列平台技术，SNAP-Tide阵列(特异性和 多肽的亲和力)，它可以在一个 单一玻片，是目前商业产品的100倍多肽密度。这种多肽的增加 密度是可能的，因为我们的创新合成过程，在一个肽编码序列 标准的DNA微阵列在体外被转化为RNA条形码的多肽，并被寻址回阵列。 在本计划中，我们将在SNAP-Tide平台上进行创新，以创建第一个可用的高密度阵列 它显示了含有每种氨基酸替代的蛋白质。具体来说，我们将：1)设计和合成 SNAP-X系统生成三种癌症相关蛋白的所有可能的氨基酸变体；2)评估 SNAP-X阵列上合成的变异蛋白的数量和功能；3)使用FoxA1变体 通过癌症基因组图谱(TCGA)在乳腺癌样本中鉴定出的基因，验证了 通过执行二级实验分析来分离这些FoxA1变体，从而获得Snap-X数据 致病性。阵列格式允许快速、同时测定蛋白质活性，例如配体 结合、蛋白质-蛋白质相互作用和蛋白质-DNA相互作用，从而提供关于个体的信息 氨基酸侧链对这些活性的贡献。虽然还有其他显示完整蛋白质的阵列， 它们不显示蛋白质的突变版本，也没有达到SNAP-X平台的密度。 此外，我们新的无细胞诱变方法将体外生产的成本、材料和时间降低了多达 一百万个具有单一氨基酸替换的蛋白质。这项技术将架起NGS外显子组特征的桥梁， 癌症表型和临床结果。

Project Summary SNAP-X: Development of a Mutagenesis Strategy and High Density Protein Array to Comprehensively Display Protein Variants PIs: Christopher L. Warren and Mary S. Ozers Personalized genomics will be realized when the results of full exome next-generation sequencing (NGS) can be understood in terms of protein functional effects. Distinguishing causal mutations from passenger mutations that have no effect remains the crucial problem to be solved before individual patient exome sequencing can be 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 assay using current methods is costly and time-consuming, not meriting the use of limited research and clinical resources. A high-throughput methodology for systematic mutational analysis of protein function is needed to spur advancements in clinical application of personalized genomics. We have previously developed a novel high density and high throughput peptide microarray platform technology, the SNAP-Tide array (Specificity and Affinity for PepTides), which can display up to one million unique peptides from the human proteome on a single glass slide, 100 times the peptide density of current commercial products. This increase in peptide density is possible because of our innovative synthesis process, in which peptide coding sequences on a standard 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 array that displays proteins containing every amino acid substitution. Specifically, we will: 1) Design and synthesize the SNAP-X system to generate all possible amino acid variants of three cancer-related proteins; 2) Assess the quantity and functionality of the variant proteins synthesized on the SNAP-X array; 3) Using FoxA1 variants that have been identified through The Cancer Genome Atlas (TCGA) in breast cancer samples, validate the SNAP-X data by performing secondary experimental assays that segregate these FoxA1 variants by pathogenicity. The array format allows for rapid, simultaneous determination of protein activities such as ligand binding, protein-protein interactions, and protein-DNA interactions, thus providing information about individual amino 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 to a million proteins with single amino acid substitutions. This technology will bridge NGS exome characterization, cancer phenotypes, and clinical outcomes.