10.1021/ja308629w.s001
Nathaniel
I. Shank
Nathaniel
I.
Shank
Ha H. Pham
Ha H.
Pham
Alan S. Waggoner
Alan S.
Waggoner
Bruce A. Armitage
Bruce A.
Armitage
Twisted Cyanines: A Non-Planar Fluorogenic Dye with
Superior Photostability and its Use in a Protein-Based Fluoromodule
American Chemical Society
2013
Twisted Cyanines
protein binding partners
fluorogenic stain
methine hydrogen
fluorogenic dye
activates fluorescence
RNA
cyano group
nonplanar conformations
parent dye
DNA
HeLa cell culture
singlet oxygen
fluorogenic behavior
fluorescence activation
Superior Photostability
cationic dyes
background fluorescence
2013-01-09 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Twisted_Cyanines_A_Non_Planar_Fluorogenic_Dye_with_Superior_Photostability_and_its_Use_in_a_Protein_Based_Fluoromodule/2453749
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.