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：
9094425
负责人：
MARTIN R SCHILLER
金额：
$ 18.5万
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 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9094425
关键词：
Address Back Biological Assay Cell model Cell physiology Cells Chimera organism Complementary DNA Coupled Data Databases Dependency Development Disease 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 RNA interference screen 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 new therapeutic target novel protein aminoacid sequence protein function protein protein interaction red fluorescent protein research study response screening

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、遗传相互作用等大型网络进行建模来理解细胞和人类作为一个系统。这些技术创建了正交知识，可以整合以提供细胞和生物体的系统模型。目前，从有关蛋白质功能的高通量筛选中获得的知识存在脱节。用于识别细胞过程所需基因的RNAi筛选通常与分子途径知识相结合，以在细胞中构建细胞过程所需的途径和网络。然而，目前还没有高通量技术来通过实验鉴定介导这些基因相互作用的分子功能，而这些功能在许多论文中都是普遍推断的。在这里，我们建议开发和测试一种新型嵌合小基序诱饵（CMD）屏幕，该屏幕可识别不同分子功能在可检测细胞过程中的作用。该筛选基于我们的 Minimotif Miner 数据库，其中包含约 600,000 个短功能肽序列，并具有经过实验确定的分子功能。在此筛选中，表达质粒文库是由按读码框附加到红色荧光蛋白 cDNA 末端的小基序随机子集的嵌合体生成的。将各个克隆转染到多孔板的不同孔中，并在任何类型的高通量测定中进行评分。对阳性克隆进行测序并关联回 Minimotif Miner 数据库，以识别分析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 感染测定中测试了这种方法。我们构建并筛选了包含 HIV 感染所需小基序的质粒库，并证明我们可以重新发现一些抑制 HIV 感染的小基序，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出色的系统。有完善的高通量荧光 HIV 感染检测方法，HIV 利用了小基序，HIV 小基序（Enfurvirtide）是 FDA 批准的药物，并且有关 HIV 感染的丰富信息有助于结果的解释。在这里，在目标 1 中，我们将优化 CMD 筛选，以重新发现阻止 HIV 感染的小基序。在目标 2 中，我们将建立一个更大的库，具有更广泛的小基序功能和基因多样性。该图书馆将筛选阻止艾滋病毒感染的新型小图案。对于 CMD 筛选中鉴定出的精选新小基序，我们将使用 siRNA 和小基序诱变来验证已鉴定的小基序。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 筛选将：(1) 提供一种独立的方法来验证 RNAi 筛选中确定的 HIV 感染宿主依赖性因子 (HDF)； (2)通过实验确定一些宿主依赖性因子之间功能相互作用的分子基础； (3) 识别新的宿主依赖性因素； (4) 确定不同组小基序的组合，这些组合将被确定为共同阻止 HIV 感染。