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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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 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 PI：Christopher L. Warren 和 Mary S. Ozers 当全外显子组下一代测序（NGS）的结果可以实现时，个性化基因组学将得以实现可以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对个体患者进行外显子组测序之前，没有效果仍然是需要解决的关键问题应用于临床。高密度蛋白质芯片是评估功能变异的新兴解决方案。制备单个突变克隆并将蛋白质变体点样到阵列上以进行功能测定使用当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。需要一种对蛋白质功能进行系统突变分析的高通量方法推动个性化基因组学临床应用的进步。我们之前开发过一部小说高密度和高通量肽微阵列平台技术，SNAP-Tide 阵列（特异性和 Affinity for PepTides），可以在计算机上显示来自人类蛋白质组的多达一百万个独特的肽单玻片，肽密度是目前商业产品的100倍。这种肽的增加由于我们创新的合成工艺，密度是可能的，其中肽编码序列在标准 DNA 微阵列在体外转化为 RNA 条形码肽并返回到阵列。在本提案中，我们将在 SNAP-Tide 平台上进行创新，创建第一个可用的高密度阵列显示包含所有氨基酸取代的蛋白质。具体来说，我们将： 1）设计和综合 SNAP-X 系统可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 SNAP-X 数据通过执行二次实验测定来分离这些 FoxA1 变体致病性。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，从而提供有关个体的信息氨基酸侧链对这些活性的贡献。虽然还有其他阵列可以显示完整的蛋白质，它们不显示蛋白质的突变版本，也未达到 SNAP-X 平台的密度。此外，我们的新型无细胞诱变方法可减少体外生产的成本、材料和时间一百万个具有单一氨基酸取代的蛋白质。该技术将连接 NGS 外显子组表征，癌症表型和临床结果。