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

SNAP-X：开发诱变策略和高密度蛋白质阵列以全面展示蛋白质变体

• 批准号: 9923621
• 负责人: Mary Szatkowski Ozers
• 金额: $ 16.84万
• 依托单位: PROTEOVISTA, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923621
• 关键词: Address; Affinity; Amino Acid Substitution; Amino Acids; Back; Bar Codes; Binding Proteins; Biochemical; Biological Assay; Cancer Patient; Cells; Clinic; Clinical; Code; Communities; Computerized Medical Record; Consumption; DNA; DNA Microarray Chip; Data; Destinations; Development; Disease; Evaluation; Generations; Genetic Transcription; Genome; Genomics; Glass; Grant; Human; In Situ; In Vitro; Individual; Lead; Length; Link; Malignant Neoplasms; Medical Care Costs; Messenger RNA; Methodology; Methods; Mutagenesis; Mutate; Mutation; Mutation Analysis; Nucleic Acids; Nucleotides; Oncogenic; Oncoproteins; Organism; Outcome; Pathogenicity; Patients; Peptides; Phenotype; Physicians; Plasmids; Positioning Attribute; Preparation; Price; Process; Protein Array; Protein Microchips; Proteins; Proteome; Publishing; RNA; Rare Diseases; Reagent; Research; Resources; Role; S-nitro-N-acetylpenicillamine; Sampling; Selection for Treatments; Side; Signal Pathway; Site-Directed Mutagenesis; Slide; Specificity; Spottings; Surface; System; Technology; Testing; The Cancer Genome Atlas; Time; Translating; Translations; Tube; Variant; base; causal variant; clinical application; cost; density; design; driver mutation; effective therapy; exome; exome sequencing; individual patient; innovation; link protein; malignant breast neoplasm; mutant; next generation sequencing; novel; personalized medicine; plasmid DNA; programs; protein function; protein protein interaction; rare variant; screening; treatment choice; tumor

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.

项目摘要 SNAP-X：一种突变策略和高密度蛋白阵列的开发，以全面展示 蛋白变体 PI：Christopher L.沃伦和玛丽S.厄泽尔 当全外显子组下一代测序（NGS）的结果可以 从蛋白质功能的角度来理解。区分因果突变和乘客突变 在个体患者外显子组测序能够 应用于临床。高密度蛋白质阵列是评估功能变体的新兴解决方案。 制备单个突变克隆并将蛋白变体点样到用于功能测定的阵列上 使用目前的方法是昂贵和耗时的，不值得使用有限的研究和临床 资源需要一种用于蛋白质功能的系统突变分析的高通量方法， 刺激了个性化基因组学临床应用的进步。我们之前开发了一种新的 高密度和高通量肽微阵列平台技术，SNAP-Tide阵列（特异性和 肽的亲和力），其可以在一个细胞上展示来自人类蛋白质组的多达一百万个独特的肽。 单个载玻片，100倍于当前商业产品的肽密度。这种肽的增加 密度是可能的，因为我们的创新合成工艺，其中肽编码序列上的一个 在体外将标准DNA微阵列转化为RNA条形码化肽，并返回到阵列。 在本提案中，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 展示包含所有氨基酸替换的蛋白质。具体来说，我们将：1）设计和合成 SNAP-X系统产生三种癌症相关蛋白的所有可能的氨基酸变体; 2）评估 在SNAP-X阵列上合成的变体蛋白的量和功能性; 3）使用FoxA 1变体 已经通过癌症基因组图谱（TCGA）在乳腺癌样本中鉴定出， SNAP-X数据，通过进行二级实验测定来分离这些FoxA 1变体， 致病性该阵列形式允许快速、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。虽然有其他的阵列显示完整的蛋白质， 它们不显示蛋白质的突变形式，也没有达到SNAP-X平台的密度。 此外，我们的新的无细胞诱变方法降低了成本，材料和时间，在体外生产高达 一百万个蛋白质都有一个氨基酸的替换该技术将桥接NGS外显子组表征， 癌症表型和临床结果。