Administrative Supplement for a Cytosurge FluidFM OMNIUM instrument: The RNA nanomachines of the gene expression machinery dissected at the single molecule level

Cytosurge FluidFM OMNIUM 仪器的行政补充：在单分子水平上解剖的基因表达机器的 RNA 纳米机器

• 批准号: 10797186
• 负责人: NILS G WALTER
• 金额: $ 25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10797186
• 关键词: Address; Administrative Supplement; Aging; Award; Biological; Biology; Biophysics; Cell physiology; Cells; Complex; Enzymatic Biochemistry; Fluorescence Microscopy; Gene Expression; Gene Silencing; Gene Silencing Pathway; Genetic Transcription; Grant; Guide RNA; Individual; Injections; Label; Lead; Learning; Mammalian Cell; Messenger RNA; MicroRNAs; Microinjections; Microscope; Microscopy; Outcome; Parents; Postdoctoral Fellow; Productivity; Proteins; RNA; RNA Splicing; Readiness; Regulation; Regulator Genes; Sampling; Speed; Spliceosomes; Structure; System; Time; Training; Transcriptional Regulation; Translations; Untranslated RNA; Update; Yeasts; empowerment; fluorophore; gene function; graduate student; instrument; instrumentation; member; nanomachine; nanoscale; parent grant; particle; single molecule; tool; transcriptome; undergraduate student

PROJECT SUMMARY: This supplement will critically update the instrumentation available for several aspects of the parent R35 MIRA award, entitled The RNA nanomachines of gene expression dissected at the single molecule level. The most critical features of the proposed instrumentation, the Cytosurge FluidFM OMNIUM, are its versatility, turnkey readiness, and ease of use for injecting, sampling and manipulating the content of live mammalian cells. These features will dramatically facilitate access by the diverse group of postdoctoral fellows, graduate students and undergraduate students in the PI's group to especially our intracellular single molecule fluorescence microscopy tools, while also empowering for the first time parallel single live cell transcriptome analyses. The injection capability of the FluidFM OMNIUM will be critical for upgrading our more difficult, aging single mammalian cell microinjector for use in the parent grant, soon to be renewed. Our MIRA grant aims to dissect the mechanisms of the nanoscale RNA machines of gene expression at the single molecule level, so far focusing on bacterial riboswitches and the yeast spliceosome. Acquisition of the FluidFM OMNIUM will empower us to include the intracellular miRNA-guided silencing machinery for the renewal. Building on our group's 23-year expertise in this space, we aim to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new functional RNAs. 2.) Push the limits of our approaches to be able to probe increasingly complex biological contexts and mechanisms since unexpected discoveries often await where individual RNA nanomachines interact. In pursuit of these aims, we will address the unifying hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, as exemplified by the fact that transient miRNA:mRNA interactions lead to the regulation of protein translation in mammalian cells. Demonstrating the power of our scientific approach to address this hypothesis, we have developed a suite of intracellular single particle tracking approaches that depend on microinjecting fluorophore labeled RNAs at defined time points, which will be uniquely enabled by the FluidFM OMNIUM. To reveal the RNA-guided gene regulatory processes of the cell, we will mechanistically probe the dynamics of miRNA-guided gene silencing complexes using a tailored combination of single cell microinjection and intracellular single molecule fluorescence microscopy. A major bottleneck in these pursuits so far has been the steep learning curve associated with our microinjection-equipped microscopes that keep new group members from making significant contributions until they have completed 1-2 years of training. We anticipate that addition of the Cytosurge FluidFM OMNIUM system to our microscopy arsenal will transform the speed of our progress in intracellular single particle tracking and functional analysis by introducing an easy-to-use cell microinjection and sampling instrument that postdocs, graduate and undergraduate students can quickly use independently.

项目概要： 本补充将严格更新的仪器可用于几个方面的父R35 MIRA 该奖项名为“在单分子水平上解剖的基因表达的RNA纳米机器”。最 建议的仪器，Cytosurge FluidFM OMNIUM的关键特征是其多功能性，交钥匙 准备就绪，并且易于用于注射、取样和操作活哺乳动物细胞的内容物。这些 功能将极大地方便了不同群体的博士后研究员，研究生和 PI组的本科生特别是我们的细胞内单分子荧光显微镜 工具，同时也首次授权并行单活细胞转录组分析。注射 FluidFM OMNIUM的能力对于升级我们更困难、老化的单个哺乳动物细胞至关重要 微型注射器用于父母补助金，不久将更新。我们的MIRA基金旨在剖析 在单分子水平上的基因表达的纳米RNA机器，到目前为止， 核糖开关和酵母剪接体。收购FluidFM OMNIUM将使我们能够将 细胞内miRNA引导的沉默机制用于更新。凭借我们集团23年的专业知识， 我们的目标是：1）将我们建立的机械酶学方法应用于更广泛的一组酶， 参与调节转录、翻译和剪接的RNA，抓住了从转录中产生的机会。 新功能RNA的不断发现。2.）的情况。突破我们方法的极限， 越来越复杂的生物背景和机制，因为意外的发现往往等待着 单个RNA纳米机器相互作用。为了实现这些目标，我们将讨论一个统一的假设， 动态RNA结构是基因表达结果的主要决定因素，如以下事实所示： 瞬时miRNA：mRNA相互作用导致哺乳动物细胞中蛋白质翻译的调节。 为了证明我们的科学方法解决这一假设的能力，我们开发了一套 细胞内单颗粒追踪方法依赖于显微注射荧光团标记的RNA， 定义的时间点，将由FluidFM OMNIUM唯一启用。为了揭示RNA引导的基因 细胞的调控过程，我们将机械地探测miRNA引导的基因沉默的动力学 使用单细胞显微注射和细胞内单分子的定制组合的复合物 荧光显微镜到目前为止，这些追求的一个主要瓶颈是陡峭的学习曲线 与我们的显微注射装备显微镜，使新的群体成员， 直到他们完成1-2年的培训。我们预计Cytosurge的加入 FluidFM OMNIUM系统将改变我们在细胞内 通过引入易于使用的细胞显微注射和取样， 博士后，研究生和本科生可以快速独立使用的仪器。