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，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进展却没有类似的进展。技术开发项目将提供新的方法，以实现廉价、高通量表达蛋白库的功能研究。目标 1：开发基于 LASSO 的 RNA 克隆 ORFeome 的方法我们建议调整我们之前建立的长接头单链寡核苷酸（“LASSO”）基于探针的 DNA 序列捕获平台，可直接从组织 RNA 克隆表达的 ORFeome。逆转录酶介导的间隙填充反应将被定义并与 RNA 模板 DNA- DNA 连接反应，以实现 ORF 序列的 LASSO 克隆，同时保持其正确的翻译捕获的 ORFeome 文库可以转移到任何选择的表达载体中，包括体外展示载体，如目标 2 中开发的载体。作为验证，完整的人类转录组 LASSO 探针在与研究共享之前，将设计、构建、测试和表征库社区。目标 2. 开发全长蛋白质的自组装条形码 RNA 显示格式体外展示技术允许对复杂蛋白质库进行基于亲和力的筛选。当应用于文库筛选时，测序提供了一种量化结合相互作用的强大方法。之前为 ParalleL 开发了一种将 DNA 测序与核糖体展示相结合的系统翻译的 ORFeomes（“PLATO”）的分析核糖体显示格式有很大的限制，然而，我们建议通过开发一种新型自组装条形码 RNA 版本来克服这个问题 PLATO（“sabr-PLATO”）将用于主要表征 sabr-PLATO 蛋白质筛选平台的性能。使用 LASSO 克隆从组织来源的 RNA 中快速生成 ORFeome 文库，并结合强大的功能使用 sabr-PLATO 筛选编码蛋白库将为任何实验室提供一种方法功能性地挖掘任何生物体任何组织的蛋白质组协同但独立的目标。该项目利用了联合首席研究员的跨学科专业知识，并以之前成功开展的研究为基础在他们的实验室里。