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

项目总结： 本补充资料将对父代R35 Mira的几个方面的可用仪器进行重要更新 奖，题为在单分子水平上剖析的基因表达的RNA纳米机器。最多的 建议的仪器的关键特征是它的通用性，交钥匙 对于注射、采样和处理活的哺乳动物细胞的内容物，准备就绪并且易于使用。这些 这些功能将极大地方便不同的博士后研究员、研究生和 皮氏组的本科生特别是我们的细胞内单分子荧光显微镜 工具，同时也首次使并行的单个活细胞转录组分析成为可能。注射用 对于升级我们更困难、更老化的单个哺乳动物细胞来说，FluidFM泛素的能力将是关键 父母助学金中使用的微型注射器，即将续期。我们的米拉基金旨在剖析 在单分子水平上基因表达的纳米级RNA机器，到目前为止主要集中在细菌 核糖开关和酵母剪接体。收购FluidFM全能将使我们能够包括 细胞内miRNA引导的更新沉默机制。以我们集团23年的专业知识为基础 在这个领域，我们的目标是：1)将我们已建立的机械酶学方法应用于更广泛的 参与调控转录、翻译和剪接的RNA，抓住 新的功能RNA的不断发现。2.)突破我们方法的极限，以便能够探测到 日益复杂的生物学背景和机制，因为意想不到的发现往往等待着 单个RNA纳米机器相互作用。为了追求这些目标，我们将解决统一的假设，即 动态的RNA结构是基因表达结果的主要决定因素，这一事实就是例证 这种短暂的miRNA：mRNA相互作用导致了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力量，我们开发了一套 依赖于微量注射荧光团标记RNA的细胞内单粒子示踪方法 定义的时间点，这将由FluidFM omnium唯一启用。揭示RNA引导的基因 细胞的调控过程，我们将机械地探索miRNA引导的基因沉默的动力学 利用单细胞显微注射和细胞内单分子量身定制组合的复合体 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 与我们配备的显微注射显微镜相关联，防止新的小组成员显著 在他们完成1-2年的培训之前，他们必须缴纳费用。我们期待着细胞清除器的加入 我们的显微镜武器库将改变我们在细胞内的进展速度 通过引入一种简单易用的细胞显微注射和采样进行单粒子跟踪和功能分析 博士后、研究生和本科生可以快速独立使用的仪器。

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.