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

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、但功能强大且通用的技术，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。全面了解各种组蛋白修饰和负责任的修饰酶如何发挥作用，需要对它们在活细胞中的定位、运动和动力学有精确的了解。目前的技术缺乏在其本国环境中跟踪这些目标所需的敏感性和多功能性。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适配子：i)多肽/蛋白质靶向适配子，ii)多个拷贝的适配子，它结合并增强正常高荧光小分子的弱荧光类似物(称为荧光分子类似物(FMA))，以及iii)核心多路RNA连接，它在紧凑的结构中结合了前面类型的适配子。我们建议在细胞或整个动物中表达这种多价适配子。MPFA还将结合外源性供应给细胞的FMA。由于FMA结合适体将被选择来显著提高FMA分子结合时的量子产率，MPFA将使目标可见，以便用荧光显微镜进行检测。多肽/蛋白质靶向和FMA结合适体与光谱可分离的FMA的不同组合将被用于同一细胞内多个蛋白质的多重成像。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，因为共价连接会干扰目标蛋白的定位和功能。多价适配子可以在成像前表达，因此对目标蛋白的功能或细胞的一般生理影响很小。然后，设计的MPFA将被仔细测试，用于在果蝇唾液腺细胞和二倍体细胞中对选定的组蛋白修饰和修饰酶靶标进行体内成像。建议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进行体内单分子检测/成像。该项目汇集了在以下方面具有互补专长的主要研究人员：化学；荧光分子及其衍生物的设计、合成和表征(LIN Lab)，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适配子的选择以及多价RNA的设计和表达(Lis Lab)；生物医学工程中微纳射流机械电子器件的设计和制造(Craighead Lab)；荧光共焦和多光子显微镜及FMA的光物理表征(Zipfel Lab)以及经证实的富有成效的合作记录。 公共卫生相关性： 项目简介：本申请题为“使用多价RNA适配子对表观遗传组蛋白修饰和修饰酶进行活体检测和成像”，介绍了RFA-RM-09-016：开发用于改进活体表观遗传学成像或分析的技术。该项目有望对医学和生命科学研究产生重大影响，开发一种基于多价RNA适配子的灵敏检测/成像技术，允许在体内对两个或更多表观遗传组蛋白修饰和/或修饰酶进行多重成像。这项新的、强大和通用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，从而提供更深入的见解和更全面的理解与人类健康和疾病相关的组蛋白修饰的表观遗传调节。

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.