Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule
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- 作者：Nathaniel I. Shank ; Ha H. Pham ; Alan S. Waggoner ; Bruce A. Armitage
- 刊名：The Journal of the American Chemical Society
- 出版年：2013
- 出版时间：January 9, 2013
- 年：2013
- 卷：135
- 期：1
- 页码：242-251
- 全文大小：626K
- 年卷期：v.135,no.1(January 9, 2013)
- ISSN：1520-5126
文摘
The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here, we report the synthesis of a bridge-substituted version of TO named 伪-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that 伪-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. 伪-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that 伪-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of 伪-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. 伪-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new 伪-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that 伪-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules.