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.

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