UV−vis and CD spectroscopy reveal that a tricationic cyanine dye spontaneously assembles into a helical J-aggregate in the presence of a double-helical DNA template. The stability of the J-aggregate is strongly dependent on the dye concentration and DNA length in a manner that reflects a high degree of cooperativity in formation of the aggregate. Slight changes in environmental conditions such as temperature and ionic strength result in interconversion between J- and H-aggregates. The aggregate likely consists of dimeric units assembled in an offset, face-to-face orientation within the minor groove of the DNA template, analogous to an earlier report of H-aggregation on DNA by a related cyanine dye. A model is proposed that relates the two aggregate structures by translation of one monomer from a given dimer along the floor of the minor groove. This translation requires adjacent monomers to also translate, leading to the observed cooperativity.
Unsymmetrical cyanine dyes are widely used in biomolecular detection due to their fluorogenic behavior, whereby fluorescence quantum yields can be very low in fluid solution but are significantly enhanced in conformationally restricted environments. Herein we describe a series of fluorinated analogues of the dye thiazole orange that exhibit improved fluorescence quantum yields and photostabilities. In addition, computational studies on these dyes revealed that twisting about the monomethine bridge beyond an interplanar angle of 60° leads to a dark state that decays nonradiatively to the ground state, accounting for the observed fluorogenic behavior. The effects of position and number of fluorine substituents correlates with both observed quantum yield and calculated activation energy for twisting beyond this critical angle.
A new fluorogenic cyanine dye was synthesized and found to have low fluorescence quantum yield in fluid solution and in the presence of double-stranded DNA but 80-fold enhanced fluorescence in viscous glycerol solution. An RNA aptamer selected for binding to the new dye exhibits K(d) = 87 nM and 60-fold fluorescence enhancement. The dye-aptamer pair is a fluoromodule that can be incorporated into fluorescent sensors and labels.
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...
Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye. Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes covering much of the visible and near-IR spectrum. The current work expands our repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic blue-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles. The dye binds to both yeast cell surface-displayed and soluble scFvs with low nanomolar Kd values. These dye-protein fluoromodules exhibit high quantum yields, approaching unity for the brightest system. The promiscuity of these scFvs with other fluorogenic cyanine dyes was also examined. Fluorescence microscopy demonstrates that the yeast cell surface-displayed scFvs can be used for multicolor imaging. The prevalence of 405 nm lasers on confocal imaging and flow cytometry systems make these new reagents potentially valuable for cell biological studies.
Thirty-seven naturally occurring withanolides (1-37), previously isolated in our laboratories, were evaluated for their potential to induce quinone reductase with cultured murine hepatoma cells (Hepa 1c1c7). Spiranoid (29, 32) and 18-functionalized withanolides (2-5, 7-9, 24) were found to be potent inducers of the enzyme, while 5alpha-substituted derivatives exhibited weak activity. Preliminary studies were performed with compound 29 to evaluate enzyme-inducing capacity in multiple organ sites of BALB/c mice. Significant induction was observed in liver and colon, but not in lung, stomach, or mammary gland.
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