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:
  • 金额:
    $ 30.3万
  • 依托单位:
  • 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

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.
摘要 信使RNA和非编码RNA的复杂时空动力学几乎影响了基因表达的所有方面。 细胞功能除了作为DNA和蛋白质之间的中间媒介外,RNA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 物理支架组装和整合大分子复合物,具有重要意义, 正常发育,以及疾病的病因。然而,尽管RNA在生物学和生长中的重要性 复杂和动态定位模式的证据,活细胞中RNA分子可视化的强大工具 是非常有限的。最广泛使用的RNA标记系统涉及添加24个MS 2茎环, 48个分子的MS 2结合蛋白与GFP融合,添加> 1300个核苷酸和> 2.6 MDa, 感兴趣的RNA。虽然这个系统已经揭示了基因的各个步骤的诱人一瞥, 表达调控,也许并不令人惊讶,它也已被证明扰乱mRNA加工,剪接, 定位和衰变。因此,迫切需要一个强大的,互补的,和最小的干扰, 可视化活细胞中单个RNA分子的工具,以绘制复杂和不断演变的RNA景观 生物学在这项工作中,我们将通过产生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,用于活体中单个RNA分子的同时多色成像, 哺乳动物细胞我们的方法建立在我们实验室的初步工作基础上, 适体,钴胺素(Cbll)结合核糖开关作为RNA标签,结合一系列Cbll连接的荧光团 以诱导荧光开启,从而照亮感兴趣的RNA。我们把这个新的RNA标签平台 Riboglow,并证明了其能够可视化mRNA和小U1 snRNA在活的哺乳动物细胞。而 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 24 xMS 2系统,还有很大的改进空间。在本提案中,我们将创建Riboglow 2.0, 通过系统地优化RNA/探针平台的模块, (Aim 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以工程化优化的适体-接头对, 具有增强的荧光开启的RNA/探针组合和正交适体/探针对, 用光谱不同的探针同时检测多种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(Aim 2)的强大而系统的管道。 我们将定义体外生物化学和生物物理特性,细胞对比度和单分子敏感性, 在不同的细胞分析中展示标记不同RNA的功能,并确保最小的细胞毒性 和RNA功能的干扰。将目标1和目标2整合到设计-工程师- 将导致一个功能强大的Riboglow工具箱,用于各种生物应用。

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Robert T Batey其他文献

Robert T Batey的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('Robert T Batey', 18)}}的其他基金

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
  • 批准号:
    9764689
  • 财政年份:
    2019
  • 资助金额:
    $ 30.3万
  • 项目类别:
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
  • 批准号:
    10374881
  • 财政年份:
    2019
  • 资助金额:
    $ 30.3万
  • 项目类别:
lncRNAs as Organizers of and Bridges Between Proteins and DNA
lncRNA 作为蛋白质和 DNA 的组织者和桥梁
  • 批准号:
    9356528
  • 财政年份:
    2016
  • 资助金额:
    $ 30.3万
  • 项目类别:
lncRNAs as Organizers of and Bridges Between Proteins and DNA
lncRNA 作为蛋白质和 DNA 的组织者和桥梁
  • 批准号:
    9158537
  • 财政年份:
    2016
  • 资助金额:
    $ 30.3万
  • 项目类别:
Purchase of an Isothermal Titration Calorimeter
购买等温滴定量热计
  • 批准号:
    7792160
  • 财政年份:
    2010
  • 资助金额:
    $ 30.3万
  • 项目类别:
Structure and Mechanism of SAM-responsive Riboswitches
SAM响应核糖开关的结构和机制
  • 批准号:
    7434273
  • 财政年份:
    2008
  • 资助金额:
    $ 30.3万
  • 项目类别:
Structure and Mechanism of SAM-responsive Riboswitches
SAM响应核糖开关的结构和机制
  • 批准号:
    8036043
  • 财政年份:
    2008
  • 资助金额:
    $ 30.3万
  • 项目类别:
Structure and Mechanism of SAM-responsive Riboswitches
SAM响应核糖开关的结构和机制
  • 批准号:
    8369542
  • 财政年份:
    2008
  • 资助金额:
    $ 30.3万
  • 项目类别:
Structure and Mechanism of SAM-responsive Riboswitches
SAM响应核糖开关的结构和机制
  • 批准号:
    8516526
  • 财政年份:
    2008
  • 资助金额:
    $ 30.3万
  • 项目类别:
Structure and Mechanism of SAM-responsive Riboswitches
SAM响应核糖开关的结构和机制
  • 批准号:
    7616428
  • 财政年份:
    2008
  • 资助金额:
    $ 30.3万
  • 项目类别:

相似海外基金

Construction of affinity sensors using high-speed oscillation of nanomaterials
利用纳米材料高速振荡构建亲和传感器
  • 批准号:
    23H01982
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Affinity evaluation for development of polymer nanocomposites with high thermal conductivity and interfacial molecular design
高导热率聚合物纳米复合材料开发和界面分子设计的亲和力评估
  • 批准号:
    23KJ0116
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Grant-in-Aid for JSPS Fellows
Development of High-Affinity and Selective Ligands as a Pharmacological Tool for the Dopamine D4 Receptor (D4R) Subtype Variants
开发高亲和力和选择性配体作为多巴胺 D4 受体 (D4R) 亚型变体的药理学工具
  • 批准号:
    10682794
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
Platform for the High Throughput Generation and Validation of Affinity Reagents
用于高通量生成和亲和试剂验证的平台
  • 批准号:
    10598276
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
  • 批准号:
    2233343
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Standard Grant
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
  • 批准号:
    2233342
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Standard Grant
Molecular mechanisms underlying high-affinity and isotype switched antibody responses
高亲和力和同种型转换抗体反应的分子机制
  • 批准号:
    479363
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Operating Grants
Deconstructed T cell antigen recognition: Separation of affinity from bond lifetime
解构 T 细胞抗原识别:亲和力与键寿命的分离
  • 批准号:
    10681989
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
CAREER: Engineered Affinity-Based Biomaterials for Harnessing the Stem Cell Secretome
职业:基于亲和力的工程生物材料用于利用干细胞分泌组
  • 批准号:
    2237240
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Continuing Grant
ADVANCE Partnership: Leveraging Intersectionality and Engineering Affinity groups in Industrial Engineering and Operations Research (LINEAGE)
ADVANCE 合作伙伴关系:利用工业工程和运筹学 (LINEAGE) 领域的交叉性和工程亲和力团体
  • 批准号:
    2305592
  • 财政年份:
    2023
  • 资助金额:
    $ 30.3万
  • 项目类别:
    Continuing Grant
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了