LARGE PARTICLE SORTING FOR THE SELECTION OF OPTIMAL APTAMER BINDERS

用于选择最佳适体结合剂的大颗粒分选

• 批准号: 7724269
• 负责人: David G Gorenstein
• 金额: $ 1.61万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724269
• 关键词: Affinity; Asthma; Biological Markers; Bioterrorism; Bronchoalveolar Lavage; Caliber; Cell model; Chemokine, Other; Collaborations; Color; Complex; Computer Retrieval of Information on Scientific Projects Database; Country; Development; Diagnostic; Disease Progression; Drug Design; Early Diagnosis; Flow Cytometry; Funding; Generations; Goals; Grant; Human; Immune response; Infection; Inflammatory Response; Institution; Laboratories; Libraries; Ligands; Natural Immunity; Numbers; Particle Size; Performance; Pichinde; Process; Progressive Disease; Proteins; Proteome; Proteomics; Range; Rate; Research; Research Personnel; Resources; Rodent Model; Sampling; Serum; Shock; Sorting - Cell Movement; Source; Speed; Technology; Testing; Therapeutic; Time; United States National Institutes of Health; Virus; Virus Diseases; Work; aptamer; base; biodefense; biosafety level 4 facility; cytokine; hemorrhagic fever virus; immunological intervention; improved; instrument; interest; outcome forecast; particle; pathogen; programs; protein expression; prototype; response; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While current droplet sorters can be used to sort the particles 20-70 microns in diameter, the size of the particles limit the sort speed to about a thousand per second. As aptamer libraries could contain >109 beads, sorting speed of larger particles will be at a premium. The technologies developed in this grant (Project 2) will result in the development of a high speed sorter (with initial target rates of 100,000 per second) that would greatly assist in sorting one bead one compound aptamer libraries (or library of libraries). This would greatly improve the ability of investigators to select aptamer and thioaptamer high affinity ligands to critical protein targets. The NFCR will develop sorting technologies as described in project 2. The sorting technologies will be integrated into a functional sorter, which will be initially tested at the NFCR to sort aptamer libraries provided by Dr. Gorenstein. Once instrument performance has been determined to be sufficient, a second generation prototype will be assembled in Dr. Gorenstein's laboratory for use by his lab. Dr. Gorenstein will provide aptamer libraries and protein targets that interest his programs for selection and sorting at the NFCR. Our goal in this project is to utilize our bead-based technology for selecting thioaptamers utilizing multi-color flow cytometry targeting the differentially expressed proteins within the host proteomes in two major projects funded by NIH. In DARPA and more recently an NIAID-funded U01 partnership grant focused on biodefense, Dr. Gorenstein (PI) is developing a new thioaptamer proteomics chip technology to investigate protein expression associated with pathogen-induced inflammatory responses. He will utilize the NFCR facility to select thioaptamers targeting specific cytokines and chemokines and other identified proteins to dissect immune responses of selected viruses of concern in bioterrorism by analyzing the dynamic changes in protein expression. Changes in protein expression are being determined in human cell models as well as rodent models of immune responses to arena viruses such as Lassa and Pichinde (hemorrhagic fever viruses. These results will aid in the design of drugs to inhibit the identified protein interactions underlying immune response processes