Bio-imaging with Isothermal DNA Self-Assembly

利用等温 DNA 自组装进行生物成像

基本信息

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
项目类别：
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

项目摘要

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.

项目概要核酸在细胞内发挥重要的遗传和调节作用，核酸的光学成像酸带来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灵敏度、定量精度、特异性和多重性方面受到限制。脱氧核糖核酸原则上，纳米技术可以提高所有四个类别的生物成像性能，但传统 DNA 纳米技术需要热退火，并且不能轻易应用于生物系统。在该提案中，等温组装和操作的 DNA 和 RNA 纳米结构和纳米器件是作为生物成像工具进行展示和测试。用于原位全胚胎 mRNA 成像、几何精确的 DNA 纳米结构将充当针对每个感兴趣的 mRNA 目标的明亮光学“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副本编号。此外，大量荧光团共定位于每个目标分子将有助于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来模拟熔化温度条件的特定成像跨越一系列温度、盐度和浓度。通过采用这种机制来荧光核酸将酸性探针显微注射到活细胞中，可以实现内源核酸的高度特异性成像。这对于成像 microRNA（发挥重要调节作用的短 RNA 分子）尤其重要在细胞内部，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 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揭示激活基于 GFP 的条件荧光团的荧光的适体。这条件荧光团足够小，可以扩散到活细胞中，因此可以对内源性 RNA 进行成像不使用任何外源引入的探针。初步的体外研究已经取得了有希望的结果。等温组装的 DNA 纳米结构天然和变性条件已通过凝胶电泳、原子力显微镜和总内反射荧光显微镜，很快就会对整个果蝇进行原位成像研究黑腹果蝇（果蝇）胚胎。确保高特异性核酸杂交的机制已被证实在各种温度和盐度以及目标序列中典型的单碱基变化中得到证明导致杂交受损 26 倍。