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

描述(由申请人提供)：科学家试图通过开发高通量技术和对代谢产物、转录反应、蛋白质-蛋白质相互作用、遗传相互作用等大型网络进行建模，将细胞和人类作为一个系统来理解。这些技术创建了可以集成的正交知识，以提供细胞和有机体的系统模型。目前，从高通量筛选获得的关于蛋白质功能的知识存在脱节。用于识别细胞过程所需基因的RNAi筛选通常与分子通路知识相结合，以在细胞中构建细胞过程所需的通路和网络。然而，没有高通量的技术来实验地识别介导这些基因相互作用的分子功能，这些功能通常在许多论文中被推断出来。在这里，我们建议开发和测试一种新的嵌合最小诱骗(CMD)屏幕，它可以识别不同分子功能在可分析细胞过程中的作用。这一筛选是基于我们的Minimitif Miner数据库，该数据库包含约600,000个具有实验确定的分子功能的短功能肽序列。在这个筛选中，表达质粒库是由框内附加在红色荧光蛋白cDNA端的最小核苷酸随机亚集的嵌合体产生的。单个克隆在多孔板的不同孔中进行转染，并在任何类型的高通量分析中进行评分。对阳性克隆进行测序，并将其返回到Minimtif Miner数据库，以确定参与检测过程的分子功能。在我们的原理实验证明中，我们在一种产生氟的HIV感染检测上测试了这种方法。我们构建并筛选了包含HIV感染所需的最小核苷酸的质粒库，证明我们可以重新发现一些抑制HIV感染的最小核苷酸，为这种方法提供了原理证明。HIV感染检测为开发和评估CMD筛查技术提供了一个很好的系统。已有成熟的高通量荧光HIV感染检测方法，HIV使用最小剂量，一种HIV最小剂量(恩福韦肽)是FDA批准的药物，大量关于HIV感染的信息有助于对结果的解释。在这里，在目标1中，我们将优化CMD筛查，以重新发现阻止HIV感染的最小分子。在目标2中，我们将建立一个更大的文库，具有更广泛的最小化功能和基因多样性。将对该文库进行筛选，以寻找阻止艾滋病毒感染的新的最低限度基因。对于在CMD筛选中发现的新的最小核苷酸，我们将使用siRNA和最小核苷酸的突变来验证所识别的最小核苷酸。在CMD技术开发的早期阶段，我们设想了四种立即的潜在用途。CMD筛查将：(1)提供一种独立的方法来验证RNAi筛查中确定的HIV感染宿主依赖因子(HDF)；(2)通过实验确定某些宿主依赖因子之间功能相互作用的分子基础；(3)识别新的宿主依赖因子；以及(4)确定共同阻止HIV感染的不同最小核苷酸集合的组合。

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.