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

描述（由申请人提供）：科学家们正试图通过开发高通量技术并对代谢物、转录反应、蛋白质-蛋白质相互作用、遗传相互作用等的大型网络进行建模，来将细胞和人类理解为一个系统。这些技术创建了正交知识，可以集成这些知识以提供细胞和生物体的系统模型。目前，从高通量筛选中获得的关于蛋白质功能的知识存在脱节。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，以在细胞中构建细胞过程所需的途径和网络。然而，目前还没有高通量的技术来实验性地鉴定介导这些基因相互作用的分子功能，这在许多论文中是普遍推断的。在这里，我们建议开发和测试一种新的嵌合体minimotif诱饵（CMD）屏幕，确定不同的分子功能的作用，在可测定的细胞过程。该筛选基于我们的Minimotif 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。在该筛选中，表达质粒文库由附加在红色荧光蛋白cDNA末端的符合读框的微型基序的随机子集的嵌合体产生。在多孔板的单独威尔斯孔中转染单个克隆，并在任何类型的高通量测定中评分。对阳性克隆进行测序，并将其与Minimotif Miner数据库关联，以鉴定测定过程中涉及的分子功能。 在我们的原理实验证明中，我们在荧光HIV感染测定上测试了这种方法。我们构建并筛选了一个含有HIV感染所需的minimotifs的质粒文库，并证明我们可以重新发现一些minimotifs抑制HIV感染，为这种方法提供了原理证明。HIV感染检测为CMD筛查技术的开发和评价提供了一个很好的系统。存在完善的高通量荧光HIV感染测定，HIV利用了微基序的使用，HIV微基序（Enfurvirtide）是FDA批准的药物，并且通过关于HIV感染的丰富信息促进了结果的解释。 在这里，在目标1中，我们将优化CMD筛选，以重新发现阻断HIV感染的微基序。在目标2中，我们将建立一个更大的文库，具有更广泛的微基序功能和基因多样性。该文库将筛选阻断HIV感染的新型微基序。对于在CMD筛选中鉴定的选择的新的微基序，我们将使用siRNA和微基序的诱变来验证鉴定的微基序。 在CMD技术发展的早期阶段，我们设想了四种直接的潜在用途。CMD屏幕将：（1）提供一种独立的方法来验证在RNAi筛选中鉴定的HIV感染宿主依赖性因子（HDF）;（2）实验性地鉴定一些宿主依赖性因子之间的功能相互作用的分子基础;（3）鉴定新的宿主依赖性因子;和（4）鉴定不同组的微基序的组合，其一起阻断HIV感染。

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.