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

描述（由申请人提供）： 项目概要/摘要：该项目的主要目标是开发一种简单，但功能强大和通用的技术，用于检测和成像活细胞中的表观遗传组蛋白修饰和组蛋白修饰酶。要全面了解各种组蛋白修饰和负责修饰酶的功能，需要精确了解它们在活细胞中的定位、运动和动力学。目前的技术缺乏在其原生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，我们提出构建多价RNA适体，其包括三个主要特征：i）肽/蛋白质靶向适体，ii）适体的多个拷贝，其结合正常高荧光小分子的弱荧光类似物并增强其荧光（称为荧光分子类似物（FMA）），和iii）核心多路RNA连接，其以紧凑结构组合先前类型的适体。我们建议在细胞或整个动物中表达这样的多价适体。MPFA还将结合外源提供给细胞的FMA。由于FMA结合适体将被选择为在结合时显著增强FMA分子的量子产率，因此MPFA将使靶标可见以用于荧光显微镜检测。肽/蛋白质靶向和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，这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。然后将仔细测试所设计的MPFA，用于果蝇唾液腺细胞和二倍体细胞中组蛋白修饰和修饰酶靶点的选择集的体内成像。所提出的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、荧光分子及其衍生物的设计、合成和表征（Lin实验室）、分子/细胞生物学、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的设计和表达（Lis实验室）方面具有互补专长的主要研究人员;在生物医学工程领域，他的研究领域包括微流控和纳米流控机械电子器件的设计和制造（Craighead实验室）;荧光共聚焦和多光子显微镜以及FMA的生物物理表征（Zipfel实验室），以及富有成效的合作记录。 公共卫生相关性： 项目叙述：本申请题为“使用多价RNA适体的表观遗传组蛋白修饰和修饰酶的体内检测和成像”，涉及RFA-RM-09-016：开发用于改进体内表观遗传成像或分析的技术。该项目预计将对医学和生命科学研究产生重大影响，通过开发一种灵敏的基于多价RNA适体的检测/成像技术，允许对两种或多种表观遗传组蛋白修饰和/或修饰酶进行体内多重成像。这种新的强大和多功能的技术将补充现有的试剂和技术，克服现有成像技术的许多技术限制，从而提供进一步的见解和更全面的了解与人类健康和疾病有关的组蛋白修饰的表观遗传调控。

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. PUBLIC HEALTH RELEVANCE: Project Narrative: This application titled "In vivo Detection and Imaging of Epigenetic Histone Modifications and Modifying Enzymes Using Multivalent RNA Aptamers" addresses the RFA-RM-09-016: Developing Technologies for Improved In Vivo Epigenetic Imaging or Analysis. The project is expected to have a major impact on medical and life sciences research by developing a sensitive multivalent RNA aptamer-based detection/imaging technology that allows multiplex imaging of two or more epigenetic histone modifications and/or modifying enzymes in vivo. This novel powerful and versatile technology will complement the existing reagents and technologies, overcome many of the technical limitations of existing imaging technologies, and thus provide further insights and a more comprehensive understanding of the epigenetic regulation of histone modifications that relate to human health and diseases.