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 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.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

“…TO gave weaker fluorescence in the presence of the protein HL1.0.1-TO1, compared with the propylsulfonate-substituted TO1 (Figure S33), so we used a different FAP for these experiments. Specifically, R1, a recombinant scFv originally selected for a photostable derivative of TO 21 was used for these experiments based on its ability to bind and activate TO with subnanomolar affinity (174 pM) and strongly enhanced fluorescence quantum yield (φ f = 0.89, a 4400-fold increase) (Table 1). Interestingly, R1 showed minimal activation of TO1 (Figure S33), further illustrating the sensitivity of these proteins toward the nature of the substituent on the benzothiazole heterocycle of thiazole orange dyes.…”

Relation of molecular structure to Franck–Condon bands in the visible-light absorption spectra of symmetric cationic cyanine dyes

“…The in silico design and validation of organic light receptors [1][2][3][4] requires efficient computational methods that can predict the absolute or relative energies of important vibronic transitions, and assign these to specific molecular structures [5][6][7]. A challenging test for candidate methods is found in the cyanines, a widely studied class of organic dyes whose solution spectra vary in dramatic ways depending on the solvent polarity, dye aggregation and other intermolecular interactions [8][9][10][11][12].…”

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

“…After binding, the fluorescence signal increased by 1,100-fold (17). The thiazole orange dye is well known to exhibit high cellular background fluorescence due to nonspecific binding with cellular DNA and RNA, and it exhibits rapid photobleaching (53). However, the high binding affinity of RNA Mango can partially enhance the signal-to-noise ratio when imaging, as only small amount of dye is needed.…”

“…The background fluorescence can be explained by partitioning into viscous membrane bilayers or nonspecific binding to cellular compounds. Background fluorescence due to nonspecific binding of thiazole orange to DNA was recently reduced by introducing a nitrile moiety that made the thiazole orange dye adopt a nonplanar configuration (53). Careful manipulation of fluorophore structures to minimize cellular interactions will be needed to ensure that new fluorophores used in RNA-fluorophore complexes have low background fluorescence.…”