RESUMO
Uracil-deoxyribonucleic acid glycosylase (UDG) is known to function as an important base-excision repair enzyme and eliminate uracil from DNA molecules to maintain genomic integrity. A new small organic molecule (DID-VP) with interesting structural properties was synthesized as a G-quadruplex selective ligand and was demonstrated to be a sensitive luminescent switch-on probe in a convenient luminescent assay specifically for UDG detection in fetal bovine serum samples under rapid and simple conditions. This newly developed analytical method is based on the UDG enzymatic activity to unwind a duplex DNA substrate, and comprises a G-quadruplex-forming sequence (ON1) and uracil-containing DNA strand (ON2) to generate a remarkable fluorescence signal through the specific interaction of DID-VP with ON1. This luminescent switch-on assay is able to achieve high sensitivity and specificity for UDG over other enzymes. The application range of the present analytical system is found to be 0.05 to 1.00 U mL(-1) UDG with a very low detection limit of 0.005 U mL(-1). The recovery study of UDG in real samples gave a very good performance with 75.05%-102.7% recovery. In addition, an extended application of the assay in screening of UDG inhibitors is demonstrated. A good dose-dependence of the luminescence response with respect to the concentration of UDG inhibitors in samples was observed.
Assuntos
Sondas Moleculares/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Bovinos , DNA/química , DNA/metabolismo , Corantes Fluorescentes/química , Quadruplex G , Sondas Moleculares/química , Piridinas/química , Espectrometria de Fluorescência , Especificidade por Substrato , Uracila-DNA Glicosidase/antagonistas & inibidores , Uracila-DNA Glicosidase/sangueRESUMO
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.
Assuntos
Benzotiazóis/química , Ácidos Nucleicos/metabolismo , Quinolinas/química , Transdução de Sinais , Bioensaio , Eletroforese em Gel de Poliacrilamida , Conformação de Ácido NucleicoRESUMO
A new RNA-selective fluorescent dye integrated with a thiazole orange and a p-(methylthio)styryl moiety shows better nucleolus RNA staining and imaging performance in live cells than the commercial stains. It also exhibits excellent photostability, cell tolerance, and counterstain compatibility with 4',6-diamidino-2-phenylindole for specific RNA-DNA colocalization in bioassays.