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：
9904726
负责人：
Robert T Batey
金额：
$ 30.3万
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/9904726
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 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 Visualization 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。 Messenger RNA和非编码RNA的复杂空间时间动态几乎影响细胞功能。除了作为DNA和蛋白质之间的中间中间体外，RNA还调节基因表达在多个层面上，在表观遗传调节和基因组组织中起作用，并充当物理脚手架以组装和整合大分子复合物，对正常发育以及疾病病因。然而，努力RNA在生物学和增长中的重要性复杂和动态定位模式的证据，可视化活细胞中RNA分子的强大工具高度有限。使用最广泛的RNA标记系统涉及添加24 ms2茎环和 MS2结合蛋白的48个分子的结合与GFP融合，添加> 1300个核苷酸和> 2.6 MDA 感兴趣的RNA。虽然该系统揭示了对基因各个步骤的诱人瞥见表达调控，也许不足为奇，它也已显示出扰动mRNA处理，剪接，本地化和衰减。那就是迫切需要强大，完整和微不足道的扰动可视化活细胞中单个RNA分子以绘制RNA的复合和不断发展的景观的工具生物学。在这项工作中，我们将通过生成一套基于潜水员的Riboswitch的RNA标签，并满足这一需求。同时实时RNA分子的同时多色成像的相应荧光问题哺乳动物细胞。我们的方法是基于利用自然界之一的实验室的初步工作适体，钴胺（CBL）结合核糖开关作为结合一系列CBL连接荧光团的RNA标签诱导荧光转机，从而点亮感兴趣的RNA。我们称这个新的RNA标记平台 Riboglow并证明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体和金标准相比，Riboglow的性能令人印象深刻 24xms2系统，有很大的改进空间。在此提案中，我们将创建Riboglow 2.0 通过系统地优化RNA/探针平台的模块，大大改善了属性（目标1）。在三个独立的子aim中，我们将探索核糖开关结构图案的模块化性质，即核糖开关序列的多样性和体外选择的功率对工程师优化的合并链链链对， RNA/探针组合具有增强的荧光转机和正交的pepater/探针对以启用简单检测具有频谱不同问题的多个RNA。在我们的第二个目标中，我们将创建一个可靠和系统的管道来表征，验证和基准测试Riboglow 2.0（AIM 2）。我们将定义体外生化和生物物理特性，细胞对比和单分子敏感性，展示了在各种细胞测定中标记不同RNA的功能，并确保细胞毒性最小 RNA功能的扰动。将AIM 1和AIM 2整合到设计工程师的迭代循环中特征将为潜水员生物学应用提供强大的Riboglow工具箱。