A Functional genomics platform with integrated library cloning and molecular display

具有集成文库克隆和分子展示功能的功能基因组学平台

基本信息

批准号：
10226125
负责人：
Harry Benjamin Larman
金额：
$ 35.06万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-15 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10226125
关键词：
Adoption Affinity Animal Model Autoantigens Back Bar Codes Binding Binding Sites Biological Cloning Code Communities Complementary DNA Computer software Coupled Custom DNA DNA Ligation DNA Sequence DNA biosynthesis DNA sequencing Destinations Development Disease Ensure Escherichia coli Feedback Funding Genome Genomic DNA High-Throughput DNA Sequencing Human Immune response In Vitro Initiator Codon Laboratories Length Libraries Link Mediating Medicine Messenger RNA Methodology Methods Molecular Molecular Target N-terminal Oligonucleotides Open Reading Frames Organism Performance Pharmaceutical Preparations Preparation Procedures Production Protein Isoforms Proteins Proteome Publications RNA RNA-Directed DNA Polymerase Reaction Reading Frames Research Ribosomes System Techniques Technology Terminator Codon Testing Tissues Transcript Translating Translations Validation base cDNA Library cost covalent bond design design and construction experimental study expression vector functional genomics genomic platform haloalkane instrumentation next generation novel open source protein complex protein function protein protein interaction proteogenomics screening small molecule technology development transcriptome transcriptomics vector

项目摘要

DNA sequencing technologies have transformed our ability to define genomes and transcriptomes. Techniques to study the proteins they encode, and thus our ability to define new protein functions, discover biologic medicines, and determine disease mechanisms, however, have not similarly advanced. This focused technology development project will provide new methodologies to enable inexpensive, high throughput functional studies of expressed protein libraries. Aim 1: Develop methodology for LASSO-based ORFeome cloning from RNA We propose to adapt our previously established Long Adapter Single Stranded Oligonucleotide (“LASSO”) probe-based DNA sequence capture platform to the cloning of expressed ORFeomes directly from tissue RNA. A reverse transcriptase-mediated gap filling reaction will be defined and combined with an RNA-templated DNA- DNA ligation reaction, to enable LASSO cloning of ORF sequences while maintaining their correct translational reading frame. Captured ORFeome libraries may be shuttled into any expression vector of choice, including in vitro display vectors, like that developed in Aim 2. As validation, a complete human transcriptomic LASSO probe library will be designed, constructed, tested and characterized, prior to being shared with the research community. Aim 2. Develop a self-assembling, barcoded-RNA display format for full length proteins In vitro display technologies permit affinity-based screening of complex protein libraries. High throughput DNA sequencing, when applied to library screening, provides a powerful means to quantify binding interactions. We have previously developed a system that combines DNA sequencing with ribosome display for the ParalleL Analysis of Translated ORFeomes (“PLATO”). The ribosome display format imposes significant limitations, however, which we propose to overcome by developing a novel self-assembling barcoded-RNA version of PLATO (“sabr-PLATO”). Rigorous validation experiments will be used to extensively characterize the performance of the sabr-PLATO protein screening platform. Rapid production of ORFeome libraries using LASSO cloning from tissue-derived RNA, combined with robust screening of the encoded protein libraries using sabr-PLATO, will provide a means for any laboratory to functionally mine the proteome of any tissue from any organism. The synergistic, yet independent Aims of this project utilize the interdisciplinary expertise of the co-PIs, and build upon previous studies successfully carried out in their laboratories.

DNA测序技术改变了我们定义基因组和转录组的能力。研究它们编码的蛋白质的技术，因此我们定义新蛋白质功能的能力，发现但是，生物药物并确定疾病机制并非同样采用。这是集中的技术开发项目将提供新的方法来实现廉价，高通量表达蛋白质文库的功能研究。 AIM 1：从RNA开发基于拉索的Orfeome克隆的方法论我们建议适应我们先前建立的长适配器单链寡核苷酸（“ Lasso”）基于探针的DNA序列捕获平台直接从组织RNA的克隆到克隆。逆转录酶介导的间隙填充反应将被定义并与RNA-示例的DNA-合并 DNA连接反应，以实现ORF序列的拉索克隆，同时保持其正确的翻译阅读框架。被捕获的Orfeome库可以穿梭到任何选择的表达向量中，包括体外显示矢量，就像在AIM 2中开发的一样。作为验证，完整的人类转录组探针在与研究共享之前，将设计，构造，测试和表征图书馆社区。目标2。为全长蛋白开发自组装，barcoded-RNA显示格式体外显示技术允许基于亲和力的复杂蛋白质文库筛选。高吞吐量DNA 测序在应用于库筛选时，提供了量化结合相互作用的强大含义。我们以前已经开发了一种将DNA测序与核糖体显示的系统翻译的ORFEOMES（“柏拉图”）的分析。核糖体显示格式不可能受到重大限制，但是，我们建议通过开发一种新颖的自组装的barcoded-rna版本来克服的柏拉图（“ sabr-plato”）。严格的验证实验将用于广泛表征 SABR-Plato蛋白筛选平台的性能。使用来自组织衍生的RNA的Lasso克隆快速生产Orfeome库，并结合强大使用SABR-Plato筛选编码的蛋白质库，将为任何实验室提供一种手段在功能上，从任何组织中挖掘任何组织的蛋白质组。协同但独立的目标项目利用Co-Pis的跨学科专业知识，并以先前的研究成功进行在他们的实验室中。