ACS PRF | ACS
All e-Annual Reports
44470-AC4
Fluorescent DNA Nanostructures for Applications in Sensors and Imaging
Introduction. The goal of this project is to create bright fluorescent labels based on a DNA nanostructure (i.e. nanotag) “loaded” with dozens of fluorescent intercalating dyes. These multichromophore assemblies would be at least 10-fold brighter than the commonly used fluorescent protein, GFP, and comparable to the brightest labels based on phycobiliproteins. Previous work in the group demonstrated that DNA nanotags could be readily assembled and used to label polymer beads and mammalian cells for analysis by flow cytometry and fluorescence microscopy [1]. However, those studies also revealed that the noncovalent nature of the association between the intercalator dyes and the DNA scaffold allowed dissociation at the low concentrations that would be used in many imaging and sensing applications. The PRF grant was funded to support synthetic efforts to covalently attach the dyes to the DNA backbone, creating more stable structures. This report describes our progress toward this objective.
Synthesis. Four different strategies were envisioned in order to achieve high density covalent attachment of intercalating dyes to DNA. In the most promising approach, several thiazole orange monoazides were prepared with different linker lengths and attachment sites. We attempted to attach these dyes to DNA bearing 5-(1-propynyl)-uracil residues via dipolar cycloaddition (“click) reactions. Both Cu2+-catalyzed and uncatalyzed reactions were unsuccessful, based on HPLC and mass spectrometry. The likely cause of these failures was the use of an internal alkyne on the DNA, which is known to react slowly with azides. However, we recently established a collaboration with Prof. Thomas Carell of Ludwig-Maximilians University in Munich. Prof. Carell reported in 2006 the successful attachment in high yield of up to 5 nonintercalating dyes at consecutive positions of a DNA strand using click chemistry [2]. The necessary DNA monomer bearing a reactive terminal alkyne group and a new catalyst for the click reaction were synthesized in the Carell laboratory. We recently sent two of the azide-functionalized thiazole orange derivatives to the Carell laboratory for covalent attachment at one position of a DNA strand. The conjugation reactions went smoothly and the purified DNA-dye products will be shipped back to our lab for the evaluation experiments described below.
Characterization. The DNA-dye conjugates synthesized in the Carell lab will be hybridized to unmodified complementary DNA strands and characterized by optical spectroscopy. First, UV melting experiments will be performed on the duplexes. We expect to find that the melting temperatures of the dye-functionalized duplexes will be higher than that of the corresponding unmodified duplex due to intercalation of the attached dye into the helix. The melting temperature of the unmodified duplex will also be measured in the presence of free thiazole orange. These experiments will help to evaluate which of the different linkers and attachment sites are optimal for permitting intercalation into the DNA helix in preparation for synthesis of the poly-functionalized DNA strands needed to make the covalent nanotags.
Fluorescence spectra will also be measured for the DNA-dye conjugates in both single- and double-stranded forms. If the covalently attached TO dye successfully intercalates into the DNA, a large fluorescence enhancement should be observed. Again, comparing the different TO analogues will help to determine the best dye for synthesis of the covalent nanotags.
Alternative Nanostructures. In addition to the synthetic efforts described above, experiments were performed to assess alternative nanostructures that would be much brighter than the assemblies tested previously. These included nanogel structures prepared from crosslinked DNA units [3] as well as a tetrahedron assembled from four partially complementary strands [4]. While we found it difficult to control the size of the gels in the first approach, the fluorescent tetrahedra were well behaved. These assemblies exhibited the efficient energy transfer previously reported for the two-dimensional nanotags and also showed significantly enhanced stability to nuclease enzymes. This latter property will be important for applications requiring imaging in complex biological media or intracellular environments. A manuscript is in preparation describing these results.
Plans. Our goal for the next year is to characterize the monofunctionalized DNA strands recently synthesized by the Carell group and determine the best dye to use for moving forward. Then, three or four partially complementary strands will be prepared, each having 5 covalently attached dyes. The resulting self-assembled nanostructures will be characterized by gel electrophoresis and fluorescence spectroscopy and then used for labeling polystyrene beads and mammalian cells as done previously.
References
1. Benvin, A. L.; et al, J. Am. Chem. Soc. 2007, 129, 2025-2034.
2. Gierlich, J.; et al, Org. Lett. 2006, 8, 3639-3642.
3. Matsuura, K.; et al, Chem. Commun. 2003, 376-377.
4. Goodman, R. P.; et al, Science 2005, 310, 1661-1665.
