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Riboglow: a robust multi-color riboswitch-based platform for imaging RNA in living cells

Riboglow：基于多色核糖开关的强大平台，用于活细胞中 RNA 成像

基本信息

批准号：
9764689
负责人：
Robert T Batey
金额：
$ 30.8万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9764689
关键词：
Affect Affinity Benchmarking Binding Binding Proteins Biochemical Biochemistry Biological Biological Assay Biology Cell physiology Cells Cellular Assay Cellular biology Chemicals Cobalamin Color Complex Cytoplasmic Granules Cytosol DNA Detection Deterioration Development Disease Dyes Engineering Ensure Environment Etiology Fluorescence Fluorescent Probes Gene Expression Gene Expression Regulation Generations Genetic Genetic Transcription Genome Goals Gold Image Imagery In Vitro Individual Ligand Binding Ligands Lighting Link Macromolecular Complexes Mammalian Cell Maps Measurement Messenger RNA Modification Molecular Probes Nature Nucleic Acids Nucleotides Pattern Performance Physiology Play Property Proteins RNA RNA Binding RNA Decay RNA Probes RNA Splicing Regulation Ribonucleoproteins Role Series Structure System Testing Toxic effect Transcript Transcription Initiation Transcriptional Regulation Translating Translations U1 small nuclear RNA Untranslated RNA Validation Variant Work aptamer base biophysical properties cytotoxicity engineering design epigenetic regulation fluorophore genetic information imaging platform improved interest live cell imaging molecular imaging portability prevent scaffold single molecule small molecule spatiotemporal stem technology development tool virtual

项目摘要

SUMMARY. The complex spatiotemporal dynamics of messenger RNAs and non-coding RNAs affect virtually all aspects of cellular function. In addition to serving as the central intermediary between DNA and proteins, RNAs regulate gene expression at multiple levels, play roles in epigenetic regulation and genome organization, and serve as physical scaffolds to assemble and integrate macromolecular complexes, with important implications for normal development, as well as disease etiology. Yet, despite the importance of RNA in biology and growing evidence of complex and dynamic localization patterns, robust tools for visualizing RNA molecules in live cells are highly limited. The most widely used RNA tagging system involves addition of 24 MS2 stem loops and binding of 48 molecules of the MS2 binding protein fused to GFP, adding > 1300 nucleotides and > 2.6 MDa to an RNA of interest. While this system has revealed tantalizing glimpses at the individual steps of gene expression regulation, perhaps not surprisingly, it has also been shown to perturb mRNA processing, splicing, localization, and decay. Thus, there is a pressing need for robust, complementary, and minimally perturbing tools to visualize individual RNA molecules in living cells to map the complex and evolving landscape of RNA biology. In this work, we will meet this need by generating a suite of diverse riboswitch-based RNA tags and corresponding fluorescent probes for simultaneous, multi-color imaging of individual RNA molecules in live mammalian cells. Our approach builds on preliminary work from our labs that exploits one of nature’s aptamers, the cobalamin (Cbl)-binding riboswitch as an RNA tag that binds a series of Cbl-linked fluorophores to induce fluorescence turn-on, thus lighting up the RNA of interest. We called this new RNA tagging platform Riboglow and demonstrated its ability to visualize mRNA and small U1 snRNA in live mammalian cells. While the performance of Riboglow was impressive compared to other dye binding aptamers and the gold standard 24xMS2 system, there is significant room for improvement. In this proposal, we will create Riboglow 2.0, with dramatically improved properties by systematically optimizing modules of the RNA/probe platform (Aim 1). In three independent subaims, we will exploit the modular nature of riboswitch structural motifs, the diversity of riboswitch sequences and power of in vitro selection to engineer optimized aptamer-linker pairs, RNA/probe combinations with enhanced fluorescence turn-on, and orthogonal aptamer/probe pairs to enable simultaneous detection of multiple RNAs with spectrally distinct probes. In our second aim, we will create a robust and systematic pipeline for characterizing, validating and benchmarking Riboglow 2.0 (Aim 2). We will define in vitro biochemical and biophysical properties, cellular contrast and single molecule sensitivity, demonstrate functionality for tagging different RNAs in diverse cellular assays, and ensure minimal cytotoxicity and perturbation of RNA function. Integration of Aim 1 and Aim 2 into an iterative cycle of design-engineer- characterize will result in a powerful Riboglow toolbox for diverse biological applications.

概括。信使 RNA 和非编码 RNA 复杂的时空动态几乎影响着生物体的所有方面。细胞功能。 RNA 除了充当 DNA 和蛋白质之间的中心中介之外，还调节基因在多个水平上表达，在表观遗传调控和基因组组织中发挥作用，并作为用于组装和整合大分子复合物的物理支架，对正常发育以及疾病病因。然而，尽管 RNA 在生物学中非常重要并且不断增长复杂和动态定位模式的证据，用于可视化活细胞中 RNA 分子的强大工具是非常有限的。最广泛使用的 RNA 标记系统涉及添加 24 个 MS2 茎环和与 GFP 融合的 MS2 结合蛋白的 48 个分子的结合，添加 > 1300 个核苷酸和 > 2.6 MDa 感兴趣的RNA。虽然这个系统揭示了基因各个步骤的诱人一瞥表达调节，也许并不奇怪，它也被证明会扰乱 mRNA 加工、剪接、本地化和衰变。因此，迫切需要强大的、互补的和最小干扰的可视化活细胞中单个 RNA 分子的工具，以绘制复杂且不断演变的 RNA 景观生物学。在这项工作中，我们将通过生成一套不同的基于核糖开关的 RNA 标签来满足这一需求相应的荧光探针，可对活体中的单个 RNA 分子进行同步、多色成像哺乳动物细胞。我们的方法建立在我们实验室的初步工作的基础上，该工作利用了自然界的一种适体，钴胺素 (Cbl) 结合核糖开关，作为 RNA 标签，结合一系列 Cbl 连接的荧光团诱导荧光开启，从而点亮感兴趣的 RNA。我们称这个新的 RNA 标记平台为 Riboglow 并展示了其在活哺乳动物细胞中可视化 mRNA 和小 U1 snRNA 的能力。尽管与其他染料结合适体和金标准相比，Riboglow 的性能令人印象深刻 24xMS2系统还有很大的改进空间。在本提案中，我们将创建 Riboglow 2.0，其中通过系统地优化 RNA/探针平台的模块，显着改善性能（目标 1）。在三个独立的子目标中，我们将利用核糖开关结构基序的模块化性质，核糖开关序列的多样性和体外选择的能力来设计优化的适体-连接子对， RNA/探针组合具有增强的荧光开启和正交适体/探针对，以实现使用光谱不同的探针同时检测多个 RNA。在我们的第二个目标中，我们将创建一个用于表征、验证和基准测试 Riboglow 2.0（目标 2）的强大而系统的管道。我们将定义体外生化和生物物理特性、细胞对比度和单分子敏感性，展示在不同细胞测定中标记不同 RNA 的功能，并确保最小的细胞毒性以及RNA功能的扰动。将目标 1 和目标 2 整合到设计-工程-的迭代循环中表征将产生一个强大的 Riboglow 工具箱，用于各种生物应用。