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

描述（由申请人提供）：科学家正试图通过开发高通量技术和模拟代谢物、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来理解细胞和人类作为一个系统。这些技术创造了相互正交的知识，可以整合起来，提供细胞和有机体的系统模型。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，以在细胞过程所需的细胞中构建途径和网络。然而，目前还没有高通量的技术来实验鉴定介导这些基因相互作用的分子功能，而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微小基序诱饵（CMD）筛选，以识别不同分子功能在可测定的细胞过程中的作用。该筛选基于我们的Minimotif Miner数据库，该数据库包含约60万个短功能肽序列，具有实验确定的分子功能。在这个屏幕中，一个表达质粒文库是由嵌合体产生的随机亚群的minimotifs附加在框内末端的红色荧光蛋白cDNA。单个克隆在多孔板的单独孔中转染，并在任何类型的高通量测定中评分。阳性克隆测序并与Minimotif Miner数据库相关，以确定参与分析过程的分子功能。在我们的原理验证实验中，我们在荧光性HIV感染试验中测试了这种方法。我们构建并筛选了一个包含HIV感染所需的minimotifs的质粒文库，并证明了我们可以重新发现一些抑制HIV感染的minimotifs，为该方法提供了原理证明。HIV感染试验为开发和评价CMD筛查技术提供了一个良好的系统。有完善的高通量荧光HIV感染检测，HIV利用minimotifs， HIV minimotif （Enfurvirtide）是FDA批准的药物，并且关于HIV感染的丰富信息有助于解释结果。在这里，在目标1中，我们将优化CMD屏幕以重新发现阻止HIV感染的最小基序。在目标2中，我们将建立一个更大的库，具有更广泛的minimotif功能和基因多样性。该图书馆将筛选阻止艾滋病毒感染的新型微小基序。对于在CMD屏幕中鉴定出的新minimotifs，我们将使用siRNA和minimotifs的诱变来验证鉴定的minimotifs。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筛查将：(1)提供一种独立的方法来验证RNAi筛查中鉴定的HIV感染宿主依赖因子（HDFs）；(2)通过实验确定一些寄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；(4)确定不同组的微小基序的组合，共同阻止HIV感染将被确定。

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.