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.

项目摘要 SNAP-X：开发诱变策略和高密度蛋白阵列以全面显示蛋白质变体 PIS：Christopher L. Warren和Mary S. Ozers 当完整的外显着下一代测序（NGS）的结果可以实现个性化基因组学可以实现从蛋白质功能效应方面可以理解。区分因果突变与乘客突变在单个患者外显子组测序可以解决的关键问题仍然可以解决被应用于诊所。高密度蛋白阵列是评估功能变体的新兴解决方案。准备单个突变克隆并将蛋白质变体斑点用于阵列以进行功能测定使用当前的方法是昂贵且耗时的，而不应使用有限的研究和临床使用资源。需要一种用于蛋白质功能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方面的进步。我们以前已经开发了一本小说高密度和高吞吐量肽微阵列平台技术，快照阵列（特异性和对肽的亲和力），它可以从人类蛋白质组中显示多达一百万个独特的肽单个载玻片，是当前商业产品的肽密度的100倍。肽的增加由于我们的创新合成过程，密度是可能的，其中肽编码序列标准DNA微阵列在体外转化为RNA-Barcoded肽，并回到阵列。在此提案中，我们将在快照平台上进行创新，以创建第一个可用的高密度阵列显示包含每个氨基酸取代的蛋白质。具体来说，我们将：1）设计和合成 SNAP-X系统生成三种与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数量和功能； 3）使用FOXA1变体已经通过癌症样品中的癌症基因组图集（TCGA）鉴定出来的，验证 SNAP-X数据通过执行辅助实验测定法将这些FOXA1变体隔离致病性。阵列格式允许快速，同时确定蛋白质活性，例如配体结合，蛋白质 - 蛋白质相互作用和蛋白-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动的贡献。虽然还有其他阵列显示完整蛋白质，但它们不显示蛋白质的突变版本，也不会达到SNAP-X平台的密度。此外，我们的新型无细胞诱变方法减少了成本，材料和时间的体外生产时间一百万个蛋白质具有单氨基酸取代。这项技术将桥接NGS外显子特征，癌症表型和临床结局。