Bio-imaging with Isothermal DNA Self-Assembly

项目摘要 核酸在细胞内起到重要的遗传和调节作用，并且核酸的光学成像是细胞内的重要组成部分。 酸导致了对生物系统行为的许多见解。目前的原位和体内方法 核酸成像在其灵敏度、定量精确度、特异性和多重性方面受到限制。DNA 原则上，纳米技术可以改善所有四类生物成像性能，但传统的DNA 纳米技术需要热退火，并且不能容易地应用于生物系统。 在这个提议中，DNA和RNA纳米结构以及等温组装和操作的纳米器件是 作为生物成像工具展示和测试。对于原位全胚胎mRNA成像，几何精确的DNA 纳米结构将充当对每个感兴趣的mRNA靶特异性的明亮的光学“标签”。每个DNA纳米结构 标签具有精确数量的功能化荧光团，因此荧光可以直接映射到浓度 或拷贝数。此外，共定位于每个靶分子的大量荧光团将促进 通过降低显微镜灵敏度要求来成像。 对于活细胞和生物体成像，提出了两种不同的方法。第一种方法确保高度 使用最近开发的分子机制模拟解链温度条件的特异性成像 在不同的温度盐度和浓度下通过采用这种机制， 通过将核酸探针显微注射到活细胞中，可以实现内源性核酸的高度特异性成像。 这与成像microRNA特别相关，microRNA是起重要调节作用的短RNA分子 在细胞内，它们与其他microRNA的差异通常只有一个碱基对。 第二种可能更强大的方法是构建基因编码的变构蛋白， RNA纳米器件。当内源性靶RNA分子与RNA纳米装置结合时，纳米装置 重新配置以显示激活基于GFP的条件荧光团的荧光的适体。的 条件荧光团足够小，可以扩散到活细胞中，因此可以对内源性RNA进行成像 而不使用任何外来引入的探针。 最初的体外研究已经取得了可喜的成果。在两种材料中等温组装的DNA纳米结构 天然和变性条件已经通过凝胶电泳、原子力显微镜和总的 内反射荧光显微镜，研究将很快在整个果蝇原位成像 果蝇（Melanogaster）胚胎。用于确保高特异性核酸杂交的机制已经被证实。 在各种温度和盐度下，以及靶序列中典型的单碱基变化， 导致杂交受损26倍。

Project Summary Nucleic acids serve important hereditary and regulatory roles within cells, and the optical imaging of nucleic acids has led to many insights on the behavior of biological systems. Current in situ and in vivo methods for nucleic acid imaging are limited in their sensitivity, quantitative precision, specificity, and multiplexing. DNA nanotechnology can, in principle, improve bio-imaging performance in all four categories, but conventional DNA nanotechnology requires thermal annealing and cannot easily be applied to biological systems. In this proposal, DNA and RNA nanostructures and nanodevices that assemble and operate isothermally are presented and tested as bio-imaging tools. For in situ whole embryo mRNA imaging, geometrically precise DNA nanostructures will act as bright optical "tags" specific to each mRNA target of interest. Each DNA nanostructure tag has a precise number of functionalized fluorophores, so fluorescence can be directly mapped to concentration or copy number. Furthermore, the large number of fluorophores colocalized to each target molecule will facilitate imaging by reducing microscope sensitivity requirements. For live cell and organism imaging, two different approaches are proposed. The first approach ensures highly specific imaging using a recently developed molecular mechanism for mimicking melting temperature conditions across a range of temperatures, salinities, and concentrations. By adopting this mechanism to fluorescent nucleic acid probes microinjected into living cells, highly specific imaging of endogenous nucleic acids can be achieved. This is particular relevant for imaging microRNAs, short RNA molecules that play important regulatory roles inside the cell, that often differ from other microRNAs by as little as a single base pair. The second, potentially much more powerful, approach is the construction of an genetically encoded allosteric RNA nanodevice. When an endogenous target RNA molecule binds to the RNA nanodevice, the nanodevice reconfigures to reveal an aptamer that activates the fluorescence of a GFP-based conditional fluorophore. The conditional fluorophore is small enough to diffuse into living cells, so it will be possible to image endogenous RNA without the use of any exogeneously introduced probes. Initial in vitro studies have yielded promising results. Isothermally assembled DNA nanostructures in both native and denaturing conditions have been verified by gel electrophoresis, atomic force microscopy, and total internal reflection fluorescence microscopy, and studies will shortly being on the in situ imaging of whole Drosophila Melanogaster (fruit fly) embryos. The mechanism for ensuring high specificity nucleic acid hybridization has been demonstrated across a variety of temperatures and salinities, and a typical single-base change in target sequence causes hybridization to be impaired by a factor of 26.