DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging

DNA 纳米标签：用于细胞内成像的明亮荧光标签和传感器

基本信息

批准号：
7818598
负责人：
Bruce A. ARMITAGE
金额：
$ 37.23万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7818598
关键词：
Antibodies Behavior Binding Biochemical Biology Cell Culture Techniques Cell surface Cells Chemistry Communities Complex Cytoplasmic Protein DNA DNA Sequence Data Detection Development Drosophila genus Dyes Elements Embryo Energy Transfer Enzymes Extinction (Psychology)Flow Cytometry Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Fluorescent Dyes Fluorescent Probes Funding Gene Expression Genomics Goals Grant Harvest Image Imaging Techniques In Vitro Incubated Injection of therapeutic agent Intercalating Agents Label Length Life Methods Microscopy Monitor Names Nanostructures Oligonucleotides Optics Pattern Photobleaching Predisposition Principal Investigator Property Protein Analysis Proteins RNA Recovery Research Research Design Resistance Signal Transduction Site Solutions Staging Technology Time Translating United States National Institutes of Health Work antibody conjugate base density design fluorescence imaging human disease in vivo interest nanoscale nanostructured nuclease parent grant programs protein complex public health relevance research study scaffold self assembly sensor single molecule tool

项目摘要

DESCRIPTION (provided by applicant): This competitive revision proposal is submitted under Notice Number (NOT-OD-09-058) and Notice Title "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications". The primary goal of the parent grant is to create a new class of bright fluorescent labels that can be used for ultrasensitive protein detection and tracking in live cells. The fluorescent component of the label consists of a large number of fluorescent dyes covalently attached to a nanostructured DNA scaffold, hence the name DNA nanotags. The high extinction coefficient of this construct will translate into bright fluorescence. The nanotags will then be attached to recognition elements such as antibodies to allow specific labeling of proteins in microinjected Drosophila embryos. This revision retains the specific aims of the original grant, but adds two new aims that will extend the utility of nanotags to RNA labeling. The nanotags will have an extended, single stranded region on one strand, allowing sequence specific hybridization to the RNA of interest. A second probe bearing an energy acceptor dye will hybridize to an adjacent site, allowing energy transfer from the light-harvesting nanotag to the acceptor probe to occur only when the two strands have hybridized to the same RNA. The nanotag detection strategy will then be validated in syncytial stage Drosophila embryos by detecting two mRNAs with known localization patterns. The two aims added to the proposal are appropriate for the two-year funding cycle for this revision. PUBLIC HEALTH RELEVANCE: The proposed research will provide new tools for detecting and tracking RNA molecules in vivo. RNA is an important intermediate between genomic DNA and expressed proteins, but it is increasingly understood to be involved in regulating gene expression. Methods that allow detection of RNA at low levels is essential for understanding how RNA functions in the cell and how this is related to human disease.

描述（由申请人提供）：此竞争性修订提案根据通知号（NOT-OD-09-058）和通知标题提交，并“ NIH宣布恢复ACT的可用性用于竞争性修订申请”。父授予的主要目标是创建一类新的明亮荧光标签，该标签可用于活细胞中的超敏蛋白检测和跟踪。标签的荧光成分由大量荧光染料共价附着在纳米结构的DNA支架上，因此名称为DNA纳米塔。该构建体的高消光系数将转化为明亮的荧光。然后，纳米菌将附着在识别元件上，例如抗体，以允许在微注射的果蝇胚胎中特异性标记蛋白。此修订保留了原始赠款的具体目标，但添加了两个新的目标，这些目标将把纳米塔的效用扩展到RNA标签。纳米塔将在一条线上具有延伸的单链区域，从而使序列特异性杂交与感兴趣的RNA。带有能量受体染料的第二个探测器将杂交与相邻位点杂交，从而使能量转移从轻度收获的纳米塔到受体探针才发生仅当两条链杂交与同一RNA时发生。然后，将通过检测两个具有已知定位模式的mRNA来验证纳米塔检测策略。提案中添加的两个目标适用于此修订的两年资金周期。公共卫生相关性：拟议的研究将提供新的工具，用于在体内检测和跟踪RNA分子。 RNA是基因组DNA和表达蛋白质之间的重要中间体，但越来越理解它参与了调节基因表达。允许在低水平上检测RNA的方法对于理解RNA在细胞中的功能以及与人类疾病的关系是至关重要的。