DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging

描述（由申请人提供）：本竞争性修订提案根据通知编号（NOT-OD-09-058）和通知标题“NIH宣布恢复法案资金可用于竞争性修订申请”提交。该基金的主要目标是创建一类新的明亮荧光标记，可用于活细胞中的超灵敏蛋白质检测和跟踪。标签的荧光成分由大量共价连接到纳米结构DNA支架的荧光染料组成，因此命名为DNA纳米标签。该构建体的高消光系数将转化为明亮的荧光。然后将纳米标签附着到识别元件上，如抗体，以允许对显微注射的果蝇胚胎中的蛋白质进行特异性标记。这次修订保留了原始拨款的具体目标，但增加了两个新的目标，将纳米标签的实用性扩展到RNA标记。纳米标签的一条链上将具有延伸的单链区域，允许与感兴趣的RNA进行序列特异性杂交。携带能量受体染料的第二探针将与相邻位点杂交，从而允许仅当两条链与相同RNA杂交时才发生从光捕获纳米标签到受体探针的能量转移。然后，通过检测具有已知定位模式的两种mRNA，在合胞期果蝇胚胎中验证纳米标签检测策略。提案中增加的两个目标适用于本次修订的两年供资周期。 公共卫生相关性：拟议中的研究将为检测和跟踪体内RNA分子提供新的工具。RNA是基因组DNA和表达蛋白质之间的重要中间体，但越来越多地理解其参与调节基因表达。允许检测低水平RNA的方法对于了解RNA在细胞中的功能以及这与人类疾病的关系至关重要。

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.