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.

Public Health Relevance

The temporal and spatial distribution of RNAs has great influence over the function of cells and development of organisms, and optical imaging of RNA is an important method by which biology can be studied. This project uses isothermal DNA and RNA self-assembly and reconfiguration methods to vastly improving the sensitivity, quantitative precision, specificity, and multiplexing capabilities of in situ and in vivo RNA imaging.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Career Transition Award (K99)
Project #
5K99EB015331-02
Application #
8449254
Study Section
Special Emphasis Panel (ZEB1-OSR-D (J2))
Program Officer
Erim, Zeynep
Project Start
2012-04-01
Project End
2013-06-30
Budget Start
2013-04-01
Budget End
2013-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$89,352
Indirect Cost
$6,619
Name
Harvard University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Sadowski, John P; Calvert, Colby R; Zhang, David Yu et al. (2014) Developmental self-assembly of a DNA tetrahedron. ACS Nano 8:3251-9
Wu, Yuqiang; Zhang, David Yu; Yin, Peng et al. (2014) Ultraspecific and highly sensitive nucleic acid detection by integrating a DNA catalytic network with a label-free microcavity. Small 10:2067-76
Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg (2013) Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA. Nat Chem 5:782-9
Zhang, David Yu; Hariadi, Rizal F; Choi, Harry M T et al. (2013) Integrating DNA strand-displacement circuitry with DNA tile self-assembly. Nat Commun 4:1965