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
- 来源:
- 关键词:AffectAffinityBenchmarkingBindingBinding ProteinsBiochemicalBiochemistryBiologicalBiological AssayBiologyCell physiologyCellsCellular AssayCellular biologyChemicalsCobalaminColorComplexCytoplasmic GranulesCytosolDNADetectionDeteriorationDevelopmentDiseaseDyesEngineeringEnsureEnvironmentEtiologyFluorescenceFluorescent ProbesGene ExpressionGene Expression RegulationGenerationsGeneticGenetic TranscriptionGenomeGoalsGoldImageIn VitroIndividualLigand BindingLigandsLightingLinkMacromolecular ComplexesMammalian CellMapsMeasurementMessenger RNAModificationMolecular ProbesNatureNucleic AcidsNucleotidesPatternPerformancePhysiologyPlayPropertyProteinsRNARNA BindingRNA DecayRNA ProbesRNA SplicingRegulationRibonucleoproteinsRoleSeriesStructureSystemTestingToxic effectTranscriptTranscription InitiationTranscriptional RegulationTranslatingTranslationsU1 small nuclear RNAUntranslated RNAValidationVariantVisualizationWorkaptamerbasebiophysical propertiescytotoxicityengineering designepigenetic regulationfluorophoregenetic informationimaging platformimprovedinterestlive cell imagingmolecular imagingportabilitypreventscaffoldsingle moleculesmall moleculespatiotemporalstemtechnology developmenttoolvirtual
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.
总结。
信使RNA和非编码RNA的复杂时空动力学几乎影响到
细胞功能。除了作为DNA和蛋白质之间的中心中介外,RNAs还调节
基因在多个水平上的表达,在表观遗传调节和基因组组织中发挥作用,并作为
组装和整合大分子络合物的物理支架,具有重要的意义
正常发育,以及疾病病因学。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,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标签系统包括添加24个MS2茎环和
48个MS2结合蛋白分子与GFP的结合,增加了1300个核苷酸和2.6个丙二醛
一种感兴趣的RNA。虽然这个系统显示了诱人的基因步骤一瞥
表达调控,也许并不令人惊讶,它也被证明扰乱了mRNA的加工,剪接,
本土化和腐朽。因此,迫切需要健壮的、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图景
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标签和
相应的荧光探针可同时对活体单个RNA分子进行多色成像
哺乳动物细胞。我们的方法建立在实验室的初步工作基础上,这些实验室利用了自然界的一种
适配子,钴胺(Cbl)结合的核糖开关,作为RNA标签与一系列Cbl连接的荧光团结合
以诱导荧光开启,从而照亮感兴趣的RNA。我们把这个新的RNA标签平台称为
Riboglow,并证明了其在活的哺乳动物细胞中显示mRNA和小的U1 SnRNA的能力。而当
与其他染料结合适配子和黄金标准相比,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,
通过系统地优化RNA/探针平台的模块,显著提高了性能
(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,即
核糖开关序列的多样性和体外选择以设计优化的适体-连接物对的能力,
具有增强荧光开启功能的RNA/探针组合,以及支持
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Riboglow 2.0(目标2)的强大和系统的流水线。
我们将定义体外生化和生物物理性质、细胞对比度和单分子敏感性,
演示在不同的细胞检测中标记不同RNA的功能,并确保将细胞毒性降至最低
和RNA函数的摄动。将目标1和目标2集成到设计工程师的迭代周期中-
Characterize将为不同的生物应用提供一个强大的Riboglow工具箱。
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:
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:
8657054 - 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:
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
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:
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:
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














{{item.name}}会员




