Nucleic Acid Specificity, Cellular Localization and Reduced Toxicities of Thiazole Orange‐Neomycin Conjugates

Selective binding of small molecule ligands to nucleic acids with high affinity and limited toxicity remains an important goal in the development of compounds that can probe DNA or RNA in cells. Thiazole orange is a cell semi-permeant, fluorescent cyanine dye, with low background noise, that binds several forms of nucleic acids. However, thiazole orange can exhibit cytotoxicity when used at high concentration and/or with prolonged exposure. Neomycin is a non-fluorescent antibiotic with affinity for several forms of nucleic acids, but does not readily enter mammalian cells. Conjugation of neomycin with thiazole orange can exploit the properties of each individual compound, yielding a small molecule that could be used for nontoxic application in cellular analysis by microscopic imaging. We demonstrate that conjugation of neomycin with thiazole orange increases the cell permeability of neomycin, decreases the cytotoxicity of thiazole orange, and exhibits a greater degree of intracellular RNA targeted localization in the nucleolus, when compared to thiazole orange. Relative to thiazole orange, the conjugated compounds showed a much higher degree of stabilization of the nucleic acids as reflected in a greater denaturation temperature. Ultimately, our studies indicate that the conjugated thiazole orange-neomycin compounds can be used as an RNA targeted, less cytotoxic alternative for cellular labeling.