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.
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.
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|Stadler, Andrea L; Delos Santos, Junriz O; Stensrud, Elizabeth S et al. (2011) Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging. Bioconjug Chem 22:1491-502|