DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging

描述(申请人提供)：DNA纳米标记：用于细胞内成像的明亮的荧光标记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疗法至关重要。健康和疾病状态背后的基本生物学过程涉及细胞内和细胞表面的瞬时分子间相互作用(如蛋白质-蛋白质或蛋白质-核酸)。这些相互作用的实时直接成像提供了对分子结合的亲和力和动力学的无与伦比的洞察。绝大多数这样的实验是使用荧光显微镜和目标蛋白与绿色荧光蛋白(GFP)或其他FP之间的融合结构来完成的。虽然这些融合蛋白已经取得了实质性的进展，但GFP相对较低的亮度和光稳定性阻碍了需要较短成像时间或由于目标蛋白丰度较低而需要高灵敏度的应用。这项提议的主要目标是创造一种新的明亮的荧光标签，相对于GFP，它将显示出极大的亮度和光稳定性。这将通过合成由分枝DNA纳米结构和数十种共价连接的插层染料组成的多发色团组件来实现。这些DNA纳米标签的设计利用了50年来关于使用荧光插层染料检测DNA的知识，以及最近在DNA纳米结构设计和合成方面的工作。纳米标签的高亮度来自于它们非常大的有效消光系数，这是由于每个DNA支架上结合了许多染料。将额外的更长波长的染料连接到DNA末端将导致有效的能量转移和整个光谱的可见光和近红外区域荧光颜色的调谐。通过合理设计染料和DNA结构，纳米标签将在其生化和光化学稳定性方面进行优化。最后，将合成纳米标签-抗体结合物，并测试用于酵母细胞表面和果蝇胚胎内的标记。总体而言，这项提议结合了有机化学、单分子光谱学和荧光显微镜，创造了一种新的通用、明亮的荧光标记。项目叙述DNA纳米标签：用于细胞内成像的明亮的荧光标签和传感器拟议的研究将导致一种新的荧光标签，这种标签将以几乎任何颜色提供，并可以附着在各种识别模块上，以允许对存在于极低浓度的细胞表面和细胞内目标进行染色。检测和跟踪细胞中单分子的能力将显著提高我们对基本生物学过程以及健康和疾病状态之间分子水平差异的理解，最终使开发出副作用更少的更有效的疗法。

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.