A novel high-throughput functional screen based upon chimeric minimotif decoys

一种基于嵌合小基序诱饵的新型高通量功能筛选

基本信息

批准号：
8924720
负责人：
MARTIN R SCHILLER
金额：
$ 22.2万
依托单位：
UNIVERSITY OF NEVADA LAS VEGAS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8924720
关键词：
Address Back Biological Assay Cell model Cell physiology Cells Chimera organism Complementary DNA Coupled Data Databases Dependency Development Disease Drug Targeting Epidemic FDA approved Future Genes Genetic HIV HIV Infections Health Human Image Individual Infection Intervention Knowledge Libraries Mediating Modeling Molecular Mutagenesis Noise Organism Paper Pathway interactions Pharmaceutical Preparations Plasmids Process Proteome RNA Interference Reproducibility Role Scientist Signal Transduction Small Interfering RNA Staging System Technology Testing base cell type combinatorial gene function gene interaction high throughput screening high throughput technology inhibitor/antagonist novel protein aminoacid sequence protein function protein protein interaction red fluorescent protein research study response screening

Address Back Biological Assay Cell model Cell physiology Cells Chimera organism Complementary DNA Coupled Data Databases Dependency Development Disease Drug Targeting Epidemic FDA approved Future Genes Genetic HIV HIV Infections Health Human Image Individual Infection Intervention Knowledge Libraries Mediating Modeling Molecular Mutagenesis Noise Organism Paper Pathway interactions Pharmaceutical Preparations Plasmids Process Proteome RNA Interference Reproducibility Role Scientist Signal Transduction Small Interfering RNA Staging System Technology Testing base cell type combinatorial gene function gene interaction high throughput screening high throughput technology inhibitor/antagonist novel protein aminoacid sequence protein function protein protein interaction red fluorescent protein research study response screening

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项目摘要

DESCRIPTION (provided by applicant): Scientists are trying to understand cells and humans as a system by developing high- throughput technologies and modeling large networks of metabolites, transcriptional responses, protein-protein interactions, genetic interactions, etc. These technologies create orthogonal knowledge that can be integrated to provide a systemic model of the cell and organism. Currently, a disconnect exists in the knowledge gained from high-throughput screens regarding protein function. RNAi screens used to identify genes that are required for a cell process are often coupled with knowledge of molecular pathways to construct pathways and networks in the cell required for a cell process. However, there is no high-throughput technology to experimentally identify the molecular functions that mediate these gene interactions, which are commonly inferred in many papers. Here, we propose to develop and test a novel chimeric minimotif decoy (CMD) screen that identifies the roles of different molecular functions in assayable cell processes. This screen is based upon our Minimotif Miner database of ~600,000 short functional peptide sequences with an experimentally determined molecular function. In this screen, an expression plasmid library is generated from chimera of random subsets of minimotifs appended in-frame to the end of a red fluorescent protein cDNA. Individual clones are transfected in separate wells of a multiwell plate and scored in any type of high-throughput assay. Positive clones are sequenced and related back to the Minimotif Miner database to identify molecular functions involved in assayed process. In our proof of principle experiments, we tested this approach on a fluorogenic HIV infection assay. We built and screened a plasmid library containing minimotifs that are required for HIV infection and demonstrated that we could rediscover some minimotifs as inhibiting HIV infection, providing proof of principle for this approach. The HIV infection assay provides an excellent system to develop and evaluate the CMD screening technology. There are well-established high-throughput fluorescent HIV infection assays, HIV exploits the use of minimotifs, a HIV minimotif (Enfurvirtide) is an FDA approved drug, and interpretation of results is facilitated by abundant information concerning HIV infection. Here, in aim 1, we will optimize the CMD screen to rediscover minimotifs that block HIV infection. In aim 2, we will build a much larger library with broader diversity of minimotif functions and genes. The library will be screened for novel minimotifs that block HIV infection. For select novel minimotifs identified in the CMD screen, we will validate the identified minimotifs using siRNA and mutagenesis of the minimotif. At this early stage of development of the CMD technology we envision four immediate potential uses. The CMD screen will: (1) provide an independent approach to validate HIV infection host dependency factors (HDFs) identified in RNAi screens; (2) experimentally identify the molecular basis of functional interactions between some host dependency factors; (3) identify novel host dependency factors; and (4) identify combinations of different sets of minimotifs that, together block HIV infection will be identified.

描述（由申请人提供）：科学家们试图通过开发高吞吐量技术并建模大型代谢物，转录反应，蛋白质 - 蛋白质相互作用，遗传相互作用等通过建模细胞和人类作为系统。这些技术可以创建正交知识，这些知识可以集成以提供细胞和生物体的系统模型。当前，从有关蛋白质功能的高通量屏幕获得的知识中存在断开连接。用于鉴定细胞过程所需的基因的RNAi筛选通常与分子途径的知识相结合，以在细胞过程中所需的细胞中构建途径和网络。但是，没有高通量技术可以在实验上识别介导这些基因相互作用的分子函数，这在许多论文中通常是在许多论文中推断出来的。在这里，我们建议开发和测试一种新型的嵌合微型诱饵（CMD）屏幕，该筛选鉴定了不同分子功能在可测细胞过程中的作用。该屏幕基于我们具有实验确定的分子功能的〜600,000个短功能肽序列的最小矿工数据库。在此屏幕中，表达质粒文库是由在框内附加到红色荧光蛋白cDNA末端的最小值的随机子集的嵌合体产生的。单个克隆被转染在多层板的单独孔中，并在任何类型的高通量测定中进行评分。阳性克隆被测序，并与最小矿工数据库相关，以识别参与测定过程的分子功能。在我们的原理实验证明中，我们在荧光HIV感染测定法上测试了这种方法。我们构建并筛选了一个质粒文库，其中包含艾滋病毒感染所需的最小拟合，并证明我们可以重新发现一些最小化的抑制艾滋病毒感染，从而为这种方法提供了原理证明。 HIV感染分析提供了一个出色的系统来开发和评估CMD筛查技术。有良好的高通量荧光HIV感染测定法，HIV利用了最小值的使用，HIV Minimotif（Enfurvirtide）是FDA批准的药物，并通过有关HIV感染的丰富信息来促进结果的解释。在这里，在AIM 1中，我们将优化CMD屏幕以重新发现阻断HIV感染的最小值。在AIM 2中，我们将建立一个更大的图书馆，具有更广泛的最小功能和基因的多样性。该库将筛选以阻止HIV感染的新型最小值。对于在CMD屏幕中鉴定出的精选新型最小值，我们将使用siRNA和最小值的诱变来验证已识别的最小值。在CMD技术开发的早期阶段，我们设想了四种直接潜在用途。 CMD屏幕将：（1）提供一种独立的方法来验证RNAi筛选中确定的HIV感染宿主依赖因子（HDF）；（2）实验确定某些宿主依赖性因素之间功能相互作用的分子基础；（3）确定新颖的宿主依赖因素；（4）确定将识别出不同的最小值集合的组合，将确定hiv感染的障碍。