DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging

描述（由申请人提供）：DNA纳米标签：用于细胞内成像的明亮荧光标签和传感器了解人类疾病的分子基础对于开发副作用最小的有效治疗方法至关重要。健康和患病状态下的基本生物学过程涉及细胞内部和细胞表面的瞬时分子间（如蛋白质-蛋白质或蛋白质-核酸）相互作用。这些相互作用的实时直接成像提供了对分子关联的亲和和动力学的无与伦比的洞察力。绝大多数这样的实验是使用荧光显微镜和感兴趣的蛋白质与绿色荧光蛋白（GFP）或其他FPs之间的融合构建完成的。虽然这些融合蛋白已经取得了实质性进展，但由于感兴趣的蛋白丰度低，GFP相对较低的亮度和光稳定性阻碍了需要短成像时间或高灵敏度的应用。本提案的主要目标是创建一类新的明亮荧光标签，相对于GFP，它将表现出大大提高的亮度和光稳定性。这将通过合成由支链DNA纳米结构和数十个共价连接的插入染料组成的多色团来完成。这些DNA纳米标签的设计利用了50年来有关使用荧光插入染料检测DNA的知识，以及最近在DNA纳米结构的设计和合成方面的工作。纳米标签的高亮度源于它们非常大的有效消光系数，这是由于存在许多与每个DNA支架结合的染料。附加的较长波长的染料到DNA末端将导致有效的能量转移和调谐整个光谱的可见和近红外区域的荧光颜色。通过对染料和DNA结构的合理设计，优化纳米标签的生化和光化学稳定性。最后，纳米标签抗体结合物将被合成并测试用于酵母细胞表面和果蝇胚胎的标记。总的来说，这个建议结合了有机化学、单分子光谱和荧光显微镜来创造一类普遍有用的、明亮的荧光标签。DNA纳米标签：用于细胞内成像的明亮荧光标签和传感器拟议的研究将导致一种新型荧光标签，它将具有几乎任何颜色，并且可以附着在各种识别模块上，以便在非常低的浓度下对细胞表面和细胞内目标进行染色。检测和跟踪细胞中单个分子的能力将大大提高我们对基本生物过程的理解，以及健康和患病状态之间的分子水平差异，最终允许开发副作用更少的更有效的治疗方法。

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.