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、生物标志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学分析方法仍然昂贵、繁琐和专业化。在这里，我们提出了一种新的测定称为MuDPAQ占领利基高通量，高度复用的蛋白质或生物标志物测定，目前服务与现有技术差。随着组织库变得更加广泛，标志物模式而不是单一标志物变得有效，以及随着对治疗的药理学反应变得更加有用，对这种类型的测定的需求日益增长。通过将蛋白质检测事件反向转换为DNA信号，MuDPAQ检测利用下一代测序的强大功能和商品化，实现大规模并行分析，可以廉价地外包给任何学术或商业机构。该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试剂，可以用超亲合力取代单价或双价亲和性。DeNAno颗粒通过环状寡核苷酸模板的滚环复制产生。我们以前已经证明，当随机模板用于产生高度多样化的文库时，特异性结合抗体包被珠的颗粒 可以通过生物淘洗容易地回收。这些颗粒由多联体重复序列组成，通过高亲和力但单独低亲和力的相互作用与同源抗体包被的微珠结合，因此可以被适当的抗原竞争性置换。可以对从珠粒释放的DeNAno颗粒进行测序，并且给定DeNAno颗粒的独特序列的计数频率被转化为其竞争性分析物的量。当与测序文库标记结合时，该技术将能够以显著低于当前技术的成本对多个多重样品进行高通量分析，并且不需要额外的仪器。对于这项I期概念验证研究，我们的目标是1）从Luminex癌症相关生物标志物组中选择并验证10个mAb包被珠中每一个的掩蔽DeNAno颗粒，以及2）证明所有10种分析物的多重分析。本研究的成功完成将验证MuDPAQ背后的核心概念，并导致MuDPAQ试剂扩展组的开发以及检测性能的进一步优化。该提案的长期目标是开发一个MuDPAQ试剂目录，可以直接出售给研究人员或授权给测序公司或试剂制造商。