Molecular Engineering of Thiazole Orange Dye: Change of Fluorescent Signaling from Universal to Specific upon Binding with Nucleic Acids in Bioassay

Abstract

The universal fluorescent staining property of thiazole orange (TO) dye was adapted in order to be specific for G-quadruplex DNA structures, through the introduction of a styrene-like substituent at the ortho-position of the TO scaffold. This extraordinary outcome was determined from experimental studies and further explored through molecular docking studies. The molecular docking studies help understand how such a small substituent leads to remarkable fluorescent signal discrimination between G-quadruplex DNA and other types of nucleic acids. The results reveal that the modified dyes bind to the G-quadruplex or duplex DNA in a similar fashion as TO, but exhibit either enhanced or quenched fluorescent signal, which is determined by the spatial length and orientation of the substituent and has never been known. The new fluorescent dye modified with a p-(dimethylamino)styryl substituent offers 10-fold more selectivity toward telomeric G-quadruplexes than double-stranded DNA substrates. In addition, native PAGE experiments, FRET, CD analysis, and live cell imaging were also studied and demonstrated the potential applications of this class of thiazole-orange-based fluorescent probes in bioassays and cell imaging.

Design of Modular G‐quadruplex Ligands

“…Fluorescent dyes 112 – 114 have been designed based on structure 110 by systematically introducing various styrene‐like substituents at the C2 position of the quinoline ring . According to the FRET results, the substituents play a critical role in the interaction with the G4.…”

“…Most of the compounds of this series present higher stabilization ability than 111 , good fluorescence discrimination between G4 and dsDNA, and high relative quantum yields upon binding with G4. In addition, the potential application of these dyes as in vitro biochemical reagents has been successfully demonstrated by native polyacrylamide gel electrophoresis (PAGE) experiments and live‐cell staining and imaging …”

Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy

“…These ligands can be regarded as a tumor-specific receptor to construct a "guided molecular missile" with different therapeutic warheads. In an effort to contribute to the development of optical imaging modalities, a variety of fluorescent dyes e.g., fluorescein, Texas Red, the rhodamines, the cyanines (indocyanine green, thiazole orange) [57][58][59], polymethine cyanine dyes (the Cy3, Cy5, Cy5.5, Cy7 series) and the Alexa Fluors have been conjugated to folic acid [60][61][62][63][64]. Nowadays, the development of fluorescent dyes with longer excitation wavelengths (λEx>500 nm) transparent to human tissues [65,66] shows great promise, because imaging of malignant cells by optical means is not hazardous for living organisms [67].…”

Combining a Ru(II) “Building Block” and Rapid Screening Approach to Identify DNA Structure-Selective “Light Switch” Compounds

“…13 Certain DNA defects and structures are more prevalent in human diseases, and the design of molecules with increasingly selective DNA recognition is an active area in drug discovery research. 7, 16–21 Two important goals involve identifying molecules that luminesce upon binding to specific DNA sequences or structures 22–26 and assay development for rapid identification of compounds that selectively bind biologically relevant DNA targets. 27 …”