Fluoromodules are discrete complexes of biomolecules and fluorogenic dyes. Binding of the dyes to their cognate biomolecule partners results in enhanced dye fluorescence. We exploited a previously reported promiscuous binding interaction between a single chain, variable fragment antibody protein and a family of cyanine dyes to create new protein-dye fluoromodules that exhibit enhanced photostability while retaining high affinity protein-dye binding. Modifications to the dye structure included electron withdrawing groups that provide resistance to photo-oxidative damage. Low nanomolar equilibrium dissociation constants were found for the new dyes. Fluorescence microscopy illustrates how yeast can be surface-labeled with three different colors based on a single protein and appropriately chosen dyes.

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 non-specific 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.

Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar K d values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.Fluoromodules are specific combinations of fluorogenic dyes and cognate protein 1-3 or nucleic acid 4-6 partners. Separately, neither component is fluorescent, but when reconstituted, strong fluorescence is observed. Rational design and straightforward synthesis allow access to fluorogenic dyes spanning the visible and near IR wavelengths while powerful in vitro and in berget@cmu.edu. army@cmu.edu. † Present address: Department of Plant Biology, University of Georgia, Athens, GA 30602.Supporting Information Available: Binding titration curves for yeast-displayed K7 with various cyanine dyes and for soluble K7 with DIR; sequence information for scFvs; experimental details for scFv selection, fluorescence microscopy and binding titrations. These reagents have already been useful in visualizing cell surface elements and certain membrane proteins within the secretory apparatus of mammalian cells. 7 Some spectral variation was generated by combining a limited set of these scFvs and fluorogen derivatives. However, the spectral range of fluorescence emission is constrained by the chromophores of the fluorogenic dyes and the methods used to select the FAPs. NIH Public AccessDimethylindole Red (DIR, Chart 1) is a fluorogenic cyanine dye. It was designed to have low nonspecific binding to DNA and RNA by using the bulky dimethylindole heterocycle to suppress intercalation and the anionic propyl sulfonate group to introduce nonspecific electrostatic repulsions from nucleic acids. 9 An RNA aptamer that was selected for binding to DIR exhibited K d = 86 nM and enhanced the fluorescence of the dye ca. 60-fold. 9 Given our earlier success in selecting scFvs for binding to the related unsymmetrical cyanine TO1-2p (Chart 1), we next subjected DIR to the two-step scFv selection procedure.A biotin analogue of DIR 9 was used to enrich the complex yeast surface display scFv library composed of ca. 10 9 clones of synthetically recombined human heavy and light chain variable regions. 8 This enrichment was accomplished by two rounds of sequential selection using streptavidin magnetic microbeads followed by anti-biotin magnetic microbeads. 10 The resulting yeast sub-library was further enriched by 3 rounds of fluorescence activated cell sorting, gating the cell sorter for cells that directly activated DIR fluorescence. Individuals from these enriched populations were automatically cloned by the cyto...