title: Twisted cyanines: a non-planar fluorogenic dye with superior photostability and its use in a protein-based fluoromodule.
abstract: 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 α-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 α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-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 α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-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. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-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.
authors.affiliation: Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Nathaniel I
authors.initials: NI
authors.lastname: Shank
authors.forename: Ha H
authors.initials: HH
authors.lastname: Pham
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waggoner
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Armitage
mesh_terms: D052160:Benzothiazoles / Q000138:chemical synthesis / Q000737:chemistry*; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D055668:Photochemical Processes; D011804:Quinolines / Q000138:chemical synthesis / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D052160:Benzothiazoles; D005456:Fluorescent Dyes; D011804:Quinolines; D057127:Single-Chain Antibodies; C051000:thiazole orange; D004247:DNA