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

• 批准号: 9756417
• 负责人: Harry Benjamin Larman
• 金额: $ 34.91万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/9756417
• 关键词: Adoption; Affinity; Animal Model; Autoantigens; Back; 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; feeding; 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

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测序技术已经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，以及我们定义新蛋白质功能的能力，发现 然而，生物药物和确定疾病机制的研究并没有取得类似的进展。这种聚焦的 技术开发项目将提供新方法， 表达蛋白质文库的功能研究。 目的1：开发基于LASSO的RNA ORFeome克隆方法 我们建议将我们先前建立的长衔接子单链寡核苷酸（“LASSO”） 基于探针的DNA序列捕获平台用于直接从组织RNA克隆表达的ORF组。 逆转录酶介导的缺口填充反应将被定义并与RNA模板化的DNA-聚合酶结合。 DNA连接反应，以使得能够LASSO克隆ORF序列，同时保持其正确的翻译 阅读帧。捕获的ORFeome文库可以穿梭到任何选择的表达载体中，包括在 体外展示载体，如Aim 2中开发的载体。作为验证，完整的人转录组LASSO探针 图书馆将被设计，建造，测试和特点，然后与研究共享 社区 目标2.为全长蛋白质开发自组装的条形码RNA展示格式 体外展示技术允许基于亲和力的复杂蛋白质文库的筛选。高通量DNA 当测序应用于文库筛选时，其提供了定量结合相互作用的有力手段。我们 我以前开发了一种系统，该系统将DNA测序与核糖体展示相结合，用于ParalleL 翻译的ORFeomes（“PLATO”）的分析。核糖体展示形式施加了显著的限制， 然而，我们建议通过开发一种新的自组装条形码RNA版本来克服， PLATO（“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。 使用来自组织来源的RNA的LASSO克隆快速产生ORFeome文库，结合稳健的 使用sabr-PLATO筛选编码蛋白质文库，将为任何实验室提供一种手段， 从任何生物体的任何组织中挖掘蛋白质组。这是一个协同的，但独立的目标， 该项目利用共同项目研究员的跨学科专业知识，并在以往成功开展的研究的基础上， 在他们的实验室里。