Multicomponent, single-molecule imaging of RNA in mammalian cells

项目摘要 RNA是细胞功能的核心。它最受赞赏的作用是携带蛋白质蓝图到核糖体 用于制造。直到最近，研究人员才开始认识到它的无数其他功能，其中许多功能， 与多种疾病有关长链非编码RNA（lncRNA）就是其中的一类 不参与中心法则的RNA。令人震惊的是，人类基因组编码了 lncRNA作为蛋白质。这些转录物通常大于200个碱基，并且已知参与转录。 通常同时结合蛋白质和核酸。然而，很少有人知道， 它们的功能。他们在何时何地与目标互动？这些相互作用持续多久，以及 还有什么其他的细胞机制这种缺乏理解的部分原因是缺乏可用的工具 来给这个生物分子成像RNA在单分子水平上的定位和RNA的多组分成像 成绩单仍然很难。现有的工具利用在哺乳动物细胞中不稳定的适体或构建体 对于成像小的转录本来说太大了。多组分RNA成像也是困难的，由于设计， 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， 单细胞分辨率的转录本。Riboglow是Palmer实验室最近开发的一种RNA成像平台。它 利用钴胺素（维生素B12）和悬垂荧光团形成的荧光猝灭对。在溶液中， 该构建体显示低荧光性。当与钴胺素核糖开关适体结构域结合时， 增加荧光度。这种工具显示出RNA成像的前景，因为它解决了许多问题， 然而，几个缺点使其无法广泛应用。拟议 工作解决了这些缺点，并寻求利用改进的Riboglow工具来研究突出的问题， 非编码RNA领域。 先前开发的Riboglow构建体遭受差的信号诱导和低亮度。首先，我的目标是 衍生化天然钴胺素结构、接头和荧光团，目标是使荧光转化率最大化， 吧将评价这些新分子的淬灭效率和信号诱导。接下来，分子I 将针对核糖开关适体文库筛选开发以进一步改善探针性质。筛选 将在哺乳动物细胞中通过帕默实验室的专长--流式细胞术进行。候选探针将是 通过活细胞中mRNA的单分子成像证实了艾德。在亮度优化的同时，我将 开发相互正交的探针，以标记同一细胞中的不同RNA转录物。功率 的SELEX来寻找选择性和紧密的结合剂，将用于筛选互斥的适体-钴胺素 对.这些对将与光谱分辨的荧光团结合，以实现对多个RNA的跟踪。 同步当lncRNA和mRNA在胞质溶胶中相互作用时，这些正交探针将用于对lncRNA和mRNA进行成像。

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.