Twisted Cyanines: A Non-Planar Fluorogenic Dye with Superior Photostability and its Use in a Protein-Based Fluoromodule

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.