|
Nucleic Acid-Based Labels for Fluorescence Imaging and Detection |
|
Fluorescent dyes have been used for decades to stain biomolecules for imaging and detection applications. Many of these dyes bind to DNA and RNA, causing the nucleic acids to become fluorescent and therefore readily detected in a fluorescence microscope, a flow cytometer or a gel. Thus, the dyes can be thought of as labels for nucleic acids. However, once they are bound together, the nucleic acid-dye complex can be thought of as a potential fluorescent label for other molecules. Two projects currently underway in our laboratory follow this theme. In one project, branched DNA nanostructures serve as a template for assembling dozens of dye molecules into bright fluorescent arrays known as DNA nanotags that can be used to label polymer beads or mammalian cells. The second project utilizes in vitro selection technology to identify RNA aptamers that bind to a nonfluorescent dye and cause it to become fluorescent. These RNA fluoromodules have potential applications in biosensor-related research. Both projects are described in more detail below.
Fluorescent DNA Nanostructures (DNA Nanotags)
The goal of this project is to develop fluorescent labels that are many times brighter than individual fluorescent dye molecules. Two parameters affect fluorophore brightness: the molar extinction coefficient (e) and the fluorescence quantum yield (ff). Many fluorescent dyes already have high f values, but there is considerable room for improvement of e values. Our approach is to bind many fluorescent dye molecules to the same DNA template by a process known as intercalation. By using branched DNA nanostructures, a very high density of fluorescent dyes can be assembled (Figure 1), yielding an extremely bright structure. These assemblies can be used as fluorescent labels for flow cytometric analysis of polymer beads or for imaging of cell surfaces by confocal microscopy (Figure 2).
Figure 1. Assembly of fluorescent intercalator arrays on nanostructured DNA templates, i.e. DNA nanotags.
Figure 2. Testing of fluorescent DNA nanotags. Left: Flow cytometry experiment in which streptavidin-coated polystyrene beads were labeled with biotinylated DNA nanotags. As the number of “arms” in the branched DNA nanostructure increases, the brightness of the fluorescence increases. Right: DNA 3WJ nanotags label surface proteins on murine T-cells.
The commercial availability of both DNA oligonucleotides and various fluorescent intercalator dyes allows the simple assembly of DNA nanotags having very high brightness and any color in the visible spectrum. Current efforts are focused on (1) developing more complex nanostructures that can bind hundreds of fluorescent dyes to further improve brightness and (2) developing chemistry to covalently attach dyes to the DNA template to avoid problems that arise in some applications from dissociation of the dye from the DNA nanostructure.
RNA Fluoromodules
Unsymmetrical cyanine dyes such as thiazole orange (TO) exhibit very low fluorescence in fluid solution but significantly enhanced fluorescence in viscous media, including when bound to DNA. Thus, TO and other “fluorogenic” dyes are widely used to detect nucleic acids in gels and also as environmentally sensitive probes that can be attached to various biomolecules.
Thiazole Orange (TO) Dimethylindole Red (DIR)
We were interested in developing dye-nucleic acid pairs that can act as fluorescent labels. The dye should be nonfluorescent at all times except when the specific nucleic acid partner is present, at which time the dye should bind to the nucleic acid and become fluorescent. The nucleic acid-dye complex is referred to as a “fluoromodule” and applications for fluoromodules include labeling of intracellular structures and as signaling components of fluorescent biosensors.
An important first step in this project was to synthesize dyes that would exhibit little nonselective binding to DNA or RNA. The long wavelength dye “Dimethylindole Red” shown above was synthesized and showed very little fluorescence enhancement in the presence of either DNA or RNA (although a strong enhancement was observed in a viscous solvent like glycerol). In vitro selection methods were then used to identify single-stranded RNA molecules that bind tightly to the dye. These RNAs were amplified by PCR and isolated by cloning in E. coli. The best of these aptamers binds to the dye at low nanomolar concentrations and causes a 40fold increase in its fluorescence (Figure 3). Addition of the dye to a different aptamer leads to no significant enhancement of fluorescence.
Figure 3. A specific RNA aptamer (W2) strongly enhances DIR fluorescence while a pool or random RNA molecules does not.
Current efforts are underway to select aptamers for other fluorogenic dyes and to incorporate existing aptamers into intracellular biosensors.
Funding and Collaboration. Our work with fluorescent DNA nanotags and RNA fluoromodules has been supported by the National Institutes of Health, the National Science Foundation and the Petroleum Research Fund of the American Chemical Society. These projects benefit from collaborations with Prof. David Yaron of the CMU, Chemistry Department, who uses computational chemistry to understand the photophysical properties of fluorescent dyes, and Prof. Alan Waggoner, an expert in the development of fluorescent dyes for labeling of biomolecules. These projects are also part of a National Technology Center for Networks and Pathways at Carnegie Mellon and the University of Pittsburgh (directed by Alan Waggoner). The goal of the TCNP is to provide fluorescence imaging and sensing reagents and technologies to researchers studying biological signaling networks and pathways. For more information, go to www.mbic.cmu.edu.
Personnel
Andrea Benvin (4th year graduate student) Tudor Constantin (4th year graduate student) Sabrina Lusvarghi (3rd year graduate student) Hayriye Özhalici (2nd year graduate student) Dr. Gloria Silva (Visiting Assistant Professor) Tamara Hamilton (Junior chemistry major) Gerentt Chan (Sophomore chemistry major) Emma Cating (1st year chemistry major)
Publications
1. Silva, G. L.; Ediz, V.; Yaron, D.; Armitage, B. A. “Experimental and Computational Investigation of Unsymmetrical Cyanine Dyes: Understanding Torsionally Responsive Fluorogenic Dyes” J. Am. Chem. Soc. 2007 ASAP Article, DOI: 10.1021/ja070025z
2. Benvin, A. L.; Creeger, Y.; Fisher, G. W.; Ballou, B.; Waggoner, A. S.; Armitage, B. A. “Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates” J. Am. Chem. Soc. 2007; 129; 2025-2034.
|
|
The Armitage Group |