and so ameliorate cytopathological immune responses resulting in shock or to enhance "innate immunity" to help mount effective immune response. Elucidating these protein expression changes will allow early diagnosis and enhanced prognosis of viral disease, and subsequent development of effective pharmacological and immunological interventions. UTMB has a BSL-4 facility for handling these select agents such as Lassa and is constructing one of two National Biocontainment Laboratories and this project is in collaboration with a number of noted virologists at UTMB such as Drs. C. J. Peters, Alan Barrett, Judy Aronson and Norbert Herzog. Dr. Gorenstein will also use the NFCR facility for selecting thioaptamers (again those involved in the immune response) to develop a thioaptamer proteomics chip as a proteomics biomarker/biosignature diagnostic tool to identify and quantify the differential expression of key proteins involved in RSV-infections related to asthma in an NHLBI-funded Proteomics Center at UTMB (one of ten in the country). The working hypothesis is that by utilizing the massive proteome HTS shown in the figure, a limited range of proteins in serum and lung lavage samples can be targeted. Accomplishing this will provide this program the opportunity to construct a biosignature "chip" which will allow identification and quantification of the various important proteins expressed during disease progression. In both projects, the long-term objectives are to develop a real-time thioaptamer chip-based identification and readout of the levels of proteins and protein+protein complexes to allow these researchers to correlate changes in protein expression to predict biomarkers/biosignatures for progressive disease and therapeutic responses. Dr. Gorenstein's lab will serve as a beta testing facility for new high speed large particle sorters and will communicate with the NFCR to optimize instrument performance.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 虽然当前的液滴划者可用于对直径20-70微米的颗粒进行排序，但颗粒的大小将排序速度限制在每秒大约一千个。由于适体库可以包含> 109个珠子，因此较大颗粒的排序速度将是高级的。这笔赠款中开发的技术（项目2）将导致开发高速分落者（初始目标率为每秒100,000个），这将极大地有助于对一个珠子进行排序一个珠子一个化合物适应性库（或库库）。这将大大提高研究人员选择适合蛋白靶标的适体和硫族人高亲和力配体的能力。 NFCR将如项目2中所述开发排序技术。排序技术将集成到功能分散器中，最初将在NFCR进行测试，以分类Gorenstein博士提供的Aptamer库。一旦确定仪器的性能就足够了，第二代原型将在戈伦斯坦博士的实验室中组装，以供他的实验室使用。 Gorenstein博士将提供适体库和蛋白质目标，使他在NFCR进行选择和分类的计划感兴趣。我们在该项目中的目标是利用基于珠子的技术在NIH资助的两个主要项目中，利用靶向宿主蛋白质组中差异表达的蛋白质的多色流式细胞仪选择硫thioaptamers。在DARPA和最近的NIAID资助的U01合作伙伴赠款的重点是Biodefense，Gorenstein博士（PI）正在开发一种新的硫代蛋白酶蛋白质组学芯片技术，以研究与病原体诱导的炎性反应相关的蛋白质表达。他将利用NFCR设施来选择靶向特定细胞因子和趋化因子和其他鉴定蛋白质的硫thioaptamers，通过分析蛋白质表达的动态变化，从而在生物恐怖主义中剖析所选病毒的免疫反应。在人类细胞模型以及对竞技场病毒（例如Lassa和Pichinde）（出血热病毒）的免疫反应的啮齿动物模型中，蛋白质表达的变化得到了确定。 这些结果将有助于设计药物，以抑制免疫反应过程中鉴定出的蛋白质相互作用，从而改善细胞病理学免疫反应，从而导致冲击或增强“先天免疫”以帮助实施有效的免疫反应。阐明这些蛋白质表达的变化将允许早期诊断和增强病毒疾病的预后，并随后发展有效的药理和免疫学干预措施。 UTMB拥有一个BSL-4设施，用于处理这些精选代理，例如LASSA，并正在构建两个国家生物疗法实验室之一，并且该项目与UTMB的许多著名病毒学家（例如DRS）合作。 C. J. Peters，Alan Barrett，Judy Aronson和Norbert Herzog。 Dr. Gorenstein will also use the NFCR facility for selecting thioaptamers (again those involved in the immune response) to develop a thioaptamer proteomics chip as a proteomics biomarker/biosignature diagnostic tool to identify and quantify the differential expression of key proteins involved in RSV-infections related to asthma in an NHLBI-funded Proteomics Center at UTMB (one of ten in这个国家）。工作假设是，通过使用图中所示的大量蛋白质组HT，可以针对血清和肺灌洗样品中的蛋白质范围有限。实现这一目标将为该计划提供构建生物签名“芯片”的机会，该“芯片”将允许识别和量化疾病进展过程中表达的各种重要蛋白质。在这两个项目中，长期目标是开发基于硫代芯片的实时硫代芯片鉴定，并读出蛋白质和蛋白质+蛋白质复合物水平的读数，以使这些研究人员能够将蛋白质表达的变化相关联，以预测生物标志物/生物签名为进行性疾病和治疗反应。 Gorenstein博士的实验室将作为新的高速大粒子分类器的Beta测试设施，并将与NFCR通信以优化仪器性能。