A Functional genomics platform with integrated library cloning and molecular display

DNA测序技术已经改变了我们定义基因组和转录体的能力。 研究它们编码的蛋白质的技术，从而发现我们定义新蛋白质功能的能力 然而，生物医学和疾病机制的确定并没有取得类似的进展。这就是焦点 技术开发项目将提供新的方法，以实现廉价、高产量 表达蛋白质文库的功能研究。 目标1：建立基于套索的RNA克隆ORFeome的方法学 我们建议修改我们之前建立的长接头单链寡核苷酸(Lasso) 基于探针的DNA序列捕获平台直接从组织RNA中克隆表达的ORFeome。 逆转录酶介导的缺口填充反应将被定义并与以RNA为模板的DNA- DNA连接反应，使套索克隆的ORF序列，同时保持其正确的翻译 阅读框。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，与在AIM 2中开发的类似。作为验证，完整的人类转录套索探针 在与研究共享之前，将设计、构建、测试和表征图书馆 社区。 目的2.开发一种用于全长蛋白质的自组装条码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 测序，当应用于文库筛选时，提供了一种强有力的手段来量化结合作用。我们 之前已经开发了一种将DNA测序与核糖体显示相结合的系统 分析翻译的ORFeome(“柏拉图”)。核糖体显示格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版本来克服这一点 柏拉图(军刀-柏拉图)。将使用严格的验证实验来广泛地表征 SABR-柏拉图蛋白质筛选平台的性能。 套索克隆组织源RNA快速构建ORFeome文库 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 从功能上挖掘任何有机体的任何组织的蛋白质组。这是协同的，但又是独立的目标 该项目利用共同绩效指标的跨学科专业知识，并建立在成功开展的先前研究的基础上 在他们的实验室里。

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.