44470-AC4
Fluorescent DNA Nanostructures for Applications in Sensors and Imaging
Introduction. The goal of this project was 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 (along with a subsequent diversity supplement) was funded to support synthetic efforts to covalently attach the dyes to the DNA backbone, creating more stable structures. Synthesis. Our approach for synthesizing covalent analogues of our DNA nanotags began with preparing several thiazole orange monoazides with different linker lengths and attachment sites for the reactive azide group. In a collaboration with Prof. Thomas Carell of Characterization. The three monofunctionalized DNA-dye conjugates synthesized in the Carell lab were hybridized to unmodified complementary DNA strands and characterized by optical spectroscopy. First, UV melting experiments were performed on the duplexes. As expected, the melting temperatures of the dye-functionalized duplexes were higher than that of the corresponding unmodified duplex due to intercalation of the attached dye into the helix. The construct in which the reactive azide group was attached on the benzothiazole ring using a longer linker gave the highest melting temperature, indicating that the dye is better accommodated into the helix by the longer linker. This was also consistent with fluorescence spectra which showed strongest fluorescence for this construct. Preliminary characterization of the first pentafunctionalized DNA strand synthesized by the Carell lab revealed a 3.5-fold increase in brightness relative to the monofunctionalized strand. Although this is lower than the theoretical maximum of 5-fold enhancement, this result shows that multiple dyes can be covalently attached and intercalated into a DNA duplex with a substantially enhanced brightness. We will soon begin testing the three-way junction bearing 15 dyes. Plans. We will prepare a manuscript describing the synthesis and characterization of the mono- and penta-functionalized DNA strands, including fluorescence labeling results. Also, In collaboration with CMU faculty members Brooke McCartney (Biological Sciences) and Linda Peteanu (Chemistry), we will attach these strands through established conjugation chemistry to antibodies against cell-surface proteins and common reporter proteins such as GFP. This will allow bright fluorescence labeling of cell membranes and internal proteins. The NIH recently awarded our team a four year grant to pursue these experiments and continue development of this promising new fluorescence labeling technology. Receiving this grant would not have been possible without the generous support of the Petroleum Research Fund and we are grateful.
