Bio-imaging with Isothermal DNA Self-Assembly

描述（由申请人提供）：核酸在细胞内起重要的遗传和调节作用，核酸的光学成像已经导致了许多关于 生物系统的行为。目前用于核酸成像的原位和体内方法在其灵敏度、定量精确度、特异性和多重性方面受到限制。原则上，DNA纳米技术可以提高所有四类生物成像性能，但传统的DNA纳米技术需要热退火，不能轻易应用于生物系统。 在这个提议中，DNA和RNA纳米结构和纳米器件，组装和等温操作的生物成像工具和测试。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靶标特异性的明亮光学“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，大量的荧光团共定位到每个目标分子将通过降低显微镜灵敏度要求来促进成像。 对于活细胞和生物体成像，提出了两种不同的方法。第一种方法使用最近开发的分子机制来确保高度特异性成像，该分子机制用于在温度、盐度和浓度范围内模拟熔化温度条件。通过采用这种机制，将荧光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是在细胞内发挥重要调节作用的短RNA分子，通常与其他microRNA的差异仅为一个碱基对。 第二，潜在 更强大的方法是构建遗传编码的变构RNA纳米装置。当内源性靶RNA分子与RNA纳米器件结合时，纳米器件重新配置以显示激活基于GFP的条件荧光团的荧光的适体。条件荧光团足够小，可以扩散到活细胞中，因此可以在不使用任何外源引入的探针的情况下对内源RNA进行成像。 最初的体外研究已经取得了可喜的成果。在天然和变性条件下的等温组装DNA纳米结构已通过凝胶电泳、原子力显微镜和全内反射荧光显微镜进行了验证，并且不久将对整个果蝇（果蝇）胚胎进行原位成像研究。确保高特异性核酸杂交的机制已在各种温度和盐度下得到证实，靶序列中典型的单碱基变化导致杂交受损26倍。 公共卫生相关性：RNA的时空分布对细胞的功能和生物体的发育有着重要的影响，RNA的光学成像是研究生物学的重要手段。该项目使用等温DNA和RNA自组装和重新配置方法，大大提高了原位和体内RNA成像的灵敏度，定量精度，特异性和多路复用能力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The temporal and spatial distribution of RNAs has great influence over the function of cells and development of organisms, and optical imaging of RNA is an important method by which biology can be studied. This project uses isothermal DNA and RNA self-assembly and reconfiguration methods to vastly improving the sensitivity, quantitative precision, specificity, and multiplexing capabilities of in situ and in vivo RNA imaging.