Genetically Encoded Small Illuminants for 4D nucleome imaging

用于 4D 核组成像的基因编码小光源

基本信息

批准号：
9003351
负责人：
R.Holland Cheng
金额：
$ 33万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9003351
关键词：
Acetylation Acylation Animals Antibodies Binding Biological Biological Process Cell Nucleus Cells Chemicals Chromatin Complementary DNA Coupled Cryoelectron Microscopy DNA Repair Deposition Development Dyes Electron Microscopy Environment Enzymes Epigenetic Process Event Exhibits Figs - dietary Fluorescence Fluorescence Resonance Energy Transfer Gene Expression Genetic Histone H2A Histones Image Implant Label Libraries Life Ligand Binding Maps Mediating Metabolic Methodology Methods Methylation Molecular N-terminal Nuclear Nucleosomes Nude Mice Peptide Library Peptides Phosphorylation Physiological Post-Translational Protein Processing Precipitation Property Proteins Regulation Reporting Series Signal Transduction Site Staining method Stains Techniques Technology Testing Time Tissues Transgenic Animals Ubiquitination Xenograft procedure cellular imaging combinatorial dalton design functional status histone modification histone-binding proteins human H2AX protein imaging system in vivo interest molecular imaging neoplastic cell new technology novel public health relevance screening small hairpin RNA spatiotemporal

项目摘要

DESCRIPTION (provided by applicant): We propose to develop a novel technology that allows one to image the spatiotemporal dynamics of the epigenetic functional status of histones within the chromatin in real time, thus enabling 4D-nucleome imaging in living cells at single cell level. The proposed molecular imaging system is developed by using a one-bead-one-compound (OBOC) combinatorial library screen to identify short peptide(s) that activate fluorescence of organic dyes or molecular rotors. Activation of fluorescence is coupled to alterations of their chemical environment including conformational change upon ligand binding and phosphorylation, acetylation, methylation, or ubiquitination of the peptide. These peptides can then be genetically fused to target proteins such as histones to enable functional cellular imaging in living cells in real time. We will adapt our newly developed technology to the epigenetics studies in this application. Hypothesis: The Genetically Encoded Small Illuminant (GESI) technology, comprised of OBOC combinatorial peptide library design and serial screening of huge arrays of immobilized bead (~1 million diversities) under pre-defined conditions, enables identification of short peptide- dye pairs that can be used as genetically encoded illuminants to probe post-translational modification of histones in nucleosomes, temporary and spatially in living cells in real time, thus enabling 4D nucleome imaging. Fluorescently activated GESI sites can be covalently marked for subsequent correlative fluorescent and electron microscopy, and chromatin precipitation via dye/GESI interaction. Impact: GESI peptides can specifically bind to and activate the fluorescence of selected organic dyes. Some GESI peptides will do so only after binding to cellular components such as Ca2+, conformational changes or post-translational modifications (PTMs). Therefore, when expressed in a living cell, they can illuminate the spatiotemporal regulation and modification of proteins of interest. The genetic illuminants are small (1200-1900 daltons), thus can be readily inserted along the sequence of the native proteins without interfering with their physiological functions. Multiplexing is possible and allows us to study cross talks of different histone PMTs and/or recruitments of histone binding proteins in real time. Specific aims of the proposed project are: Aim 1. To design and synthesize a series of organic dyes suitable for GESI reporting in the nucleus of living cells. Aim 2. To develop GESIs to track the spatiotemporal dynamics of subtype of histone H2A (H2AX and H2AZ). Aim 3. To develop GESIs to newly identify acylations status of N-terminus histone H2A/H3 inside living cells.

描述（由申请人提供）：我们建议开发一种新技术，使人们能够实时成像染色质内组蛋白表观遗传功能状态的时空动态，从而实现单细胞活细胞中的 4D 核组成像所提出的分子成像系统是通过使用一珠一化合物（OBOC）组合库筛选来识别激活有机染料或分子转子荧光的短肽。它们的化学环境包括配体结合时的构象变化以及肽的磷酸化、乙酰化、甲基化或泛素化，然后这些肽可以与组蛋白等目标蛋白进行基因融合。为了实现活细胞中的实时功能细胞成像，我们将把我们新开发的技术应用于本应用中的表观遗传学研究：遗传编码小光源（GESI）技术，包括 OBOC 组合肽库设计和序列筛选。在预定义条件下的巨大固定珠阵列（约 100 万种多样性）能够识别短肽-染料对，这些短肽-染料对可用作基因编码发光体进行探测实时和空间地实时对核小体中的组蛋白进行翻译后修饰，从而实现 4D 核组成像。可以共价标记荧光激活的 GESI 位点，用于随后的相关荧光和电子显微镜检查，以及通过染料/GESI 相互作用进行染色质沉淀。影响：GESI 肽可以特异性结合并激活选定的有机染料的荧光，某些 GESI 肽只有在与细胞结合后才会这样做。因此，当在活细胞中表达时，它们可以阐明蛋白质的时空调节和修饰。遗传发光体很小（1200-1900 道尔顿），因此可以很容易地沿着天然蛋白质的序列插入，而不干扰其生理功能，并且允许我们研究不同组蛋白 PMT 和/或的交叉对话。该项目的具体目标是：目的 1. 设计和合成一系列适合活细胞核内 GESI 报告的有机染料。 2. 开发 GESI 来追踪组蛋白 H2A 亚型（H2AX 和 H2AZ）的时空动态目标 3. 开发 GESI 来新识别活细胞内 N 末端组蛋白 H2A/H3 的酰化状态。