Multicomponent, single-molecule imaging of RNA in mammalian cells

项目总结 RNA是细胞功能的中心。它最受重视的作用是将蛋白质蓝图带到核糖体。 用于制造。直到最近，研究人员才开始欣赏它的无数其他功能，其中许多 与多种疾病状态有关。长非编码RNA(LncRNA)就是这样一个重要类别 不参与中心教条的RNA。令人担忧的是，人类基因组编码的 LncRNA作为蛋白质。这些转录本通常大于200个碱基，并且已知参与了 同时结合蛋白质和核酸，通常两者同时结合。然而，对其他方面的了解很少 它们的功能。他们在何时何地与目标互动？这些互动持续多长时间，以及 还有什么其他的细胞机制存在？这种缺乏理解的部分原因是缺乏可用的工具 来想象这种生物分子。核糖核酸在单分子水平上的定位和多组分成像 文字记录仍然不同于fi崇拜。现有的工具利用在哺乳动物细胞中不稳定的适体，或构建 它们太大了，无法对小的转录进行成像。多组分核糖核酸成像也是很难的，因为fi的设计 当前工具。 为了满足这一需求，我的目标是开发一个RNA成像平台，使之能够方便地跟踪多个 单细胞分辨率的转录本。Riboglow是Palmer实验室最近开发的RNA成像平台。它 采用fl荧光猝灭对，由钴胺(维生素B12)和悬挂式fl发光体组成。在解决方案中， 这种结构表现出较低的fl发光强度。当与钴胺核糖开关适配子结构域结合时，有一个 fl透光度增加。该工具显示了RNA成像的前景，因为它解决了许多问题 然而，面对传统的RNA探针，有几个缺点阻碍了它的广泛应用。建议数 工作解决了这些缺点，并试图利用改进的Riboglow工具来研究 非编码fi的RNA域。 以前开发的Riboglow结构存在信号感应差和亮度低的问题。首先，我的目标是 衍生天然钴胺结构、连接体和fl发光体，目标是最大限度地提高fl发光转化率。 在……上面。这些新分子将在猝灭效率和信号诱导方面进行评估。接下来，分子I 将针对核糖开关适配子文库进行筛选，以进一步改善探针性能。筛选 将通过帕尔默实验室的专业flow细胞术在哺乳动物细胞中进行。候选探测器将是 Verifi是通过活细胞中的mRNA单分子成像进行的。在亮度优化的同时，我将 开发相互正交的探针，以便能够标记同一细胞中的不同RNA转录本。那股力量 将使用SELEX和fi的选择性和紧密结合剂来筛选相互排斥的适配子-钴胺 成对的。这些对将与光谱分辨的fl载体结合，以实现对多个RNAs的跟踪 同时。当lncRNA和mRNA在细胞质中相互作用时，这些正交探针将被用来成像。

PROJECT SUMMARY RNA lies at the center of cellular function. Its most appreciated role is to carry protein blueprints to the ribosome for manufacture. Only recently have researchers begun to appreciate its myriad of other functions, many of which are implicated in a variety of disease states. Long noncoding RNA (lncRNA) comprise one such important class of RNA that does not participate in the central dogma. Alarmingly, the human genome encodes for as many lncRNA as proteins. These transcripts are typically greater than 200 bases, and are known to participate in binding both proteins and nucleic acids, often both at the same time. However, little else is understood regarding their function. Where and when do they interact with their targets? How long do these interactions occur, and what other cellular machinery is present? This lack of understanding is due in part to the lack of tools available to image this biomolecule. Localization of RNA on a single-molecule level, and multicomponent imaging of RNA transcripts remains difficult. Existing tools utilize aptamers that are unstable in mammalian cells, or constructs that are too large for imaging small transcripts. Multicomponent RNA imaging is also difficult due to the design of current tools. To address this need, I aim to develop a platform for RNA imaging that will enable facile tracking of multiple transcripts at single cell resolution. Riboglow is an RNA imaging platform recently developed in the Palmer lab. It utilizes a fluorescence-quenched pair formed by cobalamin (vitamin B12) and a pendant fluorophore. In solution, this construct shows low fluorescence. When bound to the cobalamin riboswitch aptamer domain, there is an increase in fluorescence. This tool shows promise for RNA imaging because it solves many of the problems faced by traditional RNA probes, however several drawbacks are keeping it from widespread utility. The proposed work addresses these drawbacks, and seeks to utilize improved Riboglow tools to study outstanding questions in the field of noncoding RNA. Previously developed Riboglow constructs suffered from poor signal induction and low brightness. First, I aim to derivatize the native cobalamin structure, linker and fluorophore with the goal of maximizing fluorescence turn- on. These new molecules will be evaluated for quenching efficiency and signal induction. Next, the molecules I develop will be screened against libraries of riboswitch aptamers to further improve probe properties. Screening will be carried out in mammalian cells via flow cytometry, a specialty of the Palmer lab. Candidate probes will be verified through single-molecule imaging of mRNA in living cells. In tandem with brightness optimization, I will develop mutually orthogonal probes to enable labeling of different RNA transcripts in the same cell. The power of SELEX to find selective and tight binders will be used to screen for mutually exclusive aptamer-cobalamin pairs. These pairs will be conjugated to spectrally-resolved fluorophores to enable tracking of multiple RNA simultaneously. These orthogonal probes will be used to image lncRNA and mRNA as they interact in the cytosol.