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: 8361771
• 负责人: David G Gorenstein
• 金额: $ 1.12万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361771
• 关键词: Affinity; Asthma; Biological Markers; Bioterrorism; Bronchoalveolar Lavage; Caliber; Cell model; Collaborations; Color; Country; Development; Diagnostic; Disease Progression; Drug Design; Early Diagnosis; Flow Cytometry; Funding; Generations; Goals; Grant; Human; Immune response; Infection; Inflammatory Response; Laboratories; Libraries; Ligands; National Center for Research Resources; National Heart, Lung, and Blood Institute; National Institute of Allergy and Infectious Disease; Natural Immunity; Particle Size; Performance; Pichinde; Principal Investigator; Process; Progressive Disease; Proteins; Proteome; Proteomics; Research; Research Infrastructure; 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; biosignature; chemokine; cost; cytokine; hemorrhagic fever virus; immunological intervention; improved; instrument; interest; outcome forecast; particle; pathogen; programs; protein complex; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 虽然目前的液滴分选机可以用于对直径为20-70微米的颗粒进行分选，但颗粒的大小将分选速度限制在每秒1000个左右。由于适配子文库可能包含&gt；109珠子，较大颗粒的分选速度将是一个溢价。这笔赠款(项目2)中开发的技术将导致开发一种高速分选机(初始目标速度为每秒100,000)，这将极大地帮助对一个珠粒一个化合物适配子文库(或图书馆)进行分选。这将极大地提高研究人员选择关键蛋白质靶标的适体和硫代适体高亲和力配体的能力。NFCR将开发项目2中描述的分选技术。这些分选技术将集成到一个功能分选器中，它将在NFCR进行初步测试，以对Gorenstein博士提供的适配子库进行分选。一旦确定仪器性能足够，第二代原型机将在戈伦斯坦的实验室组装，供他的实验室使用。 戈伦斯坦博士将提供他感兴趣的适体文库和蛋白质靶标，以便在NFCR进行选择和分类。我们在这个项目中的目标是利用我们的基于珠子的技术来选择硫代适配子，利用多色流式细胞术来定位于NIH资助的两个主要项目中宿主蛋白质组中差异表达的蛋白质。在DARPA和最近由NIAID资助的专注于生物防御的U01合作赠款中，Gorenstein博士(Pi)正在开发一种新的硫代适配子蛋白质组学芯片技术，以研究与病原体诱导的炎症反应相关的蛋白质表达。他将利用NFCR设施来选择针对特定细胞因子和趋化因子以及其他已识别蛋白质的硫代适配子，通过分析蛋白质表达的动态变化来剖析生物恐怖主义中所关注的选定病毒的免疫反应。人类细胞模型以及啮齿动物模型对出血热病毒如Lassa和Pichinde(出血热病毒)的免疫反应正在确定蛋白质表达的变化。 这些结果将有助于药物的设计，以抑制免疫反应过程中已识别的蛋白质相互作用，从而改善导致休克的细胞病理免疫反应，或增强“先天免疫”，以帮助建立有效的免疫反应。阐明这些蛋白表达的变化将有助于病毒疾病的早期诊断和改善预后，以及随后开发有效的药物和免疫干预措施。UTMB有一个BSL-4设施来处理这些精选的病毒剂，如LASA，并正在建设两个国家生物遏制实验室之一，该项目是与UTMB的许多著名病毒学家合作的，如C.J.彼得斯博士、艾伦·巴雷特博士、朱迪·阿伦森博士和诺伯特·赫佐格博士。Gorenstein博士还将利用NFCR的设施来选择硫代适配子(同样是那些参与免疫反应的硫代适配子)，开发一种硫代适配子蛋白质组学芯片，作为蛋白质组学生物标记/生物签名诊断工具，以识别和量化与哮喘相关的RSV感染所涉及的关键蛋白质的差异表达。工作假设是，通过利用图中所示的大量蛋白质组HTS，可以针对血清和肺灌洗样本中有限范围的蛋白质。实现这一点将为该计划提供机会，以构建一个生物签名“芯片”，它将允许识别和量化在疾病进展过程中表达的各种重要蛋白质。在这两个项目中，长期目标是开发一种基于硫代适配子芯片的实时蛋白质和蛋白质+蛋白质复合体水平的识别和读数，以使这些研究人员能够关联蛋白质表达的变化，以预测进行性疾病和治疗反应的生物标志物/生物签名。戈伦斯坦博士的实验室将作为新型高速大颗粒分选器的测试版测试设施，并将与NFCR进行通信，以优化仪器性能。