LARGE PARTICLE SORTING FOR THE SELECTION OF OPTIMAL APTAMER BINDERS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 虽然目前的液滴分选机可用于分选直径为20-70微米的颗粒，但颗粒的尺寸将分选速度限制在每秒约一千个。由于适体文库可以含有>109个珠，因此较大颗粒的分选速度将是非常重要的。在这项资助（项目2）中开发的技术将导致高速分选机的开发（初始目标速率为每秒100，000个），这将极大地有助于分选一个珠子一个化合物适体文库（或文库文库）。这将大大提高研究人员选择适体和硫代适体对关键蛋白质靶标的高亲和力配体的能力。 NFCR将开发项目2中描述的分选技术。分选技术将被整合到一个功能分选仪中，该分选仪将在NFCR进行初步测试，以分选Gorenstein博士提供的适体文库。一旦确定仪器性能足够，第二代原型将在Gorenstein博士的实验室组装，供他的实验室使用。 博士Gorenstein将提供适体库和蛋白质靶点，这些适体库和蛋白质靶点使他的项目感兴趣，以便在NFCR进行选择和分选。我们在这个项目中的目标是利用我们的基于珠子的技术，利用多色流式细胞术选择硫代适体，靶向NIH资助的两个主要项目中宿主蛋白质组内的差异表达蛋白。在DARPA和最近NIAID资助的U 01合作伙伴基金中，Gorenstein博士（PI）正在开发一种新的硫代适体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设施选择针对特定细胞因子和趋化因子以及其他已鉴定蛋白质的硫代适体，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。在人类细胞模型以及对竞技场病毒如拉沙病毒和皮钦德病毒（出血热病毒）的免疫应答的啮齿动物模型中，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，以抑制所鉴定的蛋白质相互作用的免疫应答过程，从而改善细胞病理学免疫应答，导致休克或增强“先天免疫”，以帮助建立有效的免疫应答。阐明这些蛋白质表达的变化将允许病毒性疾病的早期诊断和改善预后，以及随后开发有效的药理学和免疫学干预措施。UTMB拥有一个BSL-4设施，用于处理这些选择性病原体，如拉沙，并正在建设两个国家生物防护实验室之一，该项目与UTMB的许多著名病毒学家合作，如Drs.C。J. Peters，Alan Barrett，Judy Aronson和Norbert Herzog。 Gorenstein博士还将使用NFCR设施选择硫代适体（再次参与免疫反应），以开发硫代适体蛋白质组学芯片作为蛋白质组学生物标志物/生物特征诊断工具，以识别和量化UTMB NHLBI资助的蛋白质组学中心（全国十个之一）中与哮喘相关的RSV感染中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，可以靶向血清和肺灌洗样品中的有限范围的蛋白质。实现这一目标将为该计划提供构建生物特征“芯片”的机会，该芯片将允许识别和定量疾病进展期间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适体芯片的实时识别和蛋白质和蛋白质+蛋白质复合物水平的读数，以使这些研究人员能够关联蛋白质表达的变化，以预测进展性疾病和治疗反应的生物标志物/生物特征。 Gorenstein博士的实验室将作为新的高速大颗粒分选机的beta测试设施，并将与NFCR沟通，以优化仪器性能。

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.