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

基本信息

批准号：
8663853
负责人：
HAROLD G CRAIGHEAD
金额：
$ 50.08万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-15 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8663853
关键词：
Accounting Affinity Animals Binding Biological Assay Biomedical Engineering Buffers Cell physiology Cells Cellular biology Chemistry Chromatin Collaborations Detection Devices Diploid Cells Drosophila genus Dyes Electronics Engineering Environment Enzymes Epigenetic Process Epitopes Fluorescence Fluorescence Spectroscopy Genes Genetic Genetic Transcription Goals Histones Human Image Imagery In Vitro Individual Knowledge Libraries Life Location Measures Methods Microscopy Molecular Movement Noise Parents Peptides Physics Proteins RNA RNA Binding RNA Stability Reagent Recruitment Activity Regulation 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 in vitro testing in vivo in vivo imaging multi-photon nanofluidic positional cloning quantum single molecule small molecule tool triphenylmethane

项目摘要

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，功能强大且多才多艺的技术，用于检测和成像表观遗传组蛋白修饰和组蛋白修饰活细胞中的酶。对各种组蛋白修饰和负责任修饰酶功能的充分理解需要精确了解其定位，运动和活细胞中的动态。当前的技术缺乏在本机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）一种靶向适时的肽/蛋白质，ii）适体的多个拷贝，它们结合并增强了正常高荧光的小分子的弱荧光类似物的荧光（称为荧光分子类似物（FMA）（FMAS），以及III），将核心RNA连接组合为以前的构建类型的核心RNA结构。我们建议在细胞或整个动物中表达这种多价适体。 MPFA还将结合外源供应到细胞的FMA。由于FMA结合适体将被选择以显着增强FMA分子结合后的量子产率，因此MPFA将使靶标可通过荧光显微镜检测可见。肽/蛋白质靶向和FMA结合适体具有不同可分离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，它不取决于成像部分的共价附着，这可能会干扰目标蛋白的定位和功能。多价适体可以在成像之前表达，因此很少干扰靶蛋白的功能或细胞的一般生理学。然后，将仔细测试设计的MPFA，以在果蝇唾液腺细胞和二倍体细胞中选择的组蛋白修饰和修饰酶靶标的体内成像。将提议的技术的敏感性与现有检测/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的体内检测/成像中的单分子。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/细胞生物学中的荧光分子及其衍生物的设计，合成和表征；在应用和工程物理学中，基因和染色质调节，遗传学和反向遗传学，RNA适体选择以及多价RNA（LIS LAB）的设计和表达；生物医学工程中的微富集机械设备（Craighead Lab）的设计和制造； FMA（Zipfel Lab）的荧光共聚焦和多光子显微镜以及光物理表征以及可靠的生产性合作记录。