Bio-imaging with Isothermal DNA Self-Assembly

利用等温 DNA 自组装进行生物成像

• 批准号: 8701292
• 负责人: David Yu Zhang
• 金额: $ 24.15万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701292
• 关键词: Address; Adopted; Affinity; Atomic Force Microscopy; Base Pairing; Base Sequence; Behavior; Binding; Biology; Caenorhabditis elegans; Categories; Cell physiology; Cells; Cellular biology; DNA; DNA Probes; Development; Developmental Biology; Diffuse; Disease Marker; Drosophila genus; Drosophila melanogaster; Elements; Embryo; Ensure; Escherichia coli; Family; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Gene Expression; Genes; Goals; Heredity; Image; Imaging Device; In Situ; In Vitro; Individual; Inherited; Kinetics; Label; Larva; Life; Maps; Messenger RNA; Methods; MicroRNAs; Microscope; Molecular; Molecular Biology; Nanostructures; Nanotechnology; Nucleic Acid Hybridization; Nucleic Acid Probes; Nucleic Acids; Nucleic acid sequencing; Oligonucleotides; Optics; Organism; Pattern; Performance; Play; RNA; Reaction; Resolution; Role; Signal Transduction; Site; Spatial Distribution; Specificity; System; Technology; Temperature; Testing; Time; Work; aptamer; base; biological systems; cellular imaging; deep sequencing; design; fluorophore; gel electrophoresis; imaging modality; improved; in vivo; in vivo imaging; innovation; insight; interest; mRNA Expression; melting; monomer; nano; nanodevice; novel; nucleic acid localization; optical imaging; professor; response; self assembly; small molecule

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.

项目摘要 核酸在细胞内起重要的遗传性和调节作用，核成像的光学成像 酸导致了许多对生物系统行为的见解。当前原位和体内方法 核酸成像的敏感性，定量精度，特异性和多路复用受到限制。脱氧核糖核酸 纳米技术原则上可以改善这四个类别的生物成像性能，但是常规的DNA 纳米技术需要热退火，并且不能轻易应用于生物系统。 在此提案中，组装和运行等温度的DNA和RNA纳米结构和纳米构造是 呈现并作为生物成像工具进行了测试。对于原位整个胚胎mRNA成像，几何精确的DNA 纳米结构将充当每个感兴趣的mRNA目标特定的光学“标签”。每个DNA纳米结构 TAG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 或复制号码。此外，与每个靶分子共定位的大量荧光团将有助于 通过降低显微镜灵敏度要求进行成像。 对于活细胞和有机体成像，提出了两种不同的方法。第一种方法可确保 使用最近开发的分子机制来模仿熔化温度条件的特定成像 在一系列温度，盐度和浓度范围内。通过对荧光核的采用这种机制 酸探针微注射到活细胞中，可以实现内源性核酸的高度特异性成像。 这与成像microRNA，扮演重要调节作用的短RNA分子特别相关 在细胞内，通常与其他microRNA不同的是单个碱基对。 第二种可能更强大的方法是构建遗传编码的变构的方法 RNA纳米涂片。当内源性靶RNA分子与RNA纳米涂片结合时，纳米电视 重新配置以揭示适体激活基于GFP的条件荧光团的荧光。这 有条件的荧光团足够小，可以扩散到活细胞中，因此可以成像内源性RNA 无需使用任何外来引入的探针。 最初的体外研究产生了令人鼓舞的结果。两者中等温组装的DNA纳米结构 天然和变性条件已通过凝胶电泳，原子力显微镜和总验证 内部反射荧光显微镜，并且研究将很快就在整个果蝇的原位成像上 Melanogaster（果蝇）胚胎。确保高特异性核酸杂交的机制已有 在各种温度和盐度中都证明，目标序列的典型单基数变化 导致杂交的损害受损害26。