Multiplexed DeNAno Protein Assay and Quantitation: Sequencing Based Proteomics

多重 DeNAno 蛋白质测定和定量：基于测序的蛋白质组学

• 批准号: 8855369
• 负责人: BRADLEY T MESSMER
• 金额: $ 22.5万
• 依托单位: ABREOS BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8855369
• 关键词: Affinity; Antibodies; Antigen Targeting; Antigens; Avidity; Base Sequence; Binding; Biological Assay; Biological Markers; Cancer Patient; Cataloging; Catalogs; Coupled; DNA; DNA Sequence; Data; Detection; Development; Disease; Event; Frequencies; Genetic Code; Genomics; Goals; Immune response; Kinetics; Lead; Libraries; Licensing; Malignant Neoplasms; Manufacturer Name; Marketing; Masks; Measures; Methods; Oligonucleotides; Pattern; Performance; Phase; Population; Proteins; Proteomics; Reaction; Reagent; Relative (related person); Research Personnel; Sampling; Signal Transduction; Techniques; Technology; Tissue Banking; Tissue Banks; Translating; analytical tool; cancer cell; cost; deep sequencing; high throughput analysis; insight; instrumentation; interest; magnetic beads; nanoparticle; next generation sequencing; novel; particle; polymerization; protein quantitation/detection; public health relevance; response

 DESCRIPTION (provided by applicant): Proteomics studies have the power to deliver pivotal new insight into cancer cells, biomarkers, and host responses. Unlike genomics, however, the field of proteomics has been constrained by a lack of simple and affordable analytical tools. Massively multiplex proteomic analysis methods remain expensive, cumbersome, and specialized. Here we propose the development of a novel assay termed MuDPAQ to occupy the niche for high throughput, highly multiplexed protein or biomarker assays that is currently poorly served with existing technologies. There is a growing need for assays of this type as tissue banks become more extensive, patterns of markers rather than single markers become validated, and as pharmacologic responses to therapy become more utilized. By reverse translating a protein detection event into a DNA signal, the MuDPAQ assay leverages the power and commoditization of next generation sequencing to enable massively parallel analyses that can be cheaply outsourced to any academic or commercial facility. The core of the technology is our "DeNAno" DNA nanoparticle, a novel biomolecular affinity reagent that replaces single or bi-valent affinity with hyper-avidity. DeNAno particles are produced by rolling circle replication of circular oligonucleotide templates. We have previously shown that when random templates are used to produce highly diverse libraries, particles that bind specifically to antibody coated beads can be easily recovered by biopanning. These particles, composed of concatameric repeats, bind to the cognate antibody coated bead through highly avid but individually low affinity interactions and can thus be competitively displaced by the appropriate antigen. DeNAno particles released from the beads can be sequenced, and the count frequency of the unique sequence of a given DeNAno particle translated into the quantity of its competitive analyte. When coupled with sequencing library tagging, this technology will enable high throughput analysis of multiple, multiplexed samples at a cost significantly below current technology and with no additional instrumentation required. For this Phase I proof-of-concept study, our aims will be to 1) select and validate masking DeNAno particles for each of ten mAb coated beads from a Luminex panel of cancer relevant biomarkers, and 2) demonstrate multiplexed analysis of all ten analytes. Successful completion of this study will validate the core concept behind MuDPAQ and lead to development of expanded panels of MuDPAQ reagents as well as further optimization of the assay performance. The long term goal of this proposal is to develop a catalog of MuDPAQ reagents that can be sold directly to researchers or licensed to sequencing companies or reagent manufacturers.

 描述(由申请人提供)：蛋白质组学研究有能力提供对癌细胞、生物标记物和宿主反应的关键新见解。然而，与基因组学不同，蛋白质组学领域一直受到缺乏简单和负担得起的分析工具的限制。大规模多重蛋白质组分析方法仍然昂贵、繁琐和专业化。在这里，我们建议开发一种名为MuDPAQ的新分析方法，以占据高通量、高度多元化的蛋白质或生物标记物分析的利基市场，目前现有技术服务不足。随着组织库变得更加广泛，标志物而不是单一标志物的模式得到验证，以及对治疗的药理学反应变得更加有用，对这种类型的分析的需求越来越大。通过将蛋白质检测事件反向转换为DNA信号，MuDPAQ分析利用下一代测序的能力和商品化，实现大规模并行分析，这些分析可以廉价地外包给任何学术或商业机构。这项技术的核心是我们的“DeNAno”DNA纳米颗粒，这是一种新型的生物分子亲和剂，它用超亲和性取代了单价或双价亲和力。DeNAno颗粒是通过滚环复制环状寡核苷酸模板而产生的。我们之前已经证明，当随机模板被用来产生高度多样化的文库时，专门结合到抗体包被珠上的颗粒 可以通过生物扫描很容易地恢复。这些颗粒由串联重复序列组成，通过高度亲和力但个别低亲和力的相互作用与同源抗体包被珠结合，因此可以竞争地被适当的抗原取代。可以对从珠子中释放的DeNAno颗粒进行测序，并将给定的DeNAno颗粒的唯一序列的计数频率转化为其竞争分析物的数量。当与测序库标签相结合时，这项技术将能够以远低于当前技术的成本实现对多个多路复用样本的高通量分析，并且不需要额外的仪器。对于这项第一阶段的概念验证研究，我们的目标是：1)从Luminex癌症相关生物标志物小组中为10个单抗包裹的珠子中的每一个选择和验证掩蔽DeNAno颗粒，以及2)展示对所有10个分析物的多重分析。这项研究的成功完成将验证MuDPAQ背后的核心概念，并导致MuDPAQ试剂扩展小组的开发以及分析性能的进一步优化。这项提议的长期目标是开发一份MuDPAQ试剂目录，这些试剂可以直接出售给研究人员，或者许可给测序公司或试剂制造商。