DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging

DNA 纳米标签：用于细胞内成像的明亮荧光标签和传感器

基本信息

批准号：
7490852
负责人：
Bruce A. ARMITAGE
金额：
$ 21.56万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7490852
关键词：
Adverse effects Affinity Alkynes Antibodies Azides Binding Biochemical Biological Process Cell Extracts Cell surface Cells Chemistry Chimeric Proteins Class Color Confocal Microscopy Coupled Cultured Cells DNA DNA Structure DNA biosynthesis DNA chemical synthesis Detection Development Drosophila genus Dyes Embryo Energy Transfer Enzymes Exhibits Extinction (Psychology)Flow Cytometry Fluorescence Fluorescence Microscopy Fluorescence Spectroscopy Fluorescent Dyes Glass Goals Green Fluorescent Proteins Image Imaging Techniques Intercalating Agents Kinetics Knowledge Label Lead Microscopy Molecular Monitor Nanostructures Nucleic Acids Optics Organic Chemistry Phycobiliproteins Predisposition Principal Investigator Proteins Range Relative (related person)Research Resistance Scaffolding Protein Signal Transduction Slide Solutions Staining method Stains Structure Testing Therapeutic Time Variant Work Yeasts antibody conjugate base bioimaging cycloaddition design fluorescence imaging fluorophore human disease improved insight interest nuclease prevent programs research study scaffold sensor single molecule

项目摘要

DESCRIPTION (provided by applicant): DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging Understanding the molecular basis for human disease is essential for developing effective therapeutics with minimal side effects. The fundamental biological processes underlying both healthy and diseased states involve transient intermolecular (e.g. protein-protein or protein-nucleic acid) interactions within and at the surface of cells. Direct imaging of these interactions in real time provides unparallelled insight into the affinity and kinetics of molecular association. The overwhelming majority of such experiments are done using fluorescence microscopy and fusion constructs between proteins of interest and green fluorescent protein (GFP) or other FPs. While substantial progress has been made with these fusion proteins, the relatively low brightness and photostability of GFP hinder applications that require either short imaging times or high sensitivity due to low abundance of the protein of interest. The main objective of this proposal is to create a new class of bright fluorescent labels that will exhibit greatly improved brightness and photostability relative to GFP. This will be accomplished by synthesizing polychromophore assemblies consisting of a branched DNA nanostructure with dozens of covalently attached intercalating dyes. The design of these DNA nanotags takes advantage of 50 years of knowledge concerning the use of fluorescent intercalating dyes for the detection of DNA as well as more recent work in the design and synthesis of DNA nanostructures. The high brightness of the nanotags derives from their very large effective extinction coefficients due to the presence of many dyes bound to each DNA scaffold. Attachment of additional longer wavelength dyes to the DNA termini will lead to efficient energy transfer and tuning of the fluorescence color throughout the visible and near-IR regions of the spectrum. The nanotags will be optimized in terms of their biochemical and photochemical stability through rational design of the dye and DNA structures. Finally, nanotag-antibody conjugates will be synthesized and tested for labeling of yeast cell surfaces and within Drosophila embryos. Overall, this proposal combines organic chemistry, single- molecule spectroscopy and fluorescence microscopy to create a new class of generally useful, bright fluorescent labels. Project Narrative DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging The proposed research will lead to a new class of fluorescent labels that will be available in virtually any color and can be attached to various recognition modules to allow staining of cell surface and intracellular targets present at very low concentration. The ability to detect and track single molecules in cells will significantly advance our understanding of fundamental biological processes and the molecular-level distinctions between healthy and diseased states, ultimately allowing development of more potent therapeutics with fewer side effects.

描述（由申请人提供）：DNA纳米塔：明亮的荧光标签和传感器，用于了解人类疾病的分子基础的细胞内成像对于开发具有最小副作用的有效疗法至关重要。健康和患病状态的基本生物学过程涉及细胞表面和在细胞表面内部和处的短暂分子间（例如蛋白质 - 蛋白质或蛋白核酸）相互作用。实时对这些相互作用的直接成像为分子关联的亲和力和动力学提供了无与伦比的见解。绝大多数此类实验都是使用荧光显微镜和感兴趣蛋白质之间的融合构建体和绿色荧光蛋白（GFP）或其他FPS进行的。尽管这些融合蛋白已经取得了实质性的进展，但是由于感兴趣的蛋白质的丰富性低，因此GFP障碍的亮度和光稳定性相对较低，而GFP障碍物的应用需要短的成像时间或高灵敏度。该提案的主要目的是创建一类新的明亮荧光标签，相对于GFP，其亮度和光稳定性将大大提高。这将通过合成由分支DNA纳米结构和数十个共同附着的互化染料的分支DNA纳米结构组成。这些DNA纳米塔的设计利用了50年的知识，即使用荧光互化染料来检测DNA以及在设计和合成DNA纳米结构方面的最新工作。纳米塔的高亮度源于其非常大的有效灭绝系数，因为存在许多与每个DNA支架结合的染料。在频谱的整个可见和近红外区域中，额外的更长的波长染料的附着将导致有效的能量转移和荧光颜色调整。通过染料和DNA结构的理性设计，将根据其生化和光化学稳定性来优化纳米塔。最后，将合成纳米抗体 - 抗体结合物，并测试酵母细胞表面和果蝇胚胎中的标记。总体而言，该建议结合了有机化学，单分子光谱和荧光显微镜，以创建一类新的有用，明亮的荧光标签。项目叙述性DNA纳米塔：明亮的荧光标签和用于细胞内成像的传感器拟议的研究将导致一类新的荧光标签，这些标签几乎将以任何颜色提供，并且可以连接到各种识别模块上，以允许细胞表面和细胞内靶标的染色以非常低的浓度存在。检测和跟踪细胞中单分子的能力将显着提高我们对健康和患病状态之间的基本生物学过程以及分子级别的理解，最终允许开发更有效的疗法，具有较少的副作用。