In vivo Detection and Imaging of Epigenetic Histone Modifications and Modifying E

表观遗传组蛋白修饰和修饰 E 的体内检测和成像

• 批准号: 8471090
• 负责人: HAROLD G CRAIGHEAD
• 金额: $ 49.55万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471090
• 关键词: Accounting; Address; Affinity; Animals; Binding; Biological Assay; Biological Sciences; Biomedical Engineering; Buffers; Cell physiology; Cells; Cellular biology; Chemistry; Chromatin; Collaborations; Complement; Detection; Devices; Diploid Cells; Disease; Drosophila genus; Dyes; Electronics; Engineering; Environment; Enzymes; Epigenetic Process; Epitopes; Fluorescence; Fluorescence Spectroscopy; Genes; Genetic; Genetic Transcription; Goals; Health; Histones; Human; Image; Imagery; Imaging technology; In Vitro; Individual; Knowledge; Libraries; Life; Location; Measures; Medical; Methods; Microscopy; Molecular; Movement; Noise; Parents; Peptides; Physics; Proteins; RNA; RNA Binding; RNA Stability; Reagent; Recruitment Activity; Regulation; Research; Research Personnel; Resources; Role; Salivary Glands; Scheme; Signal Transduction; Sorting - Cell Movement; Specificity; Spectrum Analysis; Structure; System; Technology; Testing; Time; Tissues; Total Internal Reflection Fluorescent; abstracting; analog; aptamer; aqueous; base; cyanine dye; design; design and construction; fluorophore; fly; histone modification; imaging modality; improved; in vitro testing; in vivo; insight; multi-photon; nanofluidic; novel; positional cloning; public health relevance; quantum; single molecule; small molecule; tool; triphenylmethane

DESCRIPTION (provided by applicant): Project Summary/Abstract: The main goal of this project is to develop a simple, yet powerful and versatile technology for detection and imaging of epigenetic histone modifications and histone modifying enzymes in living cells. A full understanding of how various histone modifications and the responsible modifying enzymes function require precise knowledge of their localization, movement, and dynamics in a living cell. Current technologies lack the sensitivity and the versatility required to track these targets in their native environment. To achieve these goals, we propose to construct multivalent RNA aptamers comprised of three main features; i) a peptide/protein targeting aptamer, ii) multiple copies of an aptamers that bind and enhance fluorescence of weakly fluorescing analogs of normally highly-fluorescent small molecules (referred to as fluorescent molecule analogs (FMAs)), and iii) a core multi-way RNA junction that combines the previous types of aptamers in a compact structure. We propose to express such multivalent aptamers in cells or whole animals. The MPFAs will also bind FMAs exogenously supplied to cells. Since the FMA binding aptamers will have been selected to enhance dramatically the quantum yield of the FMA molecule upon binding, the MPFAs will render the target visible for detection with fluorescent microscopy. Different combinations of peptide/protein-targeting and FMA-binding aptamers with spectrally separable FMAs will be utilized for multiplex imaging of multiple proteins within the same cell. A key advantage of this approach is that it does not depend on covalent attachment of the imaging moiety, which can interfere with the localization and function of the target protein. The multivalent aptamers can be expressed just prior to imaging and therefore cause little interference with the function of the target protein or the general physiology of the cell. The designed MPFAs will then be tested carefully for in vivo imaging of the select set of histone modification and modifying enzyme targets in Drosophila salivary gland cells and diploid cells. Sensitivity of the proposed technology will be compared to existing detection/imaging methods. The ultimate goal in detection sensitivity is the single-molecule in vivo detection/imaging using MPFAs and exogenously supplied FMAs. The project brings together principle investigators that have complementary expertise in chemistry; design, synthesis and characterization of fluorescent molecules and their derivatives (Lin Lab), in molecular/cell biology; gene and chromatin regulation, genetics and reverse- genetics, RNA aptamer selections, and design and expression of multivalent RNAs (Lis Lab), in applied and engineering physics; design and fabrication of micro- and nano-fluidic mechano-electronic devices (Craighead Lab), in biomedical engineering; fluorescence confocal and multi-photon microscopy and photophysical characterization of FMAs (Zipfel Lab) and a proven record of productive collaborations.

描述（由申请人提供）： 项目摘要/摘要：该项目的主要目标是开发一种简单但强大且多功能的技术，用于活细胞中表观遗传组蛋白修饰和组蛋白修饰酶的检测和成像。要充分了解各种组蛋白修饰和负责的修饰酶如何发挥作用，需要精确了解它们在活细胞中的定位、运动和动态。当前的技术缺乏在本地环境中跟踪这些目标所需的灵敏度和多功能性。为了实现这些目标，我们建议构建由三个主要特征组成的多价RNA适体： i) 肽/蛋白质靶向适体，ii) 适体的多个拷贝，可结合并增强通常高荧光小分子的弱荧光类似物（称为荧光分子类似物 (FMA)）的荧光，以及 iii) 核心多拷贝双向RNA连接将先前类型的适体组合成紧凑的结构。我们建议在细胞或整个动物中表达此类多价适体。 MPFA 还将结合外源提供给细胞的 FMA。由于选择 FMA 结合适体来显着提高 FMA 分子结合后的量子产率，MPFA 将使目标可见，以便用荧光显微镜检测。肽/蛋白质靶向适体和 FMA 结合适体与光谱可分离的 FMA 的不同组合将用于同一细胞内多种蛋白质的多重成像。这种方法的一个关键优点是它不依赖于成像部分的共价连接，共价连接可能会干扰靶蛋白的定位和功能。多价适体可以在成像之前表达，因此对靶蛋白的功能或细胞的一般生理学几乎没有干扰。然后，将仔细测试设计的 MPFA，以对果蝇唾液腺细胞和二倍体细胞中选定的组蛋白修饰和修饰酶靶标进行体内成像。所提出技术的灵敏度将与现有的检测/成像方法进行比较。检测灵敏度的最终目标是使用 MPFA 和外源提供的 FMA 进行单分子体内检测/成像。该项目汇集了在化学方面具有互补专业知识的主要研究人员；分子/细胞生物学中荧光分子及其衍生物的设计、合成和表征（Lin Lab）；应用和工程物理学中的基因和染色质调控、遗传学和反向遗传学、RNA适体选择以及多价RNA的设计和表达（Lis实验室）；生物医学工程领域的微纳流体机械电子设备的设计和制造（Craighead 实验室）；荧光共焦和多光子显微镜以及 FMA 的光物理表征（Zipfel 实验室）以及富有成效的合作记录